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    New England Biolabs xmni
    XmnI
    XmnI 5 000 units
    https://www.bioz.com/result/xmni/product/New England Biolabs
    Average 96 stars, based on 1 article reviews
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    xmni - by Bioz Stars, 2021-05
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    1) Product Images from "The mechanism of DNA replication termination in vertebrates"

    Article Title: The mechanism of DNA replication termination in vertebrates

    Journal: Nature

    doi: 10.1038/nature14887

    Cyclin A treatment synchronizes DNA replication in Xenopus egg extracts (A–B) To synchronize DNA replication in Xenopus egg extracts, we treated extracts with Cyclin A, which probably accelerates replication initiation 45 . Plasmid DNA was incubated in High Speed Supernatant for 20 minutes, then either buffer or Cyclin A was added for a further 20 minutes. NucleoPlasmic extract was added to initiate DNA replication, along with [α- 32 P]dATP to label replication intermediates. Replication products were separated on a native agarose gel, detected by autoradiography (A), and quantified (B). In the presence of vehicle, replication was not complete by 9.5 minutes, but in the presence of Cyclin A, replication was almost complete by 4.5 minutes (B). Thus, Cyclin A treatment approximately doubles the speed of DNA replication in Xenopus egg extracts. (C–F) To test whether Cyclin A affects the ability of LacR to inhibit termination, we monitored dissolution of plasmids containing a 12x or 16x LacR array in the presence and absence of Cyclin A. p[ lacO x12], p[ lacO x16], and the parental control plasmid p[ empty ] were incubated with LacR, and then treated with buffer or Cyclin A before replication was initiated with NPE in the presence of [α- 32 P]dATP. Samples were withdrawn when dissolution of p[ empty ] plateaued (9.5 minutes in the presence of buffer, 4.5 minutes in the presence of Cyclin A). Given that Cyclin A treatment approximately doubles the speed of replication (see B), samples were withdrawn from these reactions twice as frequently as the buffer-treated samples. To measure dissolution, radiolabelled termination intermediates were cut with XmnI to monitor the conversion of double-Y molecules to linear molecules. Cut molecules were separated on a native agarose gel and detected by autoradiography (C,E). By the time the first sample was withdrawn, dissolution of p[ empty ] was essentially complete, in the absence (9.5 min, D) or presence (4.5 min, F) of Cyclin A. Importantly, dissolution of p[ lacO x12] and p[ lacO x16] was prevented in the absence (9.5 min, D) or presence (4.5 min, F) of Cyclin A. Moreover, dissolution occurred approximately twice as fast in the presence of Cyclin A (note the similarity between (D) and (F) even though samples are withdrawn twice as frequently in F.) consistent with replication being approximately twice as fast in the presence of Cyclin A. Therefore, Cyclin A does not affect the ability of a LacR array to block replication forks.
    Figure Legend Snippet: Cyclin A treatment synchronizes DNA replication in Xenopus egg extracts (A–B) To synchronize DNA replication in Xenopus egg extracts, we treated extracts with Cyclin A, which probably accelerates replication initiation 45 . Plasmid DNA was incubated in High Speed Supernatant for 20 minutes, then either buffer or Cyclin A was added for a further 20 minutes. NucleoPlasmic extract was added to initiate DNA replication, along with [α- 32 P]dATP to label replication intermediates. Replication products were separated on a native agarose gel, detected by autoradiography (A), and quantified (B). In the presence of vehicle, replication was not complete by 9.5 minutes, but in the presence of Cyclin A, replication was almost complete by 4.5 minutes (B). Thus, Cyclin A treatment approximately doubles the speed of DNA replication in Xenopus egg extracts. (C–F) To test whether Cyclin A affects the ability of LacR to inhibit termination, we monitored dissolution of plasmids containing a 12x or 16x LacR array in the presence and absence of Cyclin A. p[ lacO x12], p[ lacO x16], and the parental control plasmid p[ empty ] were incubated with LacR, and then treated with buffer or Cyclin A before replication was initiated with NPE in the presence of [α- 32 P]dATP. Samples were withdrawn when dissolution of p[ empty ] plateaued (9.5 minutes in the presence of buffer, 4.5 minutes in the presence of Cyclin A). Given that Cyclin A treatment approximately doubles the speed of replication (see B), samples were withdrawn from these reactions twice as frequently as the buffer-treated samples. To measure dissolution, radiolabelled termination intermediates were cut with XmnI to monitor the conversion of double-Y molecules to linear molecules. Cut molecules were separated on a native agarose gel and detected by autoradiography (C,E). By the time the first sample was withdrawn, dissolution of p[ empty ] was essentially complete, in the absence (9.5 min, D) or presence (4.5 min, F) of Cyclin A. Importantly, dissolution of p[ lacO x12] and p[ lacO x16] was prevented in the absence (9.5 min, D) or presence (4.5 min, F) of Cyclin A. Moreover, dissolution occurred approximately twice as fast in the presence of Cyclin A (note the similarity between (D) and (F) even though samples are withdrawn twice as frequently in F.) consistent with replication being approximately twice as fast in the presence of Cyclin A. Therefore, Cyclin A does not affect the ability of a LacR array to block replication forks.

    Techniques Used: Plasmid Preparation, Incubation, Agarose Gel Electrophoresis, Autoradiography, Blocking Assay

    Topo II-dependent decatenation of p[lacOx16] (A) The autoradiograph in primary Figure 1G is reproduced with cartoons indicating the structures of the replication and termination intermediates n-n, n-sc, sc-sc, n, and sc (see main text for definitions). The order of appearance of the different catenanes matches previous work 5 (n-n, then n-sc, then sc-sc). (B–D) To determine the role of Topo II during termination within a lacO array, termination was monitored in mock- or Topo II-depleted extracts. To confirm immunodepletion of Topo II, Mock and Topo II-depleted NPE was blotted with MCM7 and Topo II antibodies (B). p[ lacO x16] was incubated with LacR, then replicated in either mock- or Topo II-depleted egg extracts in the presence of [α- 32 P]dATP, and termination was induced with IPTG (at 7’). Untreated DNA intermediates were separated by native gel electrophoresis (C). In the mock-depleted extract, nicked and supercoiled monomers were readily produced (as in (A), albeit with slower kinetics due to nonspecific inhibition of the extracts by the immunodepletion procedure), while in the Topo II-depleted extracts, a discrete species was produced. DNA from the last time point in each reaction (lanes 4 and 8 in (C)) was purified and treated with XmnI, which cuts p[ lacO x16] once, or Nt.BspQI, which nicks p[ lacO x16] once, or recombinant Topo II, and then separated by native gel electrophoresis (D). Cleavage of the mock- and TopoII-depleted products with XmnI yielded the expected linear 3.15 kb band (lanes 2 and 6), demonstrating that in both extracts, all products were fully dissolved topoisomers of each other. Relaxation of the mock-depleted products by nicking with Nt.BspQI yielded a discrete band corresponding to nicked plasmid (lane 3), while the TopoII-depleted products were converted to a ladder of discrete topoisomers (lane 7), which we infer represent catenated dimers of different linking numbers, since the mobility difference cannot be due to differences in supercoiling. Importantly, the mobility shift following Nt.BspQI treatment (lane 5 vs lane 7) demonstrated that the Topo II-depleted products (lane 5) were covalently closed and thus in the absence of Topo II ligation of the daughter strands still occurred. Treatment of the mock- and Topo II-depleted products with recombinant human Topo II produced the same relaxed monomeric species (lanes 4 and 8), further confirming that the Topo II-depleted products contained catenanes. Collectively, these observations demonstrate that termination within a lacO array in Topo II depleted extracts produces highly catenated supercoiled-supercoiled dimers, as seen in cells lacking Topo II 16 , 17 . These data confirm that Topo II is responsible for decatenation and argue that termination within a lacO array reflects physiological termination. (E) n-n, n-sc, sc-sc, n, and sc products were also detected when plasmid lacking lacO sequences (pBlueScript) was replicated in the absence of LacR without the use of Cyclin A to synchronize replication. Therefore, these intermediates arise in the course of unperturbed DNA replication in Xenopus egg extracts.
    Figure Legend Snippet: Topo II-dependent decatenation of p[lacOx16] (A) The autoradiograph in primary Figure 1G is reproduced with cartoons indicating the structures of the replication and termination intermediates n-n, n-sc, sc-sc, n, and sc (see main text for definitions). The order of appearance of the different catenanes matches previous work 5 (n-n, then n-sc, then sc-sc). (B–D) To determine the role of Topo II during termination within a lacO array, termination was monitored in mock- or Topo II-depleted extracts. To confirm immunodepletion of Topo II, Mock and Topo II-depleted NPE was blotted with MCM7 and Topo II antibodies (B). p[ lacO x16] was incubated with LacR, then replicated in either mock- or Topo II-depleted egg extracts in the presence of [α- 32 P]dATP, and termination was induced with IPTG (at 7’). Untreated DNA intermediates were separated by native gel electrophoresis (C). In the mock-depleted extract, nicked and supercoiled monomers were readily produced (as in (A), albeit with slower kinetics due to nonspecific inhibition of the extracts by the immunodepletion procedure), while in the Topo II-depleted extracts, a discrete species was produced. DNA from the last time point in each reaction (lanes 4 and 8 in (C)) was purified and treated with XmnI, which cuts p[ lacO x16] once, or Nt.BspQI, which nicks p[ lacO x16] once, or recombinant Topo II, and then separated by native gel electrophoresis (D). Cleavage of the mock- and TopoII-depleted products with XmnI yielded the expected linear 3.15 kb band (lanes 2 and 6), demonstrating that in both extracts, all products were fully dissolved topoisomers of each other. Relaxation of the mock-depleted products by nicking with Nt.BspQI yielded a discrete band corresponding to nicked plasmid (lane 3), while the TopoII-depleted products were converted to a ladder of discrete topoisomers (lane 7), which we infer represent catenated dimers of different linking numbers, since the mobility difference cannot be due to differences in supercoiling. Importantly, the mobility shift following Nt.BspQI treatment (lane 5 vs lane 7) demonstrated that the Topo II-depleted products (lane 5) were covalently closed and thus in the absence of Topo II ligation of the daughter strands still occurred. Treatment of the mock- and Topo II-depleted products with recombinant human Topo II produced the same relaxed monomeric species (lanes 4 and 8), further confirming that the Topo II-depleted products contained catenanes. Collectively, these observations demonstrate that termination within a lacO array in Topo II depleted extracts produces highly catenated supercoiled-supercoiled dimers, as seen in cells lacking Topo II 16 , 17 . These data confirm that Topo II is responsible for decatenation and argue that termination within a lacO array reflects physiological termination. (E) n-n, n-sc, sc-sc, n, and sc products were also detected when plasmid lacking lacO sequences (pBlueScript) was replicated in the absence of LacR without the use of Cyclin A to synchronize replication. Therefore, these intermediates arise in the course of unperturbed DNA replication in Xenopus egg extracts.

    Techniques Used: Autoradiography, Incubation, Nucleic Acid Electrophoresis, Produced, Inhibition, Purification, Recombinant, Plasmid Preparation, Mobility Shift, Ligation

    Replisome progression through 12x and 32x lacO arrays (A–D) To test whether replisomes meet later in a lacO x32 array than a lacO x12 array, we monitored dissolution. LacR Block-IPTG release was performed on p[ lacO x12] and p[ lacO x32] and radiolabelled termination intermediates were digested with XmnI to monitor the conversion of double-Y molecules to linear molecules (Dissolution). Cleaved molecules were separated on a native agarose gel, detected by autoradiography (A,C), and quantified (B,D). Upon IPTG addition, dissolution was delayed by at least 1 minute within the 32x lacO array compared to the 12x lacO array (B,D). Moreover, by 6 minutes, 92% of forks had undergone dissolution on p[ lacO x12] while only 9% had dissolved on p[ lacO x32] (B,D). (E) Stall products within the 12x lacO array ( Fig. 3B , Lane 2) were quantified, signal was corrected based on size differences of the products, and the percentage of stall products at each stall point was calculated. 78% of leading strands stalled at the first three arrest points (red columns), 19% stalled at the fourth to tenth arrest points (yellow columns) and the remaining 3% stalled at the tenth to fourteenth arrest points (grey columns). The appearance of fourteen arrest points reproducible and surprising, given that the presence of only twelve lacO sequences was confirmed by sequencing in the very preparation of p[ lacO x12] that was used in Fig. 3 . The thirteenth and fourteenth arrest points cannot stem from cryptic lacO sites beyond the twelfth lacO site, as this would position the first leftward leading strand stall product ∼90 nucleotides from the lacO array, instead of the observed ∼30 nucleotides (see F-G ). At present, we do not understand the origin of these stall products. (F–G) Progression of leftward leading strands into the array. The same DNA samples used in Fig. 3 were digested with the nicking enzyme Nb.BsrDI, which released leftward leading strands (F), and separated on a denaturing polyacrylamide gel (G). The lacO sites of p[ lacO x12] are highlighted in blue on the sequencing ladder (G), which was generated using the primer JDO109 (Green arrow, F). Green circles indicate two non-specific products of digestion. These products arise because nicking enzyme activity varies between experiments, even under the same conditions. There was no significant difference in the pattern of leftward leading strand progression between the 12x lacO and 32x lacO arrays, as seen for the rightward leading strands ( Fig. 3B ). Specifically, by 5.67 minutes, the majority of leading strands had extended beyond the seventh lacO repeat within lacO x12 (lane 6) and the equivalent region of lacO x32 (lane 18). Therefore, progression of leftward leading strands is unaffected by the presence of an opposing replisome, suggesting that converging replisomes do not stall when they meet.
    Figure Legend Snippet: Replisome progression through 12x and 32x lacO arrays (A–D) To test whether replisomes meet later in a lacO x32 array than a lacO x12 array, we monitored dissolution. LacR Block-IPTG release was performed on p[ lacO x12] and p[ lacO x32] and radiolabelled termination intermediates were digested with XmnI to monitor the conversion of double-Y molecules to linear molecules (Dissolution). Cleaved molecules were separated on a native agarose gel, detected by autoradiography (A,C), and quantified (B,D). Upon IPTG addition, dissolution was delayed by at least 1 minute within the 32x lacO array compared to the 12x lacO array (B,D). Moreover, by 6 minutes, 92% of forks had undergone dissolution on p[ lacO x12] while only 9% had dissolved on p[ lacO x32] (B,D). (E) Stall products within the 12x lacO array ( Fig. 3B , Lane 2) were quantified, signal was corrected based on size differences of the products, and the percentage of stall products at each stall point was calculated. 78% of leading strands stalled at the first three arrest points (red columns), 19% stalled at the fourth to tenth arrest points (yellow columns) and the remaining 3% stalled at the tenth to fourteenth arrest points (grey columns). The appearance of fourteen arrest points reproducible and surprising, given that the presence of only twelve lacO sequences was confirmed by sequencing in the very preparation of p[ lacO x12] that was used in Fig. 3 . The thirteenth and fourteenth arrest points cannot stem from cryptic lacO sites beyond the twelfth lacO site, as this would position the first leftward leading strand stall product ∼90 nucleotides from the lacO array, instead of the observed ∼30 nucleotides (see F-G ). At present, we do not understand the origin of these stall products. (F–G) Progression of leftward leading strands into the array. The same DNA samples used in Fig. 3 were digested with the nicking enzyme Nb.BsrDI, which released leftward leading strands (F), and separated on a denaturing polyacrylamide gel (G). The lacO sites of p[ lacO x12] are highlighted in blue on the sequencing ladder (G), which was generated using the primer JDO109 (Green arrow, F). Green circles indicate two non-specific products of digestion. These products arise because nicking enzyme activity varies between experiments, even under the same conditions. There was no significant difference in the pattern of leftward leading strand progression between the 12x lacO and 32x lacO arrays, as seen for the rightward leading strands ( Fig. 3B ). Specifically, by 5.67 minutes, the majority of leading strands had extended beyond the seventh lacO repeat within lacO x12 (lane 6) and the equivalent region of lacO x32 (lane 18). Therefore, progression of leftward leading strands is unaffected by the presence of an opposing replisome, suggesting that converging replisomes do not stall when they meet.

    Techniques Used: Blocking Assay, Agarose Gel Electrophoresis, Autoradiography, Sequencing, Generated, Activity Assay

    Supplemental ChIP data (A) Cartoon depicting the LAC, FLK2 and FAR loci, which were used for ChIP. Their precise locations relative to the leftward edge of the lacO array are indicated. The LAC amplicon is present in four copies distributed across the lacOx16 array and three copies distributed across the lacOx12 array. (B–E) p[ lacO x12] was incubated with buffer or LacR and termination was induced at 5 minutes by IPTG addition. MCM7, RPA, CDC45, and Polε ChIP was performed at different time points after IPTG addition but also in the buffer control and no IPTG control. Recovery of FLK2 was measured as a percentage of input DNA. Upon IPTG addition, ChIP signal declined and by 9 minutes was comparable to the buffer control, demonstrating that unloading of replisomes was induced within 4 minutes of IPTG addition. (F) To test whether movement of the replisome into and out of the lacO array could be detected upon IPTG addition, termination was monitored within a lacO array, and we performed ChIP of the leading strand polymerase Polε, which was inferred to move into and out of the array based on the behavior of leading strands during termination ( Extended Data Fig 2B–E ). It was predicted that Polε ChIP at the LAC locus should increase slightly as Polε enters the lacO array and decline again as converging polymerases pass each other, but persist at FLK2 while the polymerases move out of the array. Prior to IPTG addition, Polε was enriched at LAC and FLK2 compared to FAR , consistent with the leading strands being positioned on either side of the lacO array ( Extended Data Fig 2C , Fig. 3 ). Upon IPTG addition, Polε became modestly enriched at LAC compared to FLK2 (5.5 min) but then declined to similar levels at both LAC and FLK2 by 6.5 min. These data are consistent with the leading strand polymerases entering the lacO array and passing each other. (G–H) To test whether CMG exhibited the same ChIP profile as Polε, MCM7 and CDC45 ChIP was performed using the same samples. Following IPTG addition, MCM7 and CDC45 were enriched at LAC compared to FLK2 (5.5 min), then declined to similar levels at both LAC and FLK2 by 6.5 min, as seen for Polε (F). These data are consistent with a model in which CMGs enter the array and pass each other during termination. A caveat of these experiments is the relatively high recovery of the FAR locus in MCM7, CDC45, and Polε ChIP. Specifically, signal was at most only ∼2-fold enriched at LAC compared to FAR . This was not due to high background binding, because by the end of the experiment (10 minute time point, not shown), we observed a decrease in signal of ∼5–7 fold. Furthermore, we observed ∼5–7 fold enrichment in binding (ChIP) of replisome components to p[ lacO x12] that had been incubated in LacR compared to a buffer control (see G-I, below). Instead, the high FAR signal was likely due to poor spatial resolution of the ChIP. Consistent with this, when a plasmid containing a DNA interstrand cross-link (ICL) was replicated, essentially all replisomes converged upon the ICL but the ChIP signal for MCM7 and CDC45 was only ∼3–4 fold enriched at the ICL compared to a control locus 41 . We speculate that the higher background observed at the control locus in our experiments is due to the decreased distance of the control locus from the experimental locus (1.3 kb for p[ lacO x16] and p[ lacO x12] vs. 2.4 kb for the ICL plasmid) and possibly due to increased catenation of the parental strands during termination. The high signal at FAR should not complicate interpretation of the MCM7, CDC45 and Polε ChIP (F), as signal at FAR was essentially unaltered between 5 and 6.5 minutes. Further evidence that the high signal seen at the FAR locus emanates from forks stalled near the lacO array is presented in panel ( K ). (I) ChIP of RPA was performed on the same chromatin samples used in B-D. As seen for pol ɛ, MCM7, and CDC45, enrichment of RPA at LAC compared to FAR was relatively low, consistent with poor spatial resolution. (J) Predicted binding of CMGs to the LAC, FLK2 and FAR loci before and after IPTG addition if CMGs converging CMG pass each other. (K) To determine whether most forks stalled at the array and not elsewhere in the plasmid, we performed a time course in which p[ lacO x16] undergoing termination was examined by 2-dimensional gel electrophoresis (2-D gel) at various time points. p[ lacO x16] was pre-bound to LacR and replicated in Xenopus egg extract containing [α- 32 P]dATP. Termination was induced by IPTG addition and samples were withdrawn at different times. Radiolabelled replication intermediates were cleaved with XmnI (as in Extended Data Fig. 1A ) and separated according to size and shape on 2-D gels 50 . A parallel reaction was performed in which samples were analyzed by ChIP, which was one of the repeats analyzed in (B)-(E). In the presence of LacR, a subset of Double Y molecules accumulated (blue arrowhead), demonstrating that 83% of replication intermediates (signal in dashed blue circle) contained two forks converged at a specific locus. Following IPTG addition, linear molecules rapidly accumulated (orange arrow) as dissolution occurred. Importantly, the vast majority of signal was present in the discrete double-Y and linear species (blue and orange arrows), demonstrating that the relatively high ChIP signal observed at FAR in panels F-I was derived from forks present at the lacO x16 array and not elsewhere.
    Figure Legend Snippet: Supplemental ChIP data (A) Cartoon depicting the LAC, FLK2 and FAR loci, which were used for ChIP. Their precise locations relative to the leftward edge of the lacO array are indicated. The LAC amplicon is present in four copies distributed across the lacOx16 array and three copies distributed across the lacOx12 array. (B–E) p[ lacO x12] was incubated with buffer or LacR and termination was induced at 5 minutes by IPTG addition. MCM7, RPA, CDC45, and Polε ChIP was performed at different time points after IPTG addition but also in the buffer control and no IPTG control. Recovery of FLK2 was measured as a percentage of input DNA. Upon IPTG addition, ChIP signal declined and by 9 minutes was comparable to the buffer control, demonstrating that unloading of replisomes was induced within 4 minutes of IPTG addition. (F) To test whether movement of the replisome into and out of the lacO array could be detected upon IPTG addition, termination was monitored within a lacO array, and we performed ChIP of the leading strand polymerase Polε, which was inferred to move into and out of the array based on the behavior of leading strands during termination ( Extended Data Fig 2B–E ). It was predicted that Polε ChIP at the LAC locus should increase slightly as Polε enters the lacO array and decline again as converging polymerases pass each other, but persist at FLK2 while the polymerases move out of the array. Prior to IPTG addition, Polε was enriched at LAC and FLK2 compared to FAR , consistent with the leading strands being positioned on either side of the lacO array ( Extended Data Fig 2C , Fig. 3 ). Upon IPTG addition, Polε became modestly enriched at LAC compared to FLK2 (5.5 min) but then declined to similar levels at both LAC and FLK2 by 6.5 min. These data are consistent with the leading strand polymerases entering the lacO array and passing each other. (G–H) To test whether CMG exhibited the same ChIP profile as Polε, MCM7 and CDC45 ChIP was performed using the same samples. Following IPTG addition, MCM7 and CDC45 were enriched at LAC compared to FLK2 (5.5 min), then declined to similar levels at both LAC and FLK2 by 6.5 min, as seen for Polε (F). These data are consistent with a model in which CMGs enter the array and pass each other during termination. A caveat of these experiments is the relatively high recovery of the FAR locus in MCM7, CDC45, and Polε ChIP. Specifically, signal was at most only ∼2-fold enriched at LAC compared to FAR . This was not due to high background binding, because by the end of the experiment (10 minute time point, not shown), we observed a decrease in signal of ∼5–7 fold. Furthermore, we observed ∼5–7 fold enrichment in binding (ChIP) of replisome components to p[ lacO x12] that had been incubated in LacR compared to a buffer control (see G-I, below). Instead, the high FAR signal was likely due to poor spatial resolution of the ChIP. Consistent with this, when a plasmid containing a DNA interstrand cross-link (ICL) was replicated, essentially all replisomes converged upon the ICL but the ChIP signal for MCM7 and CDC45 was only ∼3–4 fold enriched at the ICL compared to a control locus 41 . We speculate that the higher background observed at the control locus in our experiments is due to the decreased distance of the control locus from the experimental locus (1.3 kb for p[ lacO x16] and p[ lacO x12] vs. 2.4 kb for the ICL plasmid) and possibly due to increased catenation of the parental strands during termination. The high signal at FAR should not complicate interpretation of the MCM7, CDC45 and Polε ChIP (F), as signal at FAR was essentially unaltered between 5 and 6.5 minutes. Further evidence that the high signal seen at the FAR locus emanates from forks stalled near the lacO array is presented in panel ( K ). (I) ChIP of RPA was performed on the same chromatin samples used in B-D. As seen for pol ɛ, MCM7, and CDC45, enrichment of RPA at LAC compared to FAR was relatively low, consistent with poor spatial resolution. (J) Predicted binding of CMGs to the LAC, FLK2 and FAR loci before and after IPTG addition if CMGs converging CMG pass each other. (K) To determine whether most forks stalled at the array and not elsewhere in the plasmid, we performed a time course in which p[ lacO x16] undergoing termination was examined by 2-dimensional gel electrophoresis (2-D gel) at various time points. p[ lacO x16] was pre-bound to LacR and replicated in Xenopus egg extract containing [α- 32 P]dATP. Termination was induced by IPTG addition and samples were withdrawn at different times. Radiolabelled replication intermediates were cleaved with XmnI (as in Extended Data Fig. 1A ) and separated according to size and shape on 2-D gels 50 . A parallel reaction was performed in which samples were analyzed by ChIP, which was one of the repeats analyzed in (B)-(E). In the presence of LacR, a subset of Double Y molecules accumulated (blue arrowhead), demonstrating that 83% of replication intermediates (signal in dashed blue circle) contained two forks converged at a specific locus. Following IPTG addition, linear molecules rapidly accumulated (orange arrow) as dissolution occurred. Importantly, the vast majority of signal was present in the discrete double-Y and linear species (blue and orange arrows), demonstrating that the relatively high ChIP signal observed at FAR in panels F-I was derived from forks present at the lacO x16 array and not elsewhere.

    Techniques Used: Chromatin Immunoprecipitation, Amplification, Incubation, Recombinase Polymerase Amplification, Binding Assay, Plasmid Preparation, Nucleic Acid Electrophoresis, Derivative Assay

    Supplemental termination data for p[empty] experiments (A) Cartoon depicting the XmnI and AlwNI sites on p[ empty ], which are used for the dissolution and ligation assays, respectively, and the FLK2 locus, which is used for ChIP. (B) Plasmid DNA without a lacO array (p[ empty ]) was replicated and at different times chromatin was subjected to MCM7 and CDC45 ChIP. Percent recovery of FLK2 was quantified and used to measure dissociation of MCM7 and CDC45 (see methods). Dissolution and ligation were also quantified in parallel. mean±s.d. is plotted (n=3). The MCM7 and CDC45 dissociation data is obtained from the vehicle controls in Figure 5B–C , while the dissolution and ligation data are obtained from the vehicle controls in Fig 5D–E . (C) To seek independent evidence for the conclusions of the ChIP data presented in Figure 5B–C , we used a plasmid pull-down procedure. p[empty] was replicated in egg extracts treated with Vehicle or Ub-VS. At the indicated times, chromatin-associated proteins were captured on LacR-coated beads (which binds DNA independently of lacO sites) and analyzed by Western blotting for CDC45, MCM7, and PCNA. CDC45 and MCM7 dissociated from chromatin by 8’ in the vehicle control, but persisted following UbVS treatment. (D) To test whether the MCM7 modifications detected in panel (C) represented ubiquitylation, extracts were incubated with HIS 6 -Ubiquitin in the absence of Cyclin A, and in the absence or presence of plasmid DNA. After 15 minutes, HIS 6 -tagged proteins were captured by nickel resin pull down and blotted for MCM7. DNA replication greatly increased the levels of ubiquitylated MCM7, with the exception of a single species that was ubiquitylated independently of DNA replication (*). These data show that MCM7 is ubiquitylated during plasmid replication in egg extracts, as observed in yeast and during replication of sperm chromatin following nuclear assembly in egg extracts 24 , 25 . (E) In parallel to the plasmid pull-downs performed in (C), DNA samples were withdrawn for dissolution, ligation, and decatenation assays, none of which were perturbed by UbVS treatment. These data support our conclusion, based on ChIP experiments ( Fig. 5 ), that defective CMG unloading does not affect dissolution, ligation, or decatenation. (F) Decatenation was measured in the same reactions used to measure Dissolution and Ligation ( Fig. 5D–E ), mean±s.d. is plotted (n=3). (G–I) Given the experimental variability at the 4 minute time point in Figures 5D–F , the primary data and quantification for dissolution (G), ligation (H), and decatenation (I) for one of the three experiments summarized in Figure 5D–F is presented. This reveals that Ub-VS does not inhibit dissolution, ligation, or decatenation at the 4 minute time point. The same conclusion applies to two additional repetitions of this experiment (data not shown). (J) The primary ChIP data used to measure dissociation of MCM7 and CDC45 in Fig 5B–C is shown. Recovery of FLK2 was measured. mean±s.d. is plotted (n=3).
    Figure Legend Snippet: Supplemental termination data for p[empty] experiments (A) Cartoon depicting the XmnI and AlwNI sites on p[ empty ], which are used for the dissolution and ligation assays, respectively, and the FLK2 locus, which is used for ChIP. (B) Plasmid DNA without a lacO array (p[ empty ]) was replicated and at different times chromatin was subjected to MCM7 and CDC45 ChIP. Percent recovery of FLK2 was quantified and used to measure dissociation of MCM7 and CDC45 (see methods). Dissolution and ligation were also quantified in parallel. mean±s.d. is plotted (n=3). The MCM7 and CDC45 dissociation data is obtained from the vehicle controls in Figure 5B–C , while the dissolution and ligation data are obtained from the vehicle controls in Fig 5D–E . (C) To seek independent evidence for the conclusions of the ChIP data presented in Figure 5B–C , we used a plasmid pull-down procedure. p[empty] was replicated in egg extracts treated with Vehicle or Ub-VS. At the indicated times, chromatin-associated proteins were captured on LacR-coated beads (which binds DNA independently of lacO sites) and analyzed by Western blotting for CDC45, MCM7, and PCNA. CDC45 and MCM7 dissociated from chromatin by 8’ in the vehicle control, but persisted following UbVS treatment. (D) To test whether the MCM7 modifications detected in panel (C) represented ubiquitylation, extracts were incubated with HIS 6 -Ubiquitin in the absence of Cyclin A, and in the absence or presence of plasmid DNA. After 15 minutes, HIS 6 -tagged proteins were captured by nickel resin pull down and blotted for MCM7. DNA replication greatly increased the levels of ubiquitylated MCM7, with the exception of a single species that was ubiquitylated independently of DNA replication (*). These data show that MCM7 is ubiquitylated during plasmid replication in egg extracts, as observed in yeast and during replication of sperm chromatin following nuclear assembly in egg extracts 24 , 25 . (E) In parallel to the plasmid pull-downs performed in (C), DNA samples were withdrawn for dissolution, ligation, and decatenation assays, none of which were perturbed by UbVS treatment. These data support our conclusion, based on ChIP experiments ( Fig. 5 ), that defective CMG unloading does not affect dissolution, ligation, or decatenation. (F) Decatenation was measured in the same reactions used to measure Dissolution and Ligation ( Fig. 5D–E ), mean±s.d. is plotted (n=3). (G–I) Given the experimental variability at the 4 minute time point in Figures 5D–F , the primary data and quantification for dissolution (G), ligation (H), and decatenation (I) for one of the three experiments summarized in Figure 5D–F is presented. This reveals that Ub-VS does not inhibit dissolution, ligation, or decatenation at the 4 minute time point. The same conclusion applies to two additional repetitions of this experiment (data not shown). (J) The primary ChIP data used to measure dissociation of MCM7 and CDC45 in Fig 5B–C is shown. Recovery of FLK2 was measured. mean±s.d. is plotted (n=3).

    Techniques Used: Ligation, Chromatin Immunoprecipitation, Plasmid Preparation, Western Blot, Incubation

    2) Product Images from "Secretoglobin 1A1 and 1A1A Differentially Regulate Neutrophil Reactive Oxygen Species Production, Phagocytosis and Extracellular Trap Formation"

    Article Title: Secretoglobin 1A1 and 1A1A Differentially Regulate Neutrophil Reactive Oxygen Species Production, Phagocytosis and Extracellular Trap Formation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0096217

    Cloning, expression and purification of equine recombinant SCGB 1A1 and SCGB 1A1A proteins. (A) SCGB1A1 (“1”) and SCGB1A1A (“1A”) partial ORFs were amplified from equine lung cDNA preparations. A unique band of appropriate size (225 bp) was amplified for each gene. L = 1 Kb+ DNA ladder. (B) Fragments were digested with XmnI and SbfI restriction enzymes (purple boxes) and inserted into the multiple cloning sites (MCS) of the pMAL-c5X expression vector (top). DNA from the transformed colonies was submitted for sequencing to determine the presence, integrity, orientation and suitable translational reading frame of the insert. SCGB1A1 and SCGB1A1A sequenced (S) products showed proper orientation and 100% identity to the predicted (P) sequences. (C) Fractions collected during the purification steps of SCGB 1A1 and SCGB 1A1A were analyzed by SDS-PAGE. A fusion protein was apparent in extracts from IPTG-induced (I) but not un-induced (U) colonies. A crude extract (CE) was collected from induced cells and purified by affinity chromatography, using an amylose (A) column. The eluted fractions were pooled and incubated with Factor Xa protease (Fx) to cleave the fusion proteins. Fx was removed by FPLC (F), and MBP (42.5 kDa) was removed by additional passage on an amylose column from which pure (P) recombinant proteins (7 kDa) were collected. (D) Purified SCGB 1A1 and SCGB 1A1A proteins form dimers that dissociate under reducing and denaturing conditions. (E) Identity of dimers and monomers was confirmed by Western blot analysis. (C, D, E) S = Precision plus protein standard (dual color).
    Figure Legend Snippet: Cloning, expression and purification of equine recombinant SCGB 1A1 and SCGB 1A1A proteins. (A) SCGB1A1 (“1”) and SCGB1A1A (“1A”) partial ORFs were amplified from equine lung cDNA preparations. A unique band of appropriate size (225 bp) was amplified for each gene. L = 1 Kb+ DNA ladder. (B) Fragments were digested with XmnI and SbfI restriction enzymes (purple boxes) and inserted into the multiple cloning sites (MCS) of the pMAL-c5X expression vector (top). DNA from the transformed colonies was submitted for sequencing to determine the presence, integrity, orientation and suitable translational reading frame of the insert. SCGB1A1 and SCGB1A1A sequenced (S) products showed proper orientation and 100% identity to the predicted (P) sequences. (C) Fractions collected during the purification steps of SCGB 1A1 and SCGB 1A1A were analyzed by SDS-PAGE. A fusion protein was apparent in extracts from IPTG-induced (I) but not un-induced (U) colonies. A crude extract (CE) was collected from induced cells and purified by affinity chromatography, using an amylose (A) column. The eluted fractions were pooled and incubated with Factor Xa protease (Fx) to cleave the fusion proteins. Fx was removed by FPLC (F), and MBP (42.5 kDa) was removed by additional passage on an amylose column from which pure (P) recombinant proteins (7 kDa) were collected. (D) Purified SCGB 1A1 and SCGB 1A1A proteins form dimers that dissociate under reducing and denaturing conditions. (E) Identity of dimers and monomers was confirmed by Western blot analysis. (C, D, E) S = Precision plus protein standard (dual color).

    Techniques Used: Clone Assay, Expressing, Purification, Recombinant, Amplification, Plasmid Preparation, Transformation Assay, Sequencing, SDS Page, Affinity Chromatography, Incubation, Fast Protein Liquid Chromatography, Western Blot

    3) Product Images from "The Targeted Sequencing of Alpha Satellite DNA in Cercopithecus pogonias Provides New Insight Into the Diversity and Dynamics of Centromeric Repeats in Old World Monkeys"

    Article Title: The Targeted Sequencing of Alpha Satellite DNA in Cercopithecus pogonias Provides New Insight Into the Diversity and Dynamics of Centromeric Repeats in Old World Monkeys

    Journal: Genome Biology and Evolution

    doi: 10.1093/gbe/evy109

    —Characterization of alpha satellite DNA diversity in the HindIII monomer data set. ( A ) PCA projection on principal components 1 and 2 of the normalized 5-mer frequency vectors for all sequences from the HindIII monomer data set. Each point represents a monomer sequence. ( B ) Prediction of the position of the XmnI monomer sequences on the graph shown in ( A ). Sequences are colored according to their assignment to the C1 (purple), C2 (pastel green), C5 (red) or C6 (orange) alpha satellite families. ( C ) PCA projection shown in ( A ) with sequences colored according to their assignment to the C1′ (purple), C2′ (pastel green), C5′ (red), or C6′ (blue) alpha satellite families, using a hierarchical classification method (see Materials and methods).
    Figure Legend Snippet: —Characterization of alpha satellite DNA diversity in the HindIII monomer data set. ( A ) PCA projection on principal components 1 and 2 of the normalized 5-mer frequency vectors for all sequences from the HindIII monomer data set. Each point represents a monomer sequence. ( B ) Prediction of the position of the XmnI monomer sequences on the graph shown in ( A ). Sequences are colored according to their assignment to the C1 (purple), C2 (pastel green), C5 (red) or C6 (orange) alpha satellite families. ( C ) PCA projection shown in ( A ) with sequences colored according to their assignment to the C1′ (purple), C2′ (pastel green), C5′ (red), or C6′ (blue) alpha satellite families, using a hierarchical classification method (see Materials and methods).

    Techniques Used: Sequencing

    4) Product Images from "The mechanism of DNA replication termination in vertebrates"

    Article Title: The mechanism of DNA replication termination in vertebrates

    Journal: Nature

    doi: 10.1038/nature14887

    Cyclin A treatment synchronizes DNA replication in Xenopus egg extracts (A–B) To synchronize DNA replication in Xenopus egg extracts, we treated extracts with Cyclin A, which probably accelerates replication initiation 45 . Plasmid DNA was incubated in High Speed Supernatant for 20 minutes, then either buffer or Cyclin A was added for a further 20 minutes. NucleoPlasmic extract was added to initiate DNA replication, along with [α- 32 P]dATP to label replication intermediates. Replication products were separated on a native agarose gel, detected by autoradiography (A), and quantified (B). In the presence of vehicle, replication was not complete by 9.5 minutes, but in the presence of Cyclin A, replication was almost complete by 4.5 minutes (B). Thus, Cyclin A treatment approximately doubles the speed of DNA replication in Xenopus egg extracts. (C–F) To test whether Cyclin A affects the ability of LacR to inhibit termination, we monitored dissolution of plasmids containing a 12x or 16x LacR array in the presence and absence of Cyclin A. p[ lacO x12], p[ lacO x16], and the parental control plasmid p[ empty ] were incubated with LacR, and then treated with buffer or Cyclin A before replication was initiated with NPE in the presence of [α- 32 P]dATP. Samples were withdrawn when dissolution of p[ empty ] plateaued (9.5 minutes in the presence of buffer, 4.5 minutes in the presence of Cyclin A). Given that Cyclin A treatment approximately doubles the speed of replication (see B), samples were withdrawn from these reactions twice as frequently as the buffer-treated samples. To measure dissolution, radiolabelled termination intermediates were cut with XmnI to monitor the conversion of double-Y molecules to linear molecules. Cut molecules were separated on a native agarose gel and detected by autoradiography (C,E). By the time the first sample was withdrawn, dissolution of p[ empty ] was essentially complete, in the absence (9.5 min, D) or presence (4.5 min, F) of Cyclin A. Importantly, dissolution of p[ lacO x12] and p[ lacO x16] was prevented in the absence (9.5 min, D) or presence (4.5 min, F) of Cyclin A. Moreover, dissolution occurred approximately twice as fast in the presence of Cyclin A (note the similarity between (D) and (F) even though samples are withdrawn twice as frequently in F.) consistent with replication being approximately twice as fast in the presence of Cyclin A. Therefore, Cyclin A does not affect the ability of a LacR array to block replication forks.
    Figure Legend Snippet: Cyclin A treatment synchronizes DNA replication in Xenopus egg extracts (A–B) To synchronize DNA replication in Xenopus egg extracts, we treated extracts with Cyclin A, which probably accelerates replication initiation 45 . Plasmid DNA was incubated in High Speed Supernatant for 20 minutes, then either buffer or Cyclin A was added for a further 20 minutes. NucleoPlasmic extract was added to initiate DNA replication, along with [α- 32 P]dATP to label replication intermediates. Replication products were separated on a native agarose gel, detected by autoradiography (A), and quantified (B). In the presence of vehicle, replication was not complete by 9.5 minutes, but in the presence of Cyclin A, replication was almost complete by 4.5 minutes (B). Thus, Cyclin A treatment approximately doubles the speed of DNA replication in Xenopus egg extracts. (C–F) To test whether Cyclin A affects the ability of LacR to inhibit termination, we monitored dissolution of plasmids containing a 12x or 16x LacR array in the presence and absence of Cyclin A. p[ lacO x12], p[ lacO x16], and the parental control plasmid p[ empty ] were incubated with LacR, and then treated with buffer or Cyclin A before replication was initiated with NPE in the presence of [α- 32 P]dATP. Samples were withdrawn when dissolution of p[ empty ] plateaued (9.5 minutes in the presence of buffer, 4.5 minutes in the presence of Cyclin A). Given that Cyclin A treatment approximately doubles the speed of replication (see B), samples were withdrawn from these reactions twice as frequently as the buffer-treated samples. To measure dissolution, radiolabelled termination intermediates were cut with XmnI to monitor the conversion of double-Y molecules to linear molecules. Cut molecules were separated on a native agarose gel and detected by autoradiography (C,E). By the time the first sample was withdrawn, dissolution of p[ empty ] was essentially complete, in the absence (9.5 min, D) or presence (4.5 min, F) of Cyclin A. Importantly, dissolution of p[ lacO x12] and p[ lacO x16] was prevented in the absence (9.5 min, D) or presence (4.5 min, F) of Cyclin A. Moreover, dissolution occurred approximately twice as fast in the presence of Cyclin A (note the similarity between (D) and (F) even though samples are withdrawn twice as frequently in F.) consistent with replication being approximately twice as fast in the presence of Cyclin A. Therefore, Cyclin A does not affect the ability of a LacR array to block replication forks.

    Techniques Used: Plasmid Preparation, Incubation, Agarose Gel Electrophoresis, Autoradiography, Blocking Assay

    Topo II-dependent decatenation of p[lacOx16] (A) The autoradiograph in primary Figure 1G is reproduced with cartoons indicating the structures of the replication and termination intermediates n-n, n-sc, sc-sc, n, and sc (see main text for definitions). The order of appearance of the different catenanes matches previous work 5 (n-n, then n-sc, then sc-sc). (B–D) To determine the role of Topo II during termination within a lacO array, termination was monitored in mock- or Topo II-depleted extracts. To confirm immunodepletion of Topo II, Mock and Topo II-depleted NPE was blotted with MCM7 and Topo II antibodies (B). p[ lacO x16] was incubated with LacR, then replicated in either mock- or Topo II-depleted egg extracts in the presence of [α- 32 P]dATP, and termination was induced with IPTG (at 7’). Untreated DNA intermediates were separated by native gel electrophoresis (C). In the mock-depleted extract, nicked and supercoiled monomers were readily produced (as in (A), albeit with slower kinetics due to nonspecific inhibition of the extracts by the immunodepletion procedure), while in the Topo II-depleted extracts, a discrete species was produced. DNA from the last time point in each reaction (lanes 4 and 8 in (C)) was purified and treated with XmnI, which cuts p[ lacO x16] once, or Nt.BspQI, which nicks p[ lacO x16] once, or recombinant Topo II, and then separated by native gel electrophoresis (D). Cleavage of the mock- and TopoII-depleted products with XmnI yielded the expected linear 3.15 kb band (lanes 2 and 6), demonstrating that in both extracts, all products were fully dissolved topoisomers of each other. Relaxation of the mock-depleted products by nicking with Nt.BspQI yielded a discrete band corresponding to nicked plasmid (lane 3), while the TopoII-depleted products were converted to a ladder of discrete topoisomers (lane 7), which we infer represent catenated dimers of different linking numbers, since the mobility difference cannot be due to differences in supercoiling. Importantly, the mobility shift following Nt.BspQI treatment (lane 5 vs lane 7) demonstrated that the Topo II-depleted products (lane 5) were covalently closed and thus in the absence of Topo II ligation of the daughter strands still occurred. Treatment of the mock- and Topo II-depleted products with recombinant human Topo II produced the same relaxed monomeric species (lanes 4 and 8), further confirming that the Topo II-depleted products contained catenanes. Collectively, these observations demonstrate that termination within a lacO array in Topo II depleted extracts produces highly catenated supercoiled-supercoiled dimers, as seen in cells lacking Topo II 16 , 17 . These data confirm that Topo II is responsible for decatenation and argue that termination within a lacO array reflects physiological termination. (E) n-n, n-sc, sc-sc, n, and sc products were also detected when plasmid lacking lacO sequences (pBlueScript) was replicated in the absence of LacR without the use of Cyclin A to synchronize replication. Therefore, these intermediates arise in the course of unperturbed DNA replication in Xenopus egg extracts.
    Figure Legend Snippet: Topo II-dependent decatenation of p[lacOx16] (A) The autoradiograph in primary Figure 1G is reproduced with cartoons indicating the structures of the replication and termination intermediates n-n, n-sc, sc-sc, n, and sc (see main text for definitions). The order of appearance of the different catenanes matches previous work 5 (n-n, then n-sc, then sc-sc). (B–D) To determine the role of Topo II during termination within a lacO array, termination was monitored in mock- or Topo II-depleted extracts. To confirm immunodepletion of Topo II, Mock and Topo II-depleted NPE was blotted with MCM7 and Topo II antibodies (B). p[ lacO x16] was incubated with LacR, then replicated in either mock- or Topo II-depleted egg extracts in the presence of [α- 32 P]dATP, and termination was induced with IPTG (at 7’). Untreated DNA intermediates were separated by native gel electrophoresis (C). In the mock-depleted extract, nicked and supercoiled monomers were readily produced (as in (A), albeit with slower kinetics due to nonspecific inhibition of the extracts by the immunodepletion procedure), while in the Topo II-depleted extracts, a discrete species was produced. DNA from the last time point in each reaction (lanes 4 and 8 in (C)) was purified and treated with XmnI, which cuts p[ lacO x16] once, or Nt.BspQI, which nicks p[ lacO x16] once, or recombinant Topo II, and then separated by native gel electrophoresis (D). Cleavage of the mock- and TopoII-depleted products with XmnI yielded the expected linear 3.15 kb band (lanes 2 and 6), demonstrating that in both extracts, all products were fully dissolved topoisomers of each other. Relaxation of the mock-depleted products by nicking with Nt.BspQI yielded a discrete band corresponding to nicked plasmid (lane 3), while the TopoII-depleted products were converted to a ladder of discrete topoisomers (lane 7), which we infer represent catenated dimers of different linking numbers, since the mobility difference cannot be due to differences in supercoiling. Importantly, the mobility shift following Nt.BspQI treatment (lane 5 vs lane 7) demonstrated that the Topo II-depleted products (lane 5) were covalently closed and thus in the absence of Topo II ligation of the daughter strands still occurred. Treatment of the mock- and Topo II-depleted products with recombinant human Topo II produced the same relaxed monomeric species (lanes 4 and 8), further confirming that the Topo II-depleted products contained catenanes. Collectively, these observations demonstrate that termination within a lacO array in Topo II depleted extracts produces highly catenated supercoiled-supercoiled dimers, as seen in cells lacking Topo II 16 , 17 . These data confirm that Topo II is responsible for decatenation and argue that termination within a lacO array reflects physiological termination. (E) n-n, n-sc, sc-sc, n, and sc products were also detected when plasmid lacking lacO sequences (pBlueScript) was replicated in the absence of LacR without the use of Cyclin A to synchronize replication. Therefore, these intermediates arise in the course of unperturbed DNA replication in Xenopus egg extracts.

    Techniques Used: Autoradiography, Incubation, Nucleic Acid Electrophoresis, Produced, Inhibition, Purification, Recombinant, Plasmid Preparation, Mobility Shift, Ligation

    Replisome progression through 12x and 32x lacO arrays (A–D) To test whether replisomes meet later in a lacO x32 array than a lacO x12 array, we monitored dissolution. LacR Block-IPTG release was performed on p[ lacO x12] and p[ lacO x32] and radiolabelled termination intermediates were digested with XmnI to monitor the conversion of double-Y molecules to linear molecules (Dissolution). Cleaved molecules were separated on a native agarose gel, detected by autoradiography (A,C), and quantified (B,D). Upon IPTG addition, dissolution was delayed by at least 1 minute within the 32x lacO array compared to the 12x lacO array (B,D). Moreover, by 6 minutes, 92% of forks had undergone dissolution on p[ lacO x12] while only 9% had dissolved on p[ lacO x32] (B,D). (E) Stall products within the 12x lacO array ( Fig. 3B , Lane 2) were quantified, signal was corrected based on size differences of the products, and the percentage of stall products at each stall point was calculated. 78% of leading strands stalled at the first three arrest points (red columns), 19% stalled at the fourth to tenth arrest points (yellow columns) and the remaining 3% stalled at the tenth to fourteenth arrest points (grey columns). The appearance of fourteen arrest points reproducible and surprising, given that the presence of only twelve lacO sequences was confirmed by sequencing in the very preparation of p[ lacO x12] that was used in Fig. 3 . The thirteenth and fourteenth arrest points cannot stem from cryptic lacO sites beyond the twelfth lacO site, as this would position the first leftward leading strand stall product ∼90 nucleotides from the lacO array, instead of the observed ∼30 nucleotides (see F-G ). At present, we do not understand the origin of these stall products. (F–G) Progression of leftward leading strands into the array. The same DNA samples used in Fig. 3 were digested with the nicking enzyme Nb.BsrDI, which released leftward leading strands (F), and separated on a denaturing polyacrylamide gel (G). The lacO sites of p[ lacO x12] are highlighted in blue on the sequencing ladder (G), which was generated using the primer JDO109 (Green arrow, F). Green circles indicate two non-specific products of digestion. These products arise because nicking enzyme activity varies between experiments, even under the same conditions. There was no significant difference in the pattern of leftward leading strand progression between the 12x lacO and 32x lacO arrays, as seen for the rightward leading strands ( Fig. 3B ). Specifically, by 5.67 minutes, the majority of leading strands had extended beyond the seventh lacO repeat within lacO x12 (lane 6) and the equivalent region of lacO x32 (lane 18). Therefore, progression of leftward leading strands is unaffected by the presence of an opposing replisome, suggesting that converging replisomes do not stall when they meet.
    Figure Legend Snippet: Replisome progression through 12x and 32x lacO arrays (A–D) To test whether replisomes meet later in a lacO x32 array than a lacO x12 array, we monitored dissolution. LacR Block-IPTG release was performed on p[ lacO x12] and p[ lacO x32] and radiolabelled termination intermediates were digested with XmnI to monitor the conversion of double-Y molecules to linear molecules (Dissolution). Cleaved molecules were separated on a native agarose gel, detected by autoradiography (A,C), and quantified (B,D). Upon IPTG addition, dissolution was delayed by at least 1 minute within the 32x lacO array compared to the 12x lacO array (B,D). Moreover, by 6 minutes, 92% of forks had undergone dissolution on p[ lacO x12] while only 9% had dissolved on p[ lacO x32] (B,D). (E) Stall products within the 12x lacO array ( Fig. 3B , Lane 2) were quantified, signal was corrected based on size differences of the products, and the percentage of stall products at each stall point was calculated. 78% of leading strands stalled at the first three arrest points (red columns), 19% stalled at the fourth to tenth arrest points (yellow columns) and the remaining 3% stalled at the tenth to fourteenth arrest points (grey columns). The appearance of fourteen arrest points reproducible and surprising, given that the presence of only twelve lacO sequences was confirmed by sequencing in the very preparation of p[ lacO x12] that was used in Fig. 3 . The thirteenth and fourteenth arrest points cannot stem from cryptic lacO sites beyond the twelfth lacO site, as this would position the first leftward leading strand stall product ∼90 nucleotides from the lacO array, instead of the observed ∼30 nucleotides (see F-G ). At present, we do not understand the origin of these stall products. (F–G) Progression of leftward leading strands into the array. The same DNA samples used in Fig. 3 were digested with the nicking enzyme Nb.BsrDI, which released leftward leading strands (F), and separated on a denaturing polyacrylamide gel (G). The lacO sites of p[ lacO x12] are highlighted in blue on the sequencing ladder (G), which was generated using the primer JDO109 (Green arrow, F). Green circles indicate two non-specific products of digestion. These products arise because nicking enzyme activity varies between experiments, even under the same conditions. There was no significant difference in the pattern of leftward leading strand progression between the 12x lacO and 32x lacO arrays, as seen for the rightward leading strands ( Fig. 3B ). Specifically, by 5.67 minutes, the majority of leading strands had extended beyond the seventh lacO repeat within lacO x12 (lane 6) and the equivalent region of lacO x32 (lane 18). Therefore, progression of leftward leading strands is unaffected by the presence of an opposing replisome, suggesting that converging replisomes do not stall when they meet.

    Techniques Used: Blocking Assay, Agarose Gel Electrophoresis, Autoradiography, Sequencing, Generated, Activity Assay

    Supplemental ChIP data (A) Cartoon depicting the LAC, FLK2 and FAR loci, which were used for ChIP. Their precise locations relative to the leftward edge of the lacO array are indicated. The LAC amplicon is present in four copies distributed across the lacOx16 array and three copies distributed across the lacOx12 array. (B–E) p[ lacO x12] was incubated with buffer or LacR and termination was induced at 5 minutes by IPTG addition. MCM7, RPA, CDC45, and Polε ChIP was performed at different time points after IPTG addition but also in the buffer control and no IPTG control. Recovery of FLK2 was measured as a percentage of input DNA. Upon IPTG addition, ChIP signal declined and by 9 minutes was comparable to the buffer control, demonstrating that unloading of replisomes was induced within 4 minutes of IPTG addition. (F) To test whether movement of the replisome into and out of the lacO array could be detected upon IPTG addition, termination was monitored within a lacO array, and we performed ChIP of the leading strand polymerase Polε, which was inferred to move into and out of the array based on the behavior of leading strands during termination ( Extended Data Fig 2B–E ). It was predicted that Polε ChIP at the LAC locus should increase slightly as Polε enters the lacO array and decline again as converging polymerases pass each other, but persist at FLK2 while the polymerases move out of the array. Prior to IPTG addition, Polε was enriched at LAC and FLK2 compared to FAR , consistent with the leading strands being positioned on either side of the lacO array ( Extended Data Fig 2C , Fig. 3 ). Upon IPTG addition, Polε became modestly enriched at LAC compared to FLK2 (5.5 min) but then declined to similar levels at both LAC and FLK2 by 6.5 min. These data are consistent with the leading strand polymerases entering the lacO array and passing each other. (G–H) To test whether CMG exhibited the same ChIP profile as Polε, MCM7 and CDC45 ChIP was performed using the same samples. Following IPTG addition, MCM7 and CDC45 were enriched at LAC compared to FLK2 (5.5 min), then declined to similar levels at both LAC and FLK2 by 6.5 min, as seen for Polε (F). These data are consistent with a model in which CMGs enter the array and pass each other during termination. A caveat of these experiments is the relatively high recovery of the FAR locus in MCM7, CDC45, and Polε ChIP. Specifically, signal was at most only ∼2-fold enriched at LAC compared to FAR . This was not due to high background binding, because by the end of the experiment (10 minute time point, not shown), we observed a decrease in signal of ∼5–7 fold. Furthermore, we observed ∼5–7 fold enrichment in binding (ChIP) of replisome components to p[ lacO x12] that had been incubated in LacR compared to a buffer control (see G-I, below). Instead, the high FAR signal was likely due to poor spatial resolution of the ChIP. Consistent with this, when a plasmid containing a DNA interstrand cross-link (ICL) was replicated, essentially all replisomes converged upon the ICL but the ChIP signal for MCM7 and CDC45 was only ∼3–4 fold enriched at the ICL compared to a control locus 41 . We speculate that the higher background observed at the control locus in our experiments is due to the decreased distance of the control locus from the experimental locus (1.3 kb for p[ lacO x16] and p[ lacO x12] vs. 2.4 kb for the ICL plasmid) and possibly due to increased catenation of the parental strands during termination. The high signal at FAR should not complicate interpretation of the MCM7, CDC45 and Polε ChIP (F), as signal at FAR was essentially unaltered between 5 and 6.5 minutes. Further evidence that the high signal seen at the FAR locus emanates from forks stalled near the lacO array is presented in panel ( K ). (I) ChIP of RPA was performed on the same chromatin samples used in B-D. As seen for pol ɛ, MCM7, and CDC45, enrichment of RPA at LAC compared to FAR was relatively low, consistent with poor spatial resolution. (J) Predicted binding of CMGs to the LAC, FLK2 and FAR loci before and after IPTG addition if CMGs converging CMG pass each other. (K) To determine whether most forks stalled at the array and not elsewhere in the plasmid, we performed a time course in which p[ lacO x16] undergoing termination was examined by 2-dimensional gel electrophoresis (2-D gel) at various time points. p[ lacO x16] was pre-bound to LacR and replicated in Xenopus egg extract containing [α- 32 P]dATP. Termination was induced by IPTG addition and samples were withdrawn at different times. Radiolabelled replication intermediates were cleaved with XmnI (as in Extended Data Fig. 1A ) and separated according to size and shape on 2-D gels 50 . A parallel reaction was performed in which samples were analyzed by ChIP, which was one of the repeats analyzed in (B)-(E). In the presence of LacR, a subset of Double Y molecules accumulated (blue arrowhead), demonstrating that 83% of replication intermediates (signal in dashed blue circle) contained two forks converged at a specific locus. Following IPTG addition, linear molecules rapidly accumulated (orange arrow) as dissolution occurred. Importantly, the vast majority of signal was present in the discrete double-Y and linear species (blue and orange arrows), demonstrating that the relatively high ChIP signal observed at FAR in panels F-I was derived from forks present at the lacO x16 array and not elsewhere.
    Figure Legend Snippet: Supplemental ChIP data (A) Cartoon depicting the LAC, FLK2 and FAR loci, which were used for ChIP. Their precise locations relative to the leftward edge of the lacO array are indicated. The LAC amplicon is present in four copies distributed across the lacOx16 array and three copies distributed across the lacOx12 array. (B–E) p[ lacO x12] was incubated with buffer or LacR and termination was induced at 5 minutes by IPTG addition. MCM7, RPA, CDC45, and Polε ChIP was performed at different time points after IPTG addition but also in the buffer control and no IPTG control. Recovery of FLK2 was measured as a percentage of input DNA. Upon IPTG addition, ChIP signal declined and by 9 minutes was comparable to the buffer control, demonstrating that unloading of replisomes was induced within 4 minutes of IPTG addition. (F) To test whether movement of the replisome into and out of the lacO array could be detected upon IPTG addition, termination was monitored within a lacO array, and we performed ChIP of the leading strand polymerase Polε, which was inferred to move into and out of the array based on the behavior of leading strands during termination ( Extended Data Fig 2B–E ). It was predicted that Polε ChIP at the LAC locus should increase slightly as Polε enters the lacO array and decline again as converging polymerases pass each other, but persist at FLK2 while the polymerases move out of the array. Prior to IPTG addition, Polε was enriched at LAC and FLK2 compared to FAR , consistent with the leading strands being positioned on either side of the lacO array ( Extended Data Fig 2C , Fig. 3 ). Upon IPTG addition, Polε became modestly enriched at LAC compared to FLK2 (5.5 min) but then declined to similar levels at both LAC and FLK2 by 6.5 min. These data are consistent with the leading strand polymerases entering the lacO array and passing each other. (G–H) To test whether CMG exhibited the same ChIP profile as Polε, MCM7 and CDC45 ChIP was performed using the same samples. Following IPTG addition, MCM7 and CDC45 were enriched at LAC compared to FLK2 (5.5 min), then declined to similar levels at both LAC and FLK2 by 6.5 min, as seen for Polε (F). These data are consistent with a model in which CMGs enter the array and pass each other during termination. A caveat of these experiments is the relatively high recovery of the FAR locus in MCM7, CDC45, and Polε ChIP. Specifically, signal was at most only ∼2-fold enriched at LAC compared to FAR . This was not due to high background binding, because by the end of the experiment (10 minute time point, not shown), we observed a decrease in signal of ∼5–7 fold. Furthermore, we observed ∼5–7 fold enrichment in binding (ChIP) of replisome components to p[ lacO x12] that had been incubated in LacR compared to a buffer control (see G-I, below). Instead, the high FAR signal was likely due to poor spatial resolution of the ChIP. Consistent with this, when a plasmid containing a DNA interstrand cross-link (ICL) was replicated, essentially all replisomes converged upon the ICL but the ChIP signal for MCM7 and CDC45 was only ∼3–4 fold enriched at the ICL compared to a control locus 41 . We speculate that the higher background observed at the control locus in our experiments is due to the decreased distance of the control locus from the experimental locus (1.3 kb for p[ lacO x16] and p[ lacO x12] vs. 2.4 kb for the ICL plasmid) and possibly due to increased catenation of the parental strands during termination. The high signal at FAR should not complicate interpretation of the MCM7, CDC45 and Polε ChIP (F), as signal at FAR was essentially unaltered between 5 and 6.5 minutes. Further evidence that the high signal seen at the FAR locus emanates from forks stalled near the lacO array is presented in panel ( K ). (I) ChIP of RPA was performed on the same chromatin samples used in B-D. As seen for pol ɛ, MCM7, and CDC45, enrichment of RPA at LAC compared to FAR was relatively low, consistent with poor spatial resolution. (J) Predicted binding of CMGs to the LAC, FLK2 and FAR loci before and after IPTG addition if CMGs converging CMG pass each other. (K) To determine whether most forks stalled at the array and not elsewhere in the plasmid, we performed a time course in which p[ lacO x16] undergoing termination was examined by 2-dimensional gel electrophoresis (2-D gel) at various time points. p[ lacO x16] was pre-bound to LacR and replicated in Xenopus egg extract containing [α- 32 P]dATP. Termination was induced by IPTG addition and samples were withdrawn at different times. Radiolabelled replication intermediates were cleaved with XmnI (as in Extended Data Fig. 1A ) and separated according to size and shape on 2-D gels 50 . A parallel reaction was performed in which samples were analyzed by ChIP, which was one of the repeats analyzed in (B)-(E). In the presence of LacR, a subset of Double Y molecules accumulated (blue arrowhead), demonstrating that 83% of replication intermediates (signal in dashed blue circle) contained two forks converged at a specific locus. Following IPTG addition, linear molecules rapidly accumulated (orange arrow) as dissolution occurred. Importantly, the vast majority of signal was present in the discrete double-Y and linear species (blue and orange arrows), demonstrating that the relatively high ChIP signal observed at FAR in panels F-I was derived from forks present at the lacO x16 array and not elsewhere.

    Techniques Used: Chromatin Immunoprecipitation, Amplification, Incubation, Recombinase Polymerase Amplification, Binding Assay, Plasmid Preparation, Nucleic Acid Electrophoresis, Derivative Assay

    Supplemental termination data for p[empty] experiments (A) Cartoon depicting the XmnI and AlwNI sites on p[ empty ], which are used for the dissolution and ligation assays, respectively, and the FLK2 locus, which is used for ChIP. (B) Plasmid DNA without a lacO array (p[ empty ]) was replicated and at different times chromatin was subjected to MCM7 and CDC45 ChIP. Percent recovery of FLK2 was quantified and used to measure dissociation of MCM7 and CDC45 (see methods). Dissolution and ligation were also quantified in parallel. mean±s.d. is plotted (n=3). The MCM7 and CDC45 dissociation data is obtained from the vehicle controls in Figure 5B–C , while the dissolution and ligation data are obtained from the vehicle controls in Fig 5D–E . (C) To seek independent evidence for the conclusions of the ChIP data presented in Figure 5B–C , we used a plasmid pull-down procedure. p[empty] was replicated in egg extracts treated with Vehicle or Ub-VS. At the indicated times, chromatin-associated proteins were captured on LacR-coated beads (which binds DNA independently of lacO sites) and analyzed by Western blotting for CDC45, MCM7, and PCNA. CDC45 and MCM7 dissociated from chromatin by 8’ in the vehicle control, but persisted following UbVS treatment. (D) To test whether the MCM7 modifications detected in panel (C) represented ubiquitylation, extracts were incubated with HIS 6 -Ubiquitin in the absence of Cyclin A, and in the absence or presence of plasmid DNA. After 15 minutes, HIS 6 -tagged proteins were captured by nickel resin pull down and blotted for MCM7. DNA replication greatly increased the levels of ubiquitylated MCM7, with the exception of a single species that was ubiquitylated independently of DNA replication (*). These data show that MCM7 is ubiquitylated during plasmid replication in egg extracts, as observed in yeast and during replication of sperm chromatin following nuclear assembly in egg extracts 24 , 25 . (E) In parallel to the plasmid pull-downs performed in (C), DNA samples were withdrawn for dissolution, ligation, and decatenation assays, none of which were perturbed by UbVS treatment. These data support our conclusion, based on ChIP experiments ( Fig. 5 ), that defective CMG unloading does not affect dissolution, ligation, or decatenation. (F) Decatenation was measured in the same reactions used to measure Dissolution and Ligation ( Fig. 5D–E ), mean±s.d. is plotted (n=3). (G–I) Given the experimental variability at the 4 minute time point in Figures 5D–F , the primary data and quantification for dissolution (G), ligation (H), and decatenation (I) for one of the three experiments summarized in Figure 5D–F is presented. This reveals that Ub-VS does not inhibit dissolution, ligation, or decatenation at the 4 minute time point. The same conclusion applies to two additional repetitions of this experiment (data not shown). (J) The primary ChIP data used to measure dissociation of MCM7 and CDC45 in Fig 5B–C is shown. Recovery of FLK2 was measured. mean±s.d. is plotted (n=3).
    Figure Legend Snippet: Supplemental termination data for p[empty] experiments (A) Cartoon depicting the XmnI and AlwNI sites on p[ empty ], which are used for the dissolution and ligation assays, respectively, and the FLK2 locus, which is used for ChIP. (B) Plasmid DNA without a lacO array (p[ empty ]) was replicated and at different times chromatin was subjected to MCM7 and CDC45 ChIP. Percent recovery of FLK2 was quantified and used to measure dissociation of MCM7 and CDC45 (see methods). Dissolution and ligation were also quantified in parallel. mean±s.d. is plotted (n=3). The MCM7 and CDC45 dissociation data is obtained from the vehicle controls in Figure 5B–C , while the dissolution and ligation data are obtained from the vehicle controls in Fig 5D–E . (C) To seek independent evidence for the conclusions of the ChIP data presented in Figure 5B–C , we used a plasmid pull-down procedure. p[empty] was replicated in egg extracts treated with Vehicle or Ub-VS. At the indicated times, chromatin-associated proteins were captured on LacR-coated beads (which binds DNA independently of lacO sites) and analyzed by Western blotting for CDC45, MCM7, and PCNA. CDC45 and MCM7 dissociated from chromatin by 8’ in the vehicle control, but persisted following UbVS treatment. (D) To test whether the MCM7 modifications detected in panel (C) represented ubiquitylation, extracts were incubated with HIS 6 -Ubiquitin in the absence of Cyclin A, and in the absence or presence of plasmid DNA. After 15 minutes, HIS 6 -tagged proteins were captured by nickel resin pull down and blotted for MCM7. DNA replication greatly increased the levels of ubiquitylated MCM7, with the exception of a single species that was ubiquitylated independently of DNA replication (*). These data show that MCM7 is ubiquitylated during plasmid replication in egg extracts, as observed in yeast and during replication of sperm chromatin following nuclear assembly in egg extracts 24 , 25 . (E) In parallel to the plasmid pull-downs performed in (C), DNA samples were withdrawn for dissolution, ligation, and decatenation assays, none of which were perturbed by UbVS treatment. These data support our conclusion, based on ChIP experiments ( Fig. 5 ), that defective CMG unloading does not affect dissolution, ligation, or decatenation. (F) Decatenation was measured in the same reactions used to measure Dissolution and Ligation ( Fig. 5D–E ), mean±s.d. is plotted (n=3). (G–I) Given the experimental variability at the 4 minute time point in Figures 5D–F , the primary data and quantification for dissolution (G), ligation (H), and decatenation (I) for one of the three experiments summarized in Figure 5D–F is presented. This reveals that Ub-VS does not inhibit dissolution, ligation, or decatenation at the 4 minute time point. The same conclusion applies to two additional repetitions of this experiment (data not shown). (J) The primary ChIP data used to measure dissociation of MCM7 and CDC45 in Fig 5B–C is shown. Recovery of FLK2 was measured. mean±s.d. is plotted (n=3).

    Techniques Used: Ligation, Chromatin Immunoprecipitation, Plasmid Preparation, Western Blot, Incubation

    5) Product Images from "Cernunnos/XLF promotes the ligation of mismatched and noncohesive DNA ends"

    Article Title: Cernunnos/XLF promotes the ligation of mismatched and noncohesive DNA ends

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0702620104

    Ku, XL, and Cernunnos join mismatched ends by ligation of one strand. ( A ) Schematic of the gel-based assay for joining. We incubated Ku, XL, and Cernunnos (Ku/XL/C) or T4 ligase (T4) with a linear DNA substrate containing EcoRI–EcoRI, EcoRV–EcoRV, or EcoRV–KpnI ends; digested the DNA products with XmnI; and used T4 polynucleotide kinase to radiolabel the DNA. Concentrations of XL and Cernunnos were 1 nM and 5 nM, respectively. For EcoRV–EcoRV and EcoRV–KpnI ends, we performed a second digest with ApoI or MspA1I to remove the radiolabel from one DNA strand. To confirm that ligation created a new phosphodiester bond, we digested the products of the EcoRV–EcoRV joining reaction with EcoRV. Radiolabeled DNA strands are red with red asterisks at the 5′ end, and unlabeled DNA strands are black. DNA products were resolved with denaturing gel electrophoresis. ( B ) Ku, XL, and Cernunnos joined cohesive EcoRI–EcoRI ends. Ku, XL, and Cernunnos (lane 3) and T4 ligase (lane 4) produced a higher molecular weight band of 498 nt. The molecular weight markers consisted of a radiolabeled 50-bp ladder (lane 1). Joining efficiencies for Ku/XL/C and T4 (calculated from the ratio of intensities in the 498-nt band to the 339-nt band) were 39% and 89%, respectively. Sizes of the ligated higher molecular weight bands in B–D appear in blue typeface. ( C ) Ku, XL, and Cernunnos joined blunt EcoRV–EcoRV ends. Ku, XL, and Cernunnos (lane 3) and T4 ligase (lane 4) produced a higher molecular weight band of 470 nt, with joining efficiencies of 20% and 14%, respectively. ApoI digestion converted the 470-nt band to 413-nt and 57-nt bands and converted the 281-nt band to 224-nt and 57-nt bands (lane 6). MspA1I digestion converted the 470-nt band to 434-nt and 36-nt bands and the 189-nt band to 153-nt and 36-nt bands (lane 8). Intensities of the 413-nt and 434-nt bands were reduced to 50% of the 470-nt band because the second digestion removed the radiolabel from one strand. EcoRV digestion eliminated the 470-nt band (lane 10), demonstrating that the DNA junction contained new phosphodiester bonds. The 153-, 57-, and 36-nt bands are not shown. ( D ) Ku, XL, and Cernunnos joined only one strand from mismatched EcoRV–KpnI ends. Ku, XL, and Cernunnos (lane 3) produced a higher molecular weight band of 472 nt, with a joining efficiency of 5%. ApoI digestion converted the 472-nt band to 415-nt and 57-nt bands and the 281-nt band to 224-nt and 57-nt bands (lane 5). The intensities of the 472-nt and 415-nt bands were equivalent (lanes 3 and 5). MspA1I digestion converted the 472-nt band to a 36-nt band and converted the 191-nt band to a 36-nt band (lane 7), demonstrating the ligation of only one strand.
    Figure Legend Snippet: Ku, XL, and Cernunnos join mismatched ends by ligation of one strand. ( A ) Schematic of the gel-based assay for joining. We incubated Ku, XL, and Cernunnos (Ku/XL/C) or T4 ligase (T4) with a linear DNA substrate containing EcoRI–EcoRI, EcoRV–EcoRV, or EcoRV–KpnI ends; digested the DNA products with XmnI; and used T4 polynucleotide kinase to radiolabel the DNA. Concentrations of XL and Cernunnos were 1 nM and 5 nM, respectively. For EcoRV–EcoRV and EcoRV–KpnI ends, we performed a second digest with ApoI or MspA1I to remove the radiolabel from one DNA strand. To confirm that ligation created a new phosphodiester bond, we digested the products of the EcoRV–EcoRV joining reaction with EcoRV. Radiolabeled DNA strands are red with red asterisks at the 5′ end, and unlabeled DNA strands are black. DNA products were resolved with denaturing gel electrophoresis. ( B ) Ku, XL, and Cernunnos joined cohesive EcoRI–EcoRI ends. Ku, XL, and Cernunnos (lane 3) and T4 ligase (lane 4) produced a higher molecular weight band of 498 nt. The molecular weight markers consisted of a radiolabeled 50-bp ladder (lane 1). Joining efficiencies for Ku/XL/C and T4 (calculated from the ratio of intensities in the 498-nt band to the 339-nt band) were 39% and 89%, respectively. Sizes of the ligated higher molecular weight bands in B–D appear in blue typeface. ( C ) Ku, XL, and Cernunnos joined blunt EcoRV–EcoRV ends. Ku, XL, and Cernunnos (lane 3) and T4 ligase (lane 4) produced a higher molecular weight band of 470 nt, with joining efficiencies of 20% and 14%, respectively. ApoI digestion converted the 470-nt band to 413-nt and 57-nt bands and converted the 281-nt band to 224-nt and 57-nt bands (lane 6). MspA1I digestion converted the 470-nt band to 434-nt and 36-nt bands and the 189-nt band to 153-nt and 36-nt bands (lane 8). Intensities of the 413-nt and 434-nt bands were reduced to 50% of the 470-nt band because the second digestion removed the radiolabel from one strand. EcoRV digestion eliminated the 470-nt band (lane 10), demonstrating that the DNA junction contained new phosphodiester bonds. The 153-, 57-, and 36-nt bands are not shown. ( D ) Ku, XL, and Cernunnos joined only one strand from mismatched EcoRV–KpnI ends. Ku, XL, and Cernunnos (lane 3) produced a higher molecular weight band of 472 nt, with a joining efficiency of 5%. ApoI digestion converted the 472-nt band to 415-nt and 57-nt bands and the 281-nt band to 224-nt and 57-nt bands (lane 5). The intensities of the 472-nt and 415-nt bands were equivalent (lanes 3 and 5). MspA1I digestion converted the 472-nt band to a 36-nt band and converted the 191-nt band to a 36-nt band (lane 7), demonstrating the ligation of only one strand.

    Techniques Used: Ligation, Incubation, Nucleic Acid Electrophoresis, Produced, Molecular Weight

    6) Product Images from "The Targeted Sequencing of Alpha Satellite DNA in Cercopithecus pogonias Provides New Insight Into the Diversity and Dynamics of Centromeric Repeats in Old World Monkeys"

    Article Title: The Targeted Sequencing of Alpha Satellite DNA in Cercopithecus pogonias Provides New Insight Into the Diversity and Dynamics of Centromeric Repeats in Old World Monkeys

    Journal: Genome Biology and Evolution

    doi: 10.1093/gbe/evy109

    —Characterization of alpha satellite DNA diversity in the HindIII monomer data set. ( A ) PCA projection on principal components 1 and 2 of the normalized 5-mer frequency vectors for all sequences from the HindIII monomer data set. Each point represents a monomer sequence. ( B ) Prediction of the position of the XmnI monomer sequences on the graph shown in ( A ). Sequences are colored according to their assignment to the C1 (purple), C2 (pastel green), C5 (red) or C6 (orange) alpha satellite families. ( C ) PCA projection shown in ( A ) with sequences colored according to their assignment to the C1′ (purple), C2′ (pastel green), C5′ (red), or C6′ (blue) alpha satellite families, using a hierarchical classification method (see Materials and methods).
    Figure Legend Snippet: —Characterization of alpha satellite DNA diversity in the HindIII monomer data set. ( A ) PCA projection on principal components 1 and 2 of the normalized 5-mer frequency vectors for all sequences from the HindIII monomer data set. Each point represents a monomer sequence. ( B ) Prediction of the position of the XmnI monomer sequences on the graph shown in ( A ). Sequences are colored according to their assignment to the C1 (purple), C2 (pastel green), C5 (red) or C6 (orange) alpha satellite families. ( C ) PCA projection shown in ( A ) with sequences colored according to their assignment to the C1′ (purple), C2′ (pastel green), C5′ (red), or C6′ (blue) alpha satellite families, using a hierarchical classification method (see Materials and methods).

    Techniques Used: Sequencing

    7) Product Images from "Defining characteristics of Tn5 Transposase non-specific DNA binding"

    Article Title: Defining characteristics of Tn5 Transposase non-specific DNA binding

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl179

    Single ES substrates of differing lengths are cleaved with variable rate constants. ( A ) A partial restriction map of the plasmid (pWSR6103) used to create substrates for in vitro transposition reactions is shown. The Tnp ES is represented as a black arrow. This plasmid was digested with PflMI and either PvuII, BglI, NarI, NdeI, AatII or XmnI to create substrates varying in size from 485 to 1183 bp. Each restriction fragment contained 395 bp of transposon (Tn) DNA and varying lengths of donor backbone (dbb) DNA as shown. The location of the transposon ES in each substrate is marked with a black arrow. ( B ) A schematic of the in vitro transposition reactions with single-ended substrates is shown. Each substrate DNA was incubated (together with non-specific DNA remaining from the restriction digest) with Tnp and MgAc at 37°C. Time points were taken from 0 to 8 h. Following PEC formation, the substrate was cleaved into two products, the dbb and Tn DNA. In this figure, the single ended substrate DNA is shown as two parallel lines containing a transposon ES (gray box). The cleavage site is marked with +1. The non-specific DNA remaining from the restriction digest is shown as linear double stranded DNA. Both product DNAs are appropriately labeled and other reaction components are described as in Figure 4 . ( C ) Each time point was run on an appropriate agarose gel to separate the full-length, unreacted substrate from the dbb and Tn DNA products. In this representative gel of the 555 bp substrate, time points are shown in lanes 3–13 and DNA size markers are shown in lanes 1 and 2. The substrate, dbb and Tn DNAs are represented as in (B). ( D ) The percentage of substrates cleaved was determined for each time point as described in the Materials and Methods. The mean percentage cleaved at each time point was calculated from at least three independent experiments and was then plotted (together with error bars representing the standard error) versus time and the data were fit to a one-phase exponential equation. The plot shown here represents data for the 555 bp substrate. In vitro transposition reactions and analysis were performed in this fashion for each of the six single end substrates. ( E ) k obs,cleavage and the standard error (SE) of this value were calculated from the fits described in (D). These are shown for each of the six substrates tested. ( F ) To better visualize the effect of substrate length on k obs,cleavage , k obs,cleavage was plotted versus substrate length for each substrate. The error bars represent the standard error of k obs,cleavage for each substrate.
    Figure Legend Snippet: Single ES substrates of differing lengths are cleaved with variable rate constants. ( A ) A partial restriction map of the plasmid (pWSR6103) used to create substrates for in vitro transposition reactions is shown. The Tnp ES is represented as a black arrow. This plasmid was digested with PflMI and either PvuII, BglI, NarI, NdeI, AatII or XmnI to create substrates varying in size from 485 to 1183 bp. Each restriction fragment contained 395 bp of transposon (Tn) DNA and varying lengths of donor backbone (dbb) DNA as shown. The location of the transposon ES in each substrate is marked with a black arrow. ( B ) A schematic of the in vitro transposition reactions with single-ended substrates is shown. Each substrate DNA was incubated (together with non-specific DNA remaining from the restriction digest) with Tnp and MgAc at 37°C. Time points were taken from 0 to 8 h. Following PEC formation, the substrate was cleaved into two products, the dbb and Tn DNA. In this figure, the single ended substrate DNA is shown as two parallel lines containing a transposon ES (gray box). The cleavage site is marked with +1. The non-specific DNA remaining from the restriction digest is shown as linear double stranded DNA. Both product DNAs are appropriately labeled and other reaction components are described as in Figure 4 . ( C ) Each time point was run on an appropriate agarose gel to separate the full-length, unreacted substrate from the dbb and Tn DNA products. In this representative gel of the 555 bp substrate, time points are shown in lanes 3–13 and DNA size markers are shown in lanes 1 and 2. The substrate, dbb and Tn DNAs are represented as in (B). ( D ) The percentage of substrates cleaved was determined for each time point as described in the Materials and Methods. The mean percentage cleaved at each time point was calculated from at least three independent experiments and was then plotted (together with error bars representing the standard error) versus time and the data were fit to a one-phase exponential equation. The plot shown here represents data for the 555 bp substrate. In vitro transposition reactions and analysis were performed in this fashion for each of the six single end substrates. ( E ) k obs,cleavage and the standard error (SE) of this value were calculated from the fits described in (D). These are shown for each of the six substrates tested. ( F ) To better visualize the effect of substrate length on k obs,cleavage , k obs,cleavage was plotted versus substrate length for each substrate. The error bars represent the standard error of k obs,cleavage for each substrate.

    Techniques Used: Plasmid Preparation, In Vitro, Incubation, Labeling, Agarose Gel Electrophoresis

    8) Product Images from "The mechanism of DNA replication termination in vertebrates"

    Article Title: The mechanism of DNA replication termination in vertebrates

    Journal: Nature

    doi: 10.1038/nature14887

    Topo II-dependent decatenation of p[lacOx16] (A) The autoradiograph in primary Figure 1G is reproduced with cartoons indicating the structures of the replication and termination intermediates n-n, n-sc, sc-sc, n, and sc (see main text for definitions). The order of appearance of the different catenanes matches previous work 5 (n-n, then n-sc, then sc-sc). (B–D) To determine the role of Topo II during termination within a lacO array, termination was monitored in mock- or Topo II-depleted extracts. To confirm immunodepletion of Topo II, Mock and Topo II-depleted NPE was blotted with MCM7 and Topo II antibodies (B). p[ lacO x16] was incubated with LacR, then replicated in either mock- or Topo II-depleted egg extracts in the presence of [α- 32 P]dATP, and termination was induced with IPTG (at 7’). Untreated DNA intermediates were separated by native gel electrophoresis (C). In the mock-depleted extract, nicked and supercoiled monomers were readily produced (as in (A), albeit with slower kinetics due to nonspecific inhibition of the extracts by the immunodepletion procedure), while in the Topo II-depleted extracts, a discrete species was produced. DNA from the last time point in each reaction (lanes 4 and 8 in (C)) was purified and treated with XmnI, which cuts p[ lacO x16] once, or Nt.BspQI, which nicks p[ lacO x16] once, or recombinant Topo II, and then separated by native gel electrophoresis (D). Cleavage of the mock- and TopoII-depleted products with XmnI yielded the expected linear 3.15 kb band (lanes 2 and 6), demonstrating that in both extracts, all products were fully dissolved topoisomers of each other. Relaxation of the mock-depleted products by nicking with Nt.BspQI yielded a discrete band corresponding to nicked plasmid (lane 3), while the TopoII-depleted products were converted to a ladder of discrete topoisomers (lane 7), which we infer represent catenated dimers of different linking numbers, since the mobility difference cannot be due to differences in supercoiling. Importantly, the mobility shift following Nt.BspQI treatment (lane 5 vs lane 7) demonstrated that the Topo II-depleted products (lane 5) were covalently closed and thus in the absence of Topo II ligation of the daughter strands still occurred. Treatment of the mock- and Topo II-depleted products with recombinant human Topo II produced the same relaxed monomeric species (lanes 4 and 8), further confirming that the Topo II-depleted products contained catenanes. Collectively, these observations demonstrate that termination within a lacO array in Topo II depleted extracts produces highly catenated supercoiled-supercoiled dimers, as seen in cells lacking Topo II 16 , 17 . These data confirm that Topo II is responsible for decatenation and argue that termination within a lacO array reflects physiological termination. (E) n-n, n-sc, sc-sc, n, and sc products were also detected when plasmid lacking lacO sequences (pBlueScript) was replicated in the absence of LacR without the use of Cyclin A to synchronize replication. Therefore, these intermediates arise in the course of unperturbed DNA replication in Xenopus egg extracts.
    Figure Legend Snippet: Topo II-dependent decatenation of p[lacOx16] (A) The autoradiograph in primary Figure 1G is reproduced with cartoons indicating the structures of the replication and termination intermediates n-n, n-sc, sc-sc, n, and sc (see main text for definitions). The order of appearance of the different catenanes matches previous work 5 (n-n, then n-sc, then sc-sc). (B–D) To determine the role of Topo II during termination within a lacO array, termination was monitored in mock- or Topo II-depleted extracts. To confirm immunodepletion of Topo II, Mock and Topo II-depleted NPE was blotted with MCM7 and Topo II antibodies (B). p[ lacO x16] was incubated with LacR, then replicated in either mock- or Topo II-depleted egg extracts in the presence of [α- 32 P]dATP, and termination was induced with IPTG (at 7’). Untreated DNA intermediates were separated by native gel electrophoresis (C). In the mock-depleted extract, nicked and supercoiled monomers were readily produced (as in (A), albeit with slower kinetics due to nonspecific inhibition of the extracts by the immunodepletion procedure), while in the Topo II-depleted extracts, a discrete species was produced. DNA from the last time point in each reaction (lanes 4 and 8 in (C)) was purified and treated with XmnI, which cuts p[ lacO x16] once, or Nt.BspQI, which nicks p[ lacO x16] once, or recombinant Topo II, and then separated by native gel electrophoresis (D). Cleavage of the mock- and TopoII-depleted products with XmnI yielded the expected linear 3.15 kb band (lanes 2 and 6), demonstrating that in both extracts, all products were fully dissolved topoisomers of each other. Relaxation of the mock-depleted products by nicking with Nt.BspQI yielded a discrete band corresponding to nicked plasmid (lane 3), while the TopoII-depleted products were converted to a ladder of discrete topoisomers (lane 7), which we infer represent catenated dimers of different linking numbers, since the mobility difference cannot be due to differences in supercoiling. Importantly, the mobility shift following Nt.BspQI treatment (lane 5 vs lane 7) demonstrated that the Topo II-depleted products (lane 5) were covalently closed and thus in the absence of Topo II ligation of the daughter strands still occurred. Treatment of the mock- and Topo II-depleted products with recombinant human Topo II produced the same relaxed monomeric species (lanes 4 and 8), further confirming that the Topo II-depleted products contained catenanes. Collectively, these observations demonstrate that termination within a lacO array in Topo II depleted extracts produces highly catenated supercoiled-supercoiled dimers, as seen in cells lacking Topo II 16 , 17 . These data confirm that Topo II is responsible for decatenation and argue that termination within a lacO array reflects physiological termination. (E) n-n, n-sc, sc-sc, n, and sc products were also detected when plasmid lacking lacO sequences (pBlueScript) was replicated in the absence of LacR without the use of Cyclin A to synchronize replication. Therefore, these intermediates arise in the course of unperturbed DNA replication in Xenopus egg extracts.

    Techniques Used: Autoradiography, Incubation, Nucleic Acid Electrophoresis, Produced, Inhibition, Purification, Recombinant, Plasmid Preparation, Mobility Shift, Ligation

    9) Product Images from "Protein switches identified from diverse insertion libraries created using S1 nuclease digestion of supercoiled-form plasmid DNA"

    Article Title: Protein switches identified from diverse insertion libraries created using S1 nuclease digestion of supercoiled-form plasmid DNA

    Journal: Biotechnology and bioengineering

    doi: 10.1002/bit.23224

    S1 nuclease converts supercoiled plasmid DNA to nicked circular and linear forms through digestion at a variety of locations in the plasmid. (A) Two μg of supercoiled pRH04.152-rbsB was incubated at 22°C (top) or 37°C (bottom) with different amounts of S1 nuclease for different lengths of time and then analyzed by agarose gel electrophoresis. The sixth and seventh lanes contain undigested supercoiled control and SpeI-digested linear control, respectively. The band that runs slower than the linear band was presumed to correspond to nicked circular plasmid. In the thirteenth lane, after the DNA was digested with 10 units for 24 hours an additional 10 units of S1 nuclease was added and the DNA was incubated for an additional hour. The first and last lanes contain the λDNA/HindIII molecular weight standards. (B) Plasmids with or without the f1 origin (pDIMC8-pfMBP and pRH04-pfMBP, respectively) were incubated with or without S1 nuclease, followed by incubation with or without NcoI, EagI, and SacII and then analyzed by agarose gel electrophoresis. (C) Plasmids with or without the f1 origin (pDIMC8-rbsb and pRH04-rbsb, respectively) were incubated with S1 nuclease or DNaseI, followed by incubation with XmnI and analyzed by agarose gel electrophoresis.
    Figure Legend Snippet: S1 nuclease converts supercoiled plasmid DNA to nicked circular and linear forms through digestion at a variety of locations in the plasmid. (A) Two μg of supercoiled pRH04.152-rbsB was incubated at 22°C (top) or 37°C (bottom) with different amounts of S1 nuclease for different lengths of time and then analyzed by agarose gel electrophoresis. The sixth and seventh lanes contain undigested supercoiled control and SpeI-digested linear control, respectively. The band that runs slower than the linear band was presumed to correspond to nicked circular plasmid. In the thirteenth lane, after the DNA was digested with 10 units for 24 hours an additional 10 units of S1 nuclease was added and the DNA was incubated for an additional hour. The first and last lanes contain the λDNA/HindIII molecular weight standards. (B) Plasmids with or without the f1 origin (pDIMC8-pfMBP and pRH04-pfMBP, respectively) were incubated with or without S1 nuclease, followed by incubation with or without NcoI, EagI, and SacII and then analyzed by agarose gel electrophoresis. (C) Plasmids with or without the f1 origin (pDIMC8-rbsb and pRH04-rbsb, respectively) were incubated with S1 nuclease or DNaseI, followed by incubation with XmnI and analyzed by agarose gel electrophoresis.

    Techniques Used: Plasmid Preparation, Incubation, Agarose Gel Electrophoresis, Molecular Weight

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    Article Title: Secretoglobin 1A1 and 1A1A Differentially Regulate Neutrophil Reactive Oxygen Species Production, Phagocytosis and Extracellular Trap Formation
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    Incubation:

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    Electrophoresis:

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    Cyclin A treatment synchronizes <t>DNA</t> replication in Xenopus egg extracts (A–B) To synchronize DNA replication in Xenopus egg extracts, we treated extracts with Cyclin A, which probably accelerates replication initiation 45 . Plasmid DNA was incubated in High Speed Supernatant for 20 minutes, then either buffer or Cyclin A was added for a further 20 minutes. NucleoPlasmic extract was added to initiate DNA replication, along with [α- 32 P]dATP to label replication intermediates. Replication products were separated on a native agarose gel, detected by autoradiography (A), and quantified (B). In the presence of vehicle, replication was not complete by 9.5 minutes, but in the presence of Cyclin A, replication was almost complete by 4.5 minutes (B). Thus, Cyclin A treatment approximately doubles the speed of DNA replication in Xenopus egg extracts. (C–F) To test whether Cyclin A affects the ability of LacR to inhibit termination, we monitored dissolution of plasmids containing a 12x or 16x LacR array in the presence and absence of Cyclin A. p[ lacO x12], p[ lacO x16], and the parental control plasmid p[ empty ] were incubated with LacR, and then treated with buffer or Cyclin A before replication was initiated with NPE in the presence of [α- 32 P]dATP. Samples were withdrawn when dissolution of p[ empty ] plateaued (9.5 minutes in the presence of buffer, 4.5 minutes in the presence of Cyclin A). Given that Cyclin A treatment approximately doubles the speed of replication (see B), samples were withdrawn from these reactions twice as frequently as the buffer-treated samples. To measure dissolution, radiolabelled termination intermediates were cut with <t>XmnI</t> to monitor the conversion of double-Y molecules to linear molecules. Cut molecules were separated on a native agarose gel and detected by autoradiography (C,E). By the time the first sample was withdrawn, dissolution of p[ empty ] was essentially complete, in the absence (9.5 min, D) or presence (4.5 min, F) of Cyclin A. Importantly, dissolution of p[ lacO x12] and p[ lacO x16] was prevented in the absence (9.5 min, D) or presence (4.5 min, F) of Cyclin A. Moreover, dissolution occurred approximately twice as fast in the presence of Cyclin A (note the similarity between (D) and (F) even though samples are withdrawn twice as frequently in F.) consistent with replication being approximately twice as fast in the presence of Cyclin A. Therefore, Cyclin A does not affect the ability of a LacR array to block replication forks.
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    Cyclin A treatment synchronizes DNA replication in Xenopus egg extracts (A–B) To synchronize DNA replication in Xenopus egg extracts, we treated extracts with Cyclin A, which probably accelerates replication initiation 45 . Plasmid DNA was incubated in High Speed Supernatant for 20 minutes, then either buffer or Cyclin A was added for a further 20 minutes. NucleoPlasmic extract was added to initiate DNA replication, along with [α- 32 P]dATP to label replication intermediates. Replication products were separated on a native agarose gel, detected by autoradiography (A), and quantified (B). In the presence of vehicle, replication was not complete by 9.5 minutes, but in the presence of Cyclin A, replication was almost complete by 4.5 minutes (B). Thus, Cyclin A treatment approximately doubles the speed of DNA replication in Xenopus egg extracts. (C–F) To test whether Cyclin A affects the ability of LacR to inhibit termination, we monitored dissolution of plasmids containing a 12x or 16x LacR array in the presence and absence of Cyclin A. p[ lacO x12], p[ lacO x16], and the parental control plasmid p[ empty ] were incubated with LacR, and then treated with buffer or Cyclin A before replication was initiated with NPE in the presence of [α- 32 P]dATP. Samples were withdrawn when dissolution of p[ empty ] plateaued (9.5 minutes in the presence of buffer, 4.5 minutes in the presence of Cyclin A). Given that Cyclin A treatment approximately doubles the speed of replication (see B), samples were withdrawn from these reactions twice as frequently as the buffer-treated samples. To measure dissolution, radiolabelled termination intermediates were cut with XmnI to monitor the conversion of double-Y molecules to linear molecules. Cut molecules were separated on a native agarose gel and detected by autoradiography (C,E). By the time the first sample was withdrawn, dissolution of p[ empty ] was essentially complete, in the absence (9.5 min, D) or presence (4.5 min, F) of Cyclin A. Importantly, dissolution of p[ lacO x12] and p[ lacO x16] was prevented in the absence (9.5 min, D) or presence (4.5 min, F) of Cyclin A. Moreover, dissolution occurred approximately twice as fast in the presence of Cyclin A (note the similarity between (D) and (F) even though samples are withdrawn twice as frequently in F.) consistent with replication being approximately twice as fast in the presence of Cyclin A. Therefore, Cyclin A does not affect the ability of a LacR array to block replication forks.

    Journal: Nature

    Article Title: The mechanism of DNA replication termination in vertebrates

    doi: 10.1038/nature14887

    Figure Lengend Snippet: Cyclin A treatment synchronizes DNA replication in Xenopus egg extracts (A–B) To synchronize DNA replication in Xenopus egg extracts, we treated extracts with Cyclin A, which probably accelerates replication initiation 45 . Plasmid DNA was incubated in High Speed Supernatant for 20 minutes, then either buffer or Cyclin A was added for a further 20 minutes. NucleoPlasmic extract was added to initiate DNA replication, along with [α- 32 P]dATP to label replication intermediates. Replication products were separated on a native agarose gel, detected by autoradiography (A), and quantified (B). In the presence of vehicle, replication was not complete by 9.5 minutes, but in the presence of Cyclin A, replication was almost complete by 4.5 minutes (B). Thus, Cyclin A treatment approximately doubles the speed of DNA replication in Xenopus egg extracts. (C–F) To test whether Cyclin A affects the ability of LacR to inhibit termination, we monitored dissolution of plasmids containing a 12x or 16x LacR array in the presence and absence of Cyclin A. p[ lacO x12], p[ lacO x16], and the parental control plasmid p[ empty ] were incubated with LacR, and then treated with buffer or Cyclin A before replication was initiated with NPE in the presence of [α- 32 P]dATP. Samples were withdrawn when dissolution of p[ empty ] plateaued (9.5 minutes in the presence of buffer, 4.5 minutes in the presence of Cyclin A). Given that Cyclin A treatment approximately doubles the speed of replication (see B), samples were withdrawn from these reactions twice as frequently as the buffer-treated samples. To measure dissolution, radiolabelled termination intermediates were cut with XmnI to monitor the conversion of double-Y molecules to linear molecules. Cut molecules were separated on a native agarose gel and detected by autoradiography (C,E). By the time the first sample was withdrawn, dissolution of p[ empty ] was essentially complete, in the absence (9.5 min, D) or presence (4.5 min, F) of Cyclin A. Importantly, dissolution of p[ lacO x12] and p[ lacO x16] was prevented in the absence (9.5 min, D) or presence (4.5 min, F) of Cyclin A. Moreover, dissolution occurred approximately twice as fast in the presence of Cyclin A (note the similarity between (D) and (F) even though samples are withdrawn twice as frequently in F.) consistent with replication being approximately twice as fast in the presence of Cyclin A. Therefore, Cyclin A does not affect the ability of a LacR array to block replication forks.

    Article Snippet: Briefly, purified DNA was digested with XmnI (New England BioLabs) and then separated by native-native 2D gel electrophoresis.

    Techniques: Plasmid Preparation, Incubation, Agarose Gel Electrophoresis, Autoradiography, Blocking Assay

    Topo II-dependent decatenation of p[lacOx16] (A) The autoradiograph in primary Figure 1G is reproduced with cartoons indicating the structures of the replication and termination intermediates n-n, n-sc, sc-sc, n, and sc (see main text for definitions). The order of appearance of the different catenanes matches previous work 5 (n-n, then n-sc, then sc-sc). (B–D) To determine the role of Topo II during termination within a lacO array, termination was monitored in mock- or Topo II-depleted extracts. To confirm immunodepletion of Topo II, Mock and Topo II-depleted NPE was blotted with MCM7 and Topo II antibodies (B). p[ lacO x16] was incubated with LacR, then replicated in either mock- or Topo II-depleted egg extracts in the presence of [α- 32 P]dATP, and termination was induced with IPTG (at 7’). Untreated DNA intermediates were separated by native gel electrophoresis (C). In the mock-depleted extract, nicked and supercoiled monomers were readily produced (as in (A), albeit with slower kinetics due to nonspecific inhibition of the extracts by the immunodepletion procedure), while in the Topo II-depleted extracts, a discrete species was produced. DNA from the last time point in each reaction (lanes 4 and 8 in (C)) was purified and treated with XmnI, which cuts p[ lacO x16] once, or Nt.BspQI, which nicks p[ lacO x16] once, or recombinant Topo II, and then separated by native gel electrophoresis (D). Cleavage of the mock- and TopoII-depleted products with XmnI yielded the expected linear 3.15 kb band (lanes 2 and 6), demonstrating that in both extracts, all products were fully dissolved topoisomers of each other. Relaxation of the mock-depleted products by nicking with Nt.BspQI yielded a discrete band corresponding to nicked plasmid (lane 3), while the TopoII-depleted products were converted to a ladder of discrete topoisomers (lane 7), which we infer represent catenated dimers of different linking numbers, since the mobility difference cannot be due to differences in supercoiling. Importantly, the mobility shift following Nt.BspQI treatment (lane 5 vs lane 7) demonstrated that the Topo II-depleted products (lane 5) were covalently closed and thus in the absence of Topo II ligation of the daughter strands still occurred. Treatment of the mock- and Topo II-depleted products with recombinant human Topo II produced the same relaxed monomeric species (lanes 4 and 8), further confirming that the Topo II-depleted products contained catenanes. Collectively, these observations demonstrate that termination within a lacO array in Topo II depleted extracts produces highly catenated supercoiled-supercoiled dimers, as seen in cells lacking Topo II 16 , 17 . These data confirm that Topo II is responsible for decatenation and argue that termination within a lacO array reflects physiological termination. (E) n-n, n-sc, sc-sc, n, and sc products were also detected when plasmid lacking lacO sequences (pBlueScript) was replicated in the absence of LacR without the use of Cyclin A to synchronize replication. Therefore, these intermediates arise in the course of unperturbed DNA replication in Xenopus egg extracts.

    Journal: Nature

    Article Title: The mechanism of DNA replication termination in vertebrates

    doi: 10.1038/nature14887

    Figure Lengend Snippet: Topo II-dependent decatenation of p[lacOx16] (A) The autoradiograph in primary Figure 1G is reproduced with cartoons indicating the structures of the replication and termination intermediates n-n, n-sc, sc-sc, n, and sc (see main text for definitions). The order of appearance of the different catenanes matches previous work 5 (n-n, then n-sc, then sc-sc). (B–D) To determine the role of Topo II during termination within a lacO array, termination was monitored in mock- or Topo II-depleted extracts. To confirm immunodepletion of Topo II, Mock and Topo II-depleted NPE was blotted with MCM7 and Topo II antibodies (B). p[ lacO x16] was incubated with LacR, then replicated in either mock- or Topo II-depleted egg extracts in the presence of [α- 32 P]dATP, and termination was induced with IPTG (at 7’). Untreated DNA intermediates were separated by native gel electrophoresis (C). In the mock-depleted extract, nicked and supercoiled monomers were readily produced (as in (A), albeit with slower kinetics due to nonspecific inhibition of the extracts by the immunodepletion procedure), while in the Topo II-depleted extracts, a discrete species was produced. DNA from the last time point in each reaction (lanes 4 and 8 in (C)) was purified and treated with XmnI, which cuts p[ lacO x16] once, or Nt.BspQI, which nicks p[ lacO x16] once, or recombinant Topo II, and then separated by native gel electrophoresis (D). Cleavage of the mock- and TopoII-depleted products with XmnI yielded the expected linear 3.15 kb band (lanes 2 and 6), demonstrating that in both extracts, all products were fully dissolved topoisomers of each other. Relaxation of the mock-depleted products by nicking with Nt.BspQI yielded a discrete band corresponding to nicked plasmid (lane 3), while the TopoII-depleted products were converted to a ladder of discrete topoisomers (lane 7), which we infer represent catenated dimers of different linking numbers, since the mobility difference cannot be due to differences in supercoiling. Importantly, the mobility shift following Nt.BspQI treatment (lane 5 vs lane 7) demonstrated that the Topo II-depleted products (lane 5) were covalently closed and thus in the absence of Topo II ligation of the daughter strands still occurred. Treatment of the mock- and Topo II-depleted products with recombinant human Topo II produced the same relaxed monomeric species (lanes 4 and 8), further confirming that the Topo II-depleted products contained catenanes. Collectively, these observations demonstrate that termination within a lacO array in Topo II depleted extracts produces highly catenated supercoiled-supercoiled dimers, as seen in cells lacking Topo II 16 , 17 . These data confirm that Topo II is responsible for decatenation and argue that termination within a lacO array reflects physiological termination. (E) n-n, n-sc, sc-sc, n, and sc products were also detected when plasmid lacking lacO sequences (pBlueScript) was replicated in the absence of LacR without the use of Cyclin A to synchronize replication. Therefore, these intermediates arise in the course of unperturbed DNA replication in Xenopus egg extracts.

    Article Snippet: Briefly, purified DNA was digested with XmnI (New England BioLabs) and then separated by native-native 2D gel electrophoresis.

    Techniques: Autoradiography, Incubation, Nucleic Acid Electrophoresis, Produced, Inhibition, Purification, Recombinant, Plasmid Preparation, Mobility Shift, Ligation

    Replisome progression through 12x and 32x lacO arrays (A–D) To test whether replisomes meet later in a lacO x32 array than a lacO x12 array, we monitored dissolution. LacR Block-IPTG release was performed on p[ lacO x12] and p[ lacO x32] and radiolabelled termination intermediates were digested with XmnI to monitor the conversion of double-Y molecules to linear molecules (Dissolution). Cleaved molecules were separated on a native agarose gel, detected by autoradiography (A,C), and quantified (B,D). Upon IPTG addition, dissolution was delayed by at least 1 minute within the 32x lacO array compared to the 12x lacO array (B,D). Moreover, by 6 minutes, 92% of forks had undergone dissolution on p[ lacO x12] while only 9% had dissolved on p[ lacO x32] (B,D). (E) Stall products within the 12x lacO array ( Fig. 3B , Lane 2) were quantified, signal was corrected based on size differences of the products, and the percentage of stall products at each stall point was calculated. 78% of leading strands stalled at the first three arrest points (red columns), 19% stalled at the fourth to tenth arrest points (yellow columns) and the remaining 3% stalled at the tenth to fourteenth arrest points (grey columns). The appearance of fourteen arrest points reproducible and surprising, given that the presence of only twelve lacO sequences was confirmed by sequencing in the very preparation of p[ lacO x12] that was used in Fig. 3 . The thirteenth and fourteenth arrest points cannot stem from cryptic lacO sites beyond the twelfth lacO site, as this would position the first leftward leading strand stall product ∼90 nucleotides from the lacO array, instead of the observed ∼30 nucleotides (see F-G ). At present, we do not understand the origin of these stall products. (F–G) Progression of leftward leading strands into the array. The same DNA samples used in Fig. 3 were digested with the nicking enzyme Nb.BsrDI, which released leftward leading strands (F), and separated on a denaturing polyacrylamide gel (G). The lacO sites of p[ lacO x12] are highlighted in blue on the sequencing ladder (G), which was generated using the primer JDO109 (Green arrow, F). Green circles indicate two non-specific products of digestion. These products arise because nicking enzyme activity varies between experiments, even under the same conditions. There was no significant difference in the pattern of leftward leading strand progression between the 12x lacO and 32x lacO arrays, as seen for the rightward leading strands ( Fig. 3B ). Specifically, by 5.67 minutes, the majority of leading strands had extended beyond the seventh lacO repeat within lacO x12 (lane 6) and the equivalent region of lacO x32 (lane 18). Therefore, progression of leftward leading strands is unaffected by the presence of an opposing replisome, suggesting that converging replisomes do not stall when they meet.

    Journal: Nature

    Article Title: The mechanism of DNA replication termination in vertebrates

    doi: 10.1038/nature14887

    Figure Lengend Snippet: Replisome progression through 12x and 32x lacO arrays (A–D) To test whether replisomes meet later in a lacO x32 array than a lacO x12 array, we monitored dissolution. LacR Block-IPTG release was performed on p[ lacO x12] and p[ lacO x32] and radiolabelled termination intermediates were digested with XmnI to monitor the conversion of double-Y molecules to linear molecules (Dissolution). Cleaved molecules were separated on a native agarose gel, detected by autoradiography (A,C), and quantified (B,D). Upon IPTG addition, dissolution was delayed by at least 1 minute within the 32x lacO array compared to the 12x lacO array (B,D). Moreover, by 6 minutes, 92% of forks had undergone dissolution on p[ lacO x12] while only 9% had dissolved on p[ lacO x32] (B,D). (E) Stall products within the 12x lacO array ( Fig. 3B , Lane 2) were quantified, signal was corrected based on size differences of the products, and the percentage of stall products at each stall point was calculated. 78% of leading strands stalled at the first three arrest points (red columns), 19% stalled at the fourth to tenth arrest points (yellow columns) and the remaining 3% stalled at the tenth to fourteenth arrest points (grey columns). The appearance of fourteen arrest points reproducible and surprising, given that the presence of only twelve lacO sequences was confirmed by sequencing in the very preparation of p[ lacO x12] that was used in Fig. 3 . The thirteenth and fourteenth arrest points cannot stem from cryptic lacO sites beyond the twelfth lacO site, as this would position the first leftward leading strand stall product ∼90 nucleotides from the lacO array, instead of the observed ∼30 nucleotides (see F-G ). At present, we do not understand the origin of these stall products. (F–G) Progression of leftward leading strands into the array. The same DNA samples used in Fig. 3 were digested with the nicking enzyme Nb.BsrDI, which released leftward leading strands (F), and separated on a denaturing polyacrylamide gel (G). The lacO sites of p[ lacO x12] are highlighted in blue on the sequencing ladder (G), which was generated using the primer JDO109 (Green arrow, F). Green circles indicate two non-specific products of digestion. These products arise because nicking enzyme activity varies between experiments, even under the same conditions. There was no significant difference in the pattern of leftward leading strand progression between the 12x lacO and 32x lacO arrays, as seen for the rightward leading strands ( Fig. 3B ). Specifically, by 5.67 minutes, the majority of leading strands had extended beyond the seventh lacO repeat within lacO x12 (lane 6) and the equivalent region of lacO x32 (lane 18). Therefore, progression of leftward leading strands is unaffected by the presence of an opposing replisome, suggesting that converging replisomes do not stall when they meet.

    Article Snippet: Briefly, purified DNA was digested with XmnI (New England BioLabs) and then separated by native-native 2D gel electrophoresis.

    Techniques: Blocking Assay, Agarose Gel Electrophoresis, Autoradiography, Sequencing, Generated, Activity Assay

    Supplemental ChIP data (A) Cartoon depicting the LAC, FLK2 and FAR loci, which were used for ChIP. Their precise locations relative to the leftward edge of the lacO array are indicated. The LAC amplicon is present in four copies distributed across the lacOx16 array and three copies distributed across the lacOx12 array. (B–E) p[ lacO x12] was incubated with buffer or LacR and termination was induced at 5 minutes by IPTG addition. MCM7, RPA, CDC45, and Polε ChIP was performed at different time points after IPTG addition but also in the buffer control and no IPTG control. Recovery of FLK2 was measured as a percentage of input DNA. Upon IPTG addition, ChIP signal declined and by 9 minutes was comparable to the buffer control, demonstrating that unloading of replisomes was induced within 4 minutes of IPTG addition. (F) To test whether movement of the replisome into and out of the lacO array could be detected upon IPTG addition, termination was monitored within a lacO array, and we performed ChIP of the leading strand polymerase Polε, which was inferred to move into and out of the array based on the behavior of leading strands during termination ( Extended Data Fig 2B–E ). It was predicted that Polε ChIP at the LAC locus should increase slightly as Polε enters the lacO array and decline again as converging polymerases pass each other, but persist at FLK2 while the polymerases move out of the array. Prior to IPTG addition, Polε was enriched at LAC and FLK2 compared to FAR , consistent with the leading strands being positioned on either side of the lacO array ( Extended Data Fig 2C , Fig. 3 ). Upon IPTG addition, Polε became modestly enriched at LAC compared to FLK2 (5.5 min) but then declined to similar levels at both LAC and FLK2 by 6.5 min. These data are consistent with the leading strand polymerases entering the lacO array and passing each other. (G–H) To test whether CMG exhibited the same ChIP profile as Polε, MCM7 and CDC45 ChIP was performed using the same samples. Following IPTG addition, MCM7 and CDC45 were enriched at LAC compared to FLK2 (5.5 min), then declined to similar levels at both LAC and FLK2 by 6.5 min, as seen for Polε (F). These data are consistent with a model in which CMGs enter the array and pass each other during termination. A caveat of these experiments is the relatively high recovery of the FAR locus in MCM7, CDC45, and Polε ChIP. Specifically, signal was at most only ∼2-fold enriched at LAC compared to FAR . This was not due to high background binding, because by the end of the experiment (10 minute time point, not shown), we observed a decrease in signal of ∼5–7 fold. Furthermore, we observed ∼5–7 fold enrichment in binding (ChIP) of replisome components to p[ lacO x12] that had been incubated in LacR compared to a buffer control (see G-I, below). Instead, the high FAR signal was likely due to poor spatial resolution of the ChIP. Consistent with this, when a plasmid containing a DNA interstrand cross-link (ICL) was replicated, essentially all replisomes converged upon the ICL but the ChIP signal for MCM7 and CDC45 was only ∼3–4 fold enriched at the ICL compared to a control locus 41 . We speculate that the higher background observed at the control locus in our experiments is due to the decreased distance of the control locus from the experimental locus (1.3 kb for p[ lacO x16] and p[ lacO x12] vs. 2.4 kb for the ICL plasmid) and possibly due to increased catenation of the parental strands during termination. The high signal at FAR should not complicate interpretation of the MCM7, CDC45 and Polε ChIP (F), as signal at FAR was essentially unaltered between 5 and 6.5 minutes. Further evidence that the high signal seen at the FAR locus emanates from forks stalled near the lacO array is presented in panel ( K ). (I) ChIP of RPA was performed on the same chromatin samples used in B-D. As seen for pol ɛ, MCM7, and CDC45, enrichment of RPA at LAC compared to FAR was relatively low, consistent with poor spatial resolution. (J) Predicted binding of CMGs to the LAC, FLK2 and FAR loci before and after IPTG addition if CMGs converging CMG pass each other. (K) To determine whether most forks stalled at the array and not elsewhere in the plasmid, we performed a time course in which p[ lacO x16] undergoing termination was examined by 2-dimensional gel electrophoresis (2-D gel) at various time points. p[ lacO x16] was pre-bound to LacR and replicated in Xenopus egg extract containing [α- 32 P]dATP. Termination was induced by IPTG addition and samples were withdrawn at different times. Radiolabelled replication intermediates were cleaved with XmnI (as in Extended Data Fig. 1A ) and separated according to size and shape on 2-D gels 50 . A parallel reaction was performed in which samples were analyzed by ChIP, which was one of the repeats analyzed in (B)-(E). In the presence of LacR, a subset of Double Y molecules accumulated (blue arrowhead), demonstrating that 83% of replication intermediates (signal in dashed blue circle) contained two forks converged at a specific locus. Following IPTG addition, linear molecules rapidly accumulated (orange arrow) as dissolution occurred. Importantly, the vast majority of signal was present in the discrete double-Y and linear species (blue and orange arrows), demonstrating that the relatively high ChIP signal observed at FAR in panels F-I was derived from forks present at the lacO x16 array and not elsewhere.

    Journal: Nature

    Article Title: The mechanism of DNA replication termination in vertebrates

    doi: 10.1038/nature14887

    Figure Lengend Snippet: Supplemental ChIP data (A) Cartoon depicting the LAC, FLK2 and FAR loci, which were used for ChIP. Their precise locations relative to the leftward edge of the lacO array are indicated. The LAC amplicon is present in four copies distributed across the lacOx16 array and three copies distributed across the lacOx12 array. (B–E) p[ lacO x12] was incubated with buffer or LacR and termination was induced at 5 minutes by IPTG addition. MCM7, RPA, CDC45, and Polε ChIP was performed at different time points after IPTG addition but also in the buffer control and no IPTG control. Recovery of FLK2 was measured as a percentage of input DNA. Upon IPTG addition, ChIP signal declined and by 9 minutes was comparable to the buffer control, demonstrating that unloading of replisomes was induced within 4 minutes of IPTG addition. (F) To test whether movement of the replisome into and out of the lacO array could be detected upon IPTG addition, termination was monitored within a lacO array, and we performed ChIP of the leading strand polymerase Polε, which was inferred to move into and out of the array based on the behavior of leading strands during termination ( Extended Data Fig 2B–E ). It was predicted that Polε ChIP at the LAC locus should increase slightly as Polε enters the lacO array and decline again as converging polymerases pass each other, but persist at FLK2 while the polymerases move out of the array. Prior to IPTG addition, Polε was enriched at LAC and FLK2 compared to FAR , consistent with the leading strands being positioned on either side of the lacO array ( Extended Data Fig 2C , Fig. 3 ). Upon IPTG addition, Polε became modestly enriched at LAC compared to FLK2 (5.5 min) but then declined to similar levels at both LAC and FLK2 by 6.5 min. These data are consistent with the leading strand polymerases entering the lacO array and passing each other. (G–H) To test whether CMG exhibited the same ChIP profile as Polε, MCM7 and CDC45 ChIP was performed using the same samples. Following IPTG addition, MCM7 and CDC45 were enriched at LAC compared to FLK2 (5.5 min), then declined to similar levels at both LAC and FLK2 by 6.5 min, as seen for Polε (F). These data are consistent with a model in which CMGs enter the array and pass each other during termination. A caveat of these experiments is the relatively high recovery of the FAR locus in MCM7, CDC45, and Polε ChIP. Specifically, signal was at most only ∼2-fold enriched at LAC compared to FAR . This was not due to high background binding, because by the end of the experiment (10 minute time point, not shown), we observed a decrease in signal of ∼5–7 fold. Furthermore, we observed ∼5–7 fold enrichment in binding (ChIP) of replisome components to p[ lacO x12] that had been incubated in LacR compared to a buffer control (see G-I, below). Instead, the high FAR signal was likely due to poor spatial resolution of the ChIP. Consistent with this, when a plasmid containing a DNA interstrand cross-link (ICL) was replicated, essentially all replisomes converged upon the ICL but the ChIP signal for MCM7 and CDC45 was only ∼3–4 fold enriched at the ICL compared to a control locus 41 . We speculate that the higher background observed at the control locus in our experiments is due to the decreased distance of the control locus from the experimental locus (1.3 kb for p[ lacO x16] and p[ lacO x12] vs. 2.4 kb for the ICL plasmid) and possibly due to increased catenation of the parental strands during termination. The high signal at FAR should not complicate interpretation of the MCM7, CDC45 and Polε ChIP (F), as signal at FAR was essentially unaltered between 5 and 6.5 minutes. Further evidence that the high signal seen at the FAR locus emanates from forks stalled near the lacO array is presented in panel ( K ). (I) ChIP of RPA was performed on the same chromatin samples used in B-D. As seen for pol ɛ, MCM7, and CDC45, enrichment of RPA at LAC compared to FAR was relatively low, consistent with poor spatial resolution. (J) Predicted binding of CMGs to the LAC, FLK2 and FAR loci before and after IPTG addition if CMGs converging CMG pass each other. (K) To determine whether most forks stalled at the array and not elsewhere in the plasmid, we performed a time course in which p[ lacO x16] undergoing termination was examined by 2-dimensional gel electrophoresis (2-D gel) at various time points. p[ lacO x16] was pre-bound to LacR and replicated in Xenopus egg extract containing [α- 32 P]dATP. Termination was induced by IPTG addition and samples were withdrawn at different times. Radiolabelled replication intermediates were cleaved with XmnI (as in Extended Data Fig. 1A ) and separated according to size and shape on 2-D gels 50 . A parallel reaction was performed in which samples were analyzed by ChIP, which was one of the repeats analyzed in (B)-(E). In the presence of LacR, a subset of Double Y molecules accumulated (blue arrowhead), demonstrating that 83% of replication intermediates (signal in dashed blue circle) contained two forks converged at a specific locus. Following IPTG addition, linear molecules rapidly accumulated (orange arrow) as dissolution occurred. Importantly, the vast majority of signal was present in the discrete double-Y and linear species (blue and orange arrows), demonstrating that the relatively high ChIP signal observed at FAR in panels F-I was derived from forks present at the lacO x16 array and not elsewhere.

    Article Snippet: Briefly, purified DNA was digested with XmnI (New England BioLabs) and then separated by native-native 2D gel electrophoresis.

    Techniques: Chromatin Immunoprecipitation, Amplification, Incubation, Recombinase Polymerase Amplification, Binding Assay, Plasmid Preparation, Nucleic Acid Electrophoresis, Derivative Assay

    Supplemental termination data for p[empty] experiments (A) Cartoon depicting the XmnI and AlwNI sites on p[ empty ], which are used for the dissolution and ligation assays, respectively, and the FLK2 locus, which is used for ChIP. (B) Plasmid DNA without a lacO array (p[ empty ]) was replicated and at different times chromatin was subjected to MCM7 and CDC45 ChIP. Percent recovery of FLK2 was quantified and used to measure dissociation of MCM7 and CDC45 (see methods). Dissolution and ligation were also quantified in parallel. mean±s.d. is plotted (n=3). The MCM7 and CDC45 dissociation data is obtained from the vehicle controls in Figure 5B–C , while the dissolution and ligation data are obtained from the vehicle controls in Fig 5D–E . (C) To seek independent evidence for the conclusions of the ChIP data presented in Figure 5B–C , we used a plasmid pull-down procedure. p[empty] was replicated in egg extracts treated with Vehicle or Ub-VS. At the indicated times, chromatin-associated proteins were captured on LacR-coated beads (which binds DNA independently of lacO sites) and analyzed by Western blotting for CDC45, MCM7, and PCNA. CDC45 and MCM7 dissociated from chromatin by 8’ in the vehicle control, but persisted following UbVS treatment. (D) To test whether the MCM7 modifications detected in panel (C) represented ubiquitylation, extracts were incubated with HIS 6 -Ubiquitin in the absence of Cyclin A, and in the absence or presence of plasmid DNA. After 15 minutes, HIS 6 -tagged proteins were captured by nickel resin pull down and blotted for MCM7. DNA replication greatly increased the levels of ubiquitylated MCM7, with the exception of a single species that was ubiquitylated independently of DNA replication (*). These data show that MCM7 is ubiquitylated during plasmid replication in egg extracts, as observed in yeast and during replication of sperm chromatin following nuclear assembly in egg extracts 24 , 25 . (E) In parallel to the plasmid pull-downs performed in (C), DNA samples were withdrawn for dissolution, ligation, and decatenation assays, none of which were perturbed by UbVS treatment. These data support our conclusion, based on ChIP experiments ( Fig. 5 ), that defective CMG unloading does not affect dissolution, ligation, or decatenation. (F) Decatenation was measured in the same reactions used to measure Dissolution and Ligation ( Fig. 5D–E ), mean±s.d. is plotted (n=3). (G–I) Given the experimental variability at the 4 minute time point in Figures 5D–F , the primary data and quantification for dissolution (G), ligation (H), and decatenation (I) for one of the three experiments summarized in Figure 5D–F is presented. This reveals that Ub-VS does not inhibit dissolution, ligation, or decatenation at the 4 minute time point. The same conclusion applies to two additional repetitions of this experiment (data not shown). (J) The primary ChIP data used to measure dissociation of MCM7 and CDC45 in Fig 5B–C is shown. Recovery of FLK2 was measured. mean±s.d. is plotted (n=3).

    Journal: Nature

    Article Title: The mechanism of DNA replication termination in vertebrates

    doi: 10.1038/nature14887

    Figure Lengend Snippet: Supplemental termination data for p[empty] experiments (A) Cartoon depicting the XmnI and AlwNI sites on p[ empty ], which are used for the dissolution and ligation assays, respectively, and the FLK2 locus, which is used for ChIP. (B) Plasmid DNA without a lacO array (p[ empty ]) was replicated and at different times chromatin was subjected to MCM7 and CDC45 ChIP. Percent recovery of FLK2 was quantified and used to measure dissociation of MCM7 and CDC45 (see methods). Dissolution and ligation were also quantified in parallel. mean±s.d. is plotted (n=3). The MCM7 and CDC45 dissociation data is obtained from the vehicle controls in Figure 5B–C , while the dissolution and ligation data are obtained from the vehicle controls in Fig 5D–E . (C) To seek independent evidence for the conclusions of the ChIP data presented in Figure 5B–C , we used a plasmid pull-down procedure. p[empty] was replicated in egg extracts treated with Vehicle or Ub-VS. At the indicated times, chromatin-associated proteins were captured on LacR-coated beads (which binds DNA independently of lacO sites) and analyzed by Western blotting for CDC45, MCM7, and PCNA. CDC45 and MCM7 dissociated from chromatin by 8’ in the vehicle control, but persisted following UbVS treatment. (D) To test whether the MCM7 modifications detected in panel (C) represented ubiquitylation, extracts were incubated with HIS 6 -Ubiquitin in the absence of Cyclin A, and in the absence or presence of plasmid DNA. After 15 minutes, HIS 6 -tagged proteins were captured by nickel resin pull down and blotted for MCM7. DNA replication greatly increased the levels of ubiquitylated MCM7, with the exception of a single species that was ubiquitylated independently of DNA replication (*). These data show that MCM7 is ubiquitylated during plasmid replication in egg extracts, as observed in yeast and during replication of sperm chromatin following nuclear assembly in egg extracts 24 , 25 . (E) In parallel to the plasmid pull-downs performed in (C), DNA samples were withdrawn for dissolution, ligation, and decatenation assays, none of which were perturbed by UbVS treatment. These data support our conclusion, based on ChIP experiments ( Fig. 5 ), that defective CMG unloading does not affect dissolution, ligation, or decatenation. (F) Decatenation was measured in the same reactions used to measure Dissolution and Ligation ( Fig. 5D–E ), mean±s.d. is plotted (n=3). (G–I) Given the experimental variability at the 4 minute time point in Figures 5D–F , the primary data and quantification for dissolution (G), ligation (H), and decatenation (I) for one of the three experiments summarized in Figure 5D–F is presented. This reveals that Ub-VS does not inhibit dissolution, ligation, or decatenation at the 4 minute time point. The same conclusion applies to two additional repetitions of this experiment (data not shown). (J) The primary ChIP data used to measure dissociation of MCM7 and CDC45 in Fig 5B–C is shown. Recovery of FLK2 was measured. mean±s.d. is plotted (n=3).

    Article Snippet: Briefly, purified DNA was digested with XmnI (New England BioLabs) and then separated by native-native 2D gel electrophoresis.

    Techniques: Ligation, Chromatin Immunoprecipitation, Plasmid Preparation, Western Blot, Incubation

    Cloning, expression and purification of equine recombinant SCGB 1A1 and SCGB 1A1A proteins. (A) SCGB1A1 (“1”) and SCGB1A1A (“1A”) partial ORFs were amplified from equine lung cDNA preparations. A unique band of appropriate size (225 bp) was amplified for each gene. L = 1 Kb+ DNA ladder. (B) Fragments were digested with XmnI and SbfI restriction enzymes (purple boxes) and inserted into the multiple cloning sites (MCS) of the pMAL-c5X expression vector (top). DNA from the transformed colonies was submitted for sequencing to determine the presence, integrity, orientation and suitable translational reading frame of the insert. SCGB1A1 and SCGB1A1A sequenced (S) products showed proper orientation and 100% identity to the predicted (P) sequences. (C) Fractions collected during the purification steps of SCGB 1A1 and SCGB 1A1A were analyzed by SDS-PAGE. A fusion protein was apparent in extracts from IPTG-induced (I) but not un-induced (U) colonies. A crude extract (CE) was collected from induced cells and purified by affinity chromatography, using an amylose (A) column. The eluted fractions were pooled and incubated with Factor Xa protease (Fx) to cleave the fusion proteins. Fx was removed by FPLC (F), and MBP (42.5 kDa) was removed by additional passage on an amylose column from which pure (P) recombinant proteins (7 kDa) were collected. (D) Purified SCGB 1A1 and SCGB 1A1A proteins form dimers that dissociate under reducing and denaturing conditions. (E) Identity of dimers and monomers was confirmed by Western blot analysis. (C, D, E) S = Precision plus protein standard (dual color).

    Journal: PLoS ONE

    Article Title: Secretoglobin 1A1 and 1A1A Differentially Regulate Neutrophil Reactive Oxygen Species Production, Phagocytosis and Extracellular Trap Formation

    doi: 10.1371/journal.pone.0096217

    Figure Lengend Snippet: Cloning, expression and purification of equine recombinant SCGB 1A1 and SCGB 1A1A proteins. (A) SCGB1A1 (“1”) and SCGB1A1A (“1A”) partial ORFs were amplified from equine lung cDNA preparations. A unique band of appropriate size (225 bp) was amplified for each gene. L = 1 Kb+ DNA ladder. (B) Fragments were digested with XmnI and SbfI restriction enzymes (purple boxes) and inserted into the multiple cloning sites (MCS) of the pMAL-c5X expression vector (top). DNA from the transformed colonies was submitted for sequencing to determine the presence, integrity, orientation and suitable translational reading frame of the insert. SCGB1A1 and SCGB1A1A sequenced (S) products showed proper orientation and 100% identity to the predicted (P) sequences. (C) Fractions collected during the purification steps of SCGB 1A1 and SCGB 1A1A were analyzed by SDS-PAGE. A fusion protein was apparent in extracts from IPTG-induced (I) but not un-induced (U) colonies. A crude extract (CE) was collected from induced cells and purified by affinity chromatography, using an amylose (A) column. The eluted fractions were pooled and incubated with Factor Xa protease (Fx) to cleave the fusion proteins. Fx was removed by FPLC (F), and MBP (42.5 kDa) was removed by additional passage on an amylose column from which pure (P) recombinant proteins (7 kDa) were collected. (D) Purified SCGB 1A1 and SCGB 1A1A proteins form dimers that dissociate under reducing and denaturing conditions. (E) Identity of dimers and monomers was confirmed by Western blot analysis. (C, D, E) S = Precision plus protein standard (dual color).

    Article Snippet: Primers were designed with XmnI and SbfI restriction sites for cloning into the pMAL-c5X expression vector (New England BioLabs, Mississauga, ON).

    Techniques: Clone Assay, Expressing, Purification, Recombinant, Amplification, Plasmid Preparation, Transformation Assay, Sequencing, SDS Page, Affinity Chromatography, Incubation, Fast Protein Liquid Chromatography, Western Blot

    RFLP patterns of choE amplicons from three R. equi isolates (strains 15, 65, and 115, of equine, human, and environmental origin, respectively, in that order for the digestions with each of the enzymes) digested with Pvu I (lanes 1 to 3), Xho I (lanes 4 to 6), Xmn I (lanes 7 to 9), Ava I (lanes 10 to 12), Bam HI (lanes 13 to 15), Bgl I (lanes 16 to 18), and Hin PI (lanes 19 to 21). Lane M, DNA size marker.

    Journal: Journal of Clinical Microbiology

    Article Title: Rapid Identification of Rhodococcus equi by a PCR Assay Targeting the choE Gene

    doi: 10.1128/JCM.41.7.3241-3245.2003

    Figure Lengend Snippet: RFLP patterns of choE amplicons from three R. equi isolates (strains 15, 65, and 115, of equine, human, and environmental origin, respectively, in that order for the digestions with each of the enzymes) digested with Pvu I (lanes 1 to 3), Xho I (lanes 4 to 6), Xmn I (lanes 7 to 9), Ava I (lanes 10 to 12), Bam HI (lanes 13 to 15), Bgl I (lanes 16 to 18), and Hin PI (lanes 19 to 21). Lane M, DNA size marker.

    Article Snippet: For restriction fragment length polymorphism (RFLP) analysis, the choE amplicons were purified by use of the Gel Extraction Purification kit (Qiagen) and were subsequently digested with the following restriction enzymes: Ava I, Bam HI, Bgl I, Hin PI, Pvu I, Xho I, and Xmn I (New England Biolabs, Beverly, Mass.).

    Techniques: Antiviral Assay, Marker

    —Characterization of alpha satellite DNA diversity in the HindIII monomer data set. ( A ) PCA projection on principal components 1 and 2 of the normalized 5-mer frequency vectors for all sequences from the HindIII monomer data set. Each point represents a monomer sequence. ( B ) Prediction of the position of the XmnI monomer sequences on the graph shown in ( A ). Sequences are colored according to their assignment to the C1 (purple), C2 (pastel green), C5 (red) or C6 (orange) alpha satellite families. ( C ) PCA projection shown in ( A ) with sequences colored according to their assignment to the C1′ (purple), C2′ (pastel green), C5′ (red), or C6′ (blue) alpha satellite families, using a hierarchical classification method (see Materials and methods).

    Journal: Genome Biology and Evolution

    Article Title: The Targeted Sequencing of Alpha Satellite DNA in Cercopithecus pogonias Provides New Insight Into the Diversity and Dynamics of Centromeric Repeats in Old World Monkeys

    doi: 10.1093/gbe/evy109

    Figure Lengend Snippet: —Characterization of alpha satellite DNA diversity in the HindIII monomer data set. ( A ) PCA projection on principal components 1 and 2 of the normalized 5-mer frequency vectors for all sequences from the HindIII monomer data set. Each point represents a monomer sequence. ( B ) Prediction of the position of the XmnI monomer sequences on the graph shown in ( A ). Sequences are colored according to their assignment to the C1 (purple), C2 (pastel green), C5 (red) or C6 (orange) alpha satellite families. ( C ) PCA projection shown in ( A ) with sequences colored according to their assignment to the C1′ (purple), C2′ (pastel green), C5′ (red), or C6′ (blue) alpha satellite families, using a hierarchical classification method (see Materials and methods).

    Article Snippet: 10 µg of CPO genomic DNA were incubated for 6 h at 37 °C with 70 units of XmnI or HindIII (New England Biolabs) in a total volume of 35 µl.

    Techniques: Sequencing