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  • 99
    New England Biolabs endoh
    Asn86 is the N-glycosylation site of Panx2. ( A ) Based on sequence analysis, Panx2 (Uniprot ID: Q6IMP4-1) is predicted to contain four transmembrane domains, one intracellular (IL) and two extracellular loops (EL). The predicted N-glycosylation site is located at Asn86 in the first extracellular loop (EL1) (red residue). ( B ) Western blot (WB) comparing wildtype Panx2 and mutant N86Q, the latter shows a faster migrating band than the wildtype counterpart, indicative of decreased molecular weight. ( C ) Cell lysates of HEK293T transiently expressing Panx2 and N86Q mutant were subjected to enzymatic digestions with <t>PNGase</t> F and <t>EndoH</t> N-glycosidases. WB analysis confirmed that N86 is the only glycosylation site for Panx2 since only the wildtype protein exhibited a band shift after treatment with both glycosidases, and the de-glycosylated Panx2 band ran to the same position as the N86Q mutant. GAPDH was used as loading control. Molecular weights are noted in kDa.
    Endoh, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1173 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    New England Biolabs mspji
    Characterization of phage <t>T4</t> DNA modification. (A) Phage T4(glc-HMC), T4(HMC), and T4(C) DNA left untreated (−) or treated with (+) restriction enzymes AluI (top), which cleaves unmodified DNA; <t>MspJI</t> (middle), which cleaves HMC-containing DNA; or T4 glucosyltransferase (bottom), which increases the mobility of HMC-containing DNA by the addition of glucose groups. The arrows indicate the mobility shift due to glucose attachment. (B) Analysis of phage T4 DNA modification by single-molecule sequencing. Results are summarized for each genome by mapping IPD ratios at each base for each of the T4 strains studied. The coloration of each base is shown by the key at the bottom left. The T4 nucleotide sequence runs from top to bottom for each of the four genomes. The distance each colored point is displaced from the center indicates the IPD ratio (scale at bottom; leftward for the reverse strand, rightward for the forward strand). Examples of interpulse distances (indicative of modification) are shown to the right for a short segment of the T4 genome. Bars indicate the magnitude of the IPD ratio (upward for the forward strand and downward for the reverse strand). A 5′ GATC 3′ site of DAM methylation is highlighted in yellow. (C) Violin plot showing IPD ratios of A residues at 5′ GATC 3′ sequences.
    Mspji, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 107 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    New England Biolabs dsrna ladder
    Confocal imaging of <t>dsRNA</t> reporter N. benthamiana infected with TBSV, PVX, TCV, TRV, TuMV, and GFLV. Virus infection resulted in a variety of patterns of intracellular relocation of <t>B2:GFP.</t> Profound modifications of B2:GFP localization to large cytoplasmic aggregates were observed upon TBSV and PVX infections. Smaller cytoplasmic aggregates were observed upon TCV and TRV-infections and almost no modification in B2:GFP localization occurred upon TuMV and GFLV infection except for the near depletion of B2:GFP from the nucleoli (arrows, compare with Figure 4 ). Scale bars: 50 μm (upper panels) and 10 μm (lower panels).
    Dsrna Ladder, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 75 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs alui
    Characterization of phage <t>T4</t> DNA modification. (A) Phage T4(glc-HMC), T4(HMC), and T4(C) DNA left untreated (−) or treated with (+) restriction enzymes <t>AluI</t> (top), which cleaves unmodified DNA; MspJI (middle), which cleaves HMC-containing DNA; or T4 glucosyltransferase (bottom), which increases the mobility of HMC-containing DNA by the addition of glucose groups. The arrows indicate the mobility shift due to glucose attachment. (B) Analysis of phage T4 DNA modification by single-molecule sequencing. Results are summarized for each genome by mapping IPD ratios at each base for each of the T4 strains studied. The coloration of each base is shown by the key at the bottom left. The T4 nucleotide sequence runs from top to bottom for each of the four genomes. The distance each colored point is displaced from the center indicates the IPD ratio (scale at bottom; leftward for the reverse strand, rightward for the forward strand). Examples of interpulse distances (indicative of modification) are shown to the right for a short segment of the T4 genome. Bars indicate the magnitude of the IPD ratio (upward for the forward strand and downward for the reverse strand). A 5′ GATC 3′ site of DAM methylation is highlighted in yellow. (C) Violin plot showing IPD ratios of A residues at 5′ GATC 3′ sequences.
    Alui, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1225 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs smai
    Characterization of phage <t>T4</t> DNA modification. (A) Phage T4(glc-HMC), T4(HMC), and T4(C) DNA left untreated (−) or treated with (+) restriction enzymes <t>AluI</t> (top), which cleaves unmodified DNA; MspJI (middle), which cleaves HMC-containing DNA; or T4 glucosyltransferase (bottom), which increases the mobility of HMC-containing DNA by the addition of glucose groups. The arrows indicate the mobility shift due to glucose attachment. (B) Analysis of phage T4 DNA modification by single-molecule sequencing. Results are summarized for each genome by mapping IPD ratios at each base for each of the T4 strains studied. The coloration of each base is shown by the key at the bottom left. The T4 nucleotide sequence runs from top to bottom for each of the four genomes. The distance each colored point is displaced from the center indicates the IPD ratio (scale at bottom; leftward for the reverse strand, rightward for the forward strand). Examples of interpulse distances (indicative of modification) are shown to the right for a short segment of the T4 genome. Bars indicate the magnitude of the IPD ratio (upward for the forward strand and downward for the reverse strand). A 5′ GATC 3′ site of DAM methylation is highlighted in yellow. (C) Violin plot showing IPD ratios of A residues at 5′ GATC 3′ sequences.
    Smai, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1694 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs t4 pnk
    5′-adenylation of long RNA substrates. ( A ) Schematic diagram of the experimental strategy. The  > 100-mer RNA substrate is too long for 5′-AppRNA formation to induce a measurable gel shift relative to a 5′-monophosphate. Therefore, an appropriate 8–17 deoxyribozyme is used to cleave the 5′-portion of the RNA substrate, leaving a small fragment for which 5′-AppRNA formation does cause a gel shift. ( B ) The strategy in  A  applied to the 160-nt P4–P6 domain of the  Tetrahymena  group I intron RNA. Blocking oligos were uncapped. The three time points are at 0.5 min, 10 min, and 1 h (6% PAGE). The RNA substrate was internally radiolabeled by transcription incorporating α- 32 P-ATP; the 5′-monophosphate was provided by performing the transcription in the presence of excess GMP (see Materials and Methods). Although the side products have not been studied in great detail, the side product formed in the first experiment (P4–P6 with no DNA blocking oligo) is tentatively assigned as circularized P4–P6 on the basis of attempted 5′- 32 P-radiolabeling with T4 polynucleotide kinase and γ- 32 P-ATP; no reaction was observed alongside a positive control. Only the  lower  band (a mixture of 5′-monophosphate and 5′-AppRNA) was carried to the 8–17 deoxyribozyme cleavage experiment. std, P4–P6 standard RNA carried through all reactions with no blocking oligo, except that T4 RNA ligase was omitted. ( C ) The strategy in  A ).
    T4 Pnk, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 4190 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs ape1
    Assessment of the removal of rotationally and translationally positioned uracils by UDG and <t>APE1.</t> A, NCPs containing a single uracil at different sites were incubated with UDG and APE1. Open symbols represent in uracils as follows: red square , NCP-UI
    Ape1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 529 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs λ phosphatase
    Identification of a Hyperactive Phosphomutant ATOH1 (A) Diagram depicting the location of proline-directed kinase motifs (serine-proline [SP] or threonine-proline [TP]) in Atoh1 protein and mutations of these sites into alanine in ATOH1 phosphomutants. (B) In vitro -translated Atoh1 protein band-shift following incubation with different cyclin-dependent kinases (CDKs). Ngn3 was used as a positive control. (C) WT ATOH1 bands (arrows) collapse following <t>λ</t> phosphatase treatment, demonstrating phosphorylation. (D) DLD-1 cell proliferation following doxycycline (Dox)-induced expression of WT or phosphomutant Atoh1 (n = 3 biological replicates, 2 technical replicates, mean ± SEM). (E) Cell cycle profile of uninduced and Dox-treated cells showing increased G1 and decreased S/M populations upon induction of 9S/T-A Atoh1 . (F) Gene expression of Atoh1 and its target and secretory differentiation genes 72 hr after Dox induction of DLD-1 cells (n = 3 biological replicates, 2 technical replicates; Gapdh-normalized, mean ± SEM). Two-way ANOVA was used for statistical analysis; ∗ p
    λ Phosphatase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2639 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    New England Biolabs drai
    Extrusion of dsDNA produced by RT from ssDNA-NCp7 co-aggregates. Progression of DNA synthesis analyzed by TEM after 2 min. (A), 10 min. (B) and 40 min. (C) from reactions with ssDNA (5 nM), RT (50 nM) and NCp7 (3.4 µM). Disaggregation after 10 min. (B) appeared both at the periphery and within the aggregates. A few individual molecules were visible close to the aggregate after 40 min. (C). (D) A typical DNA product visualized by TEM after 40 min. of DNA synthesis with subsequent incubation for 15 min. at 70°C in the presence of 0.4 M NaCl. (E) Band shift analysis of DNA flap synthesis within the dsDNA produced by RT after 40 min. at 37°C with 50 nM RT, with or without 3.4 µM NCp7. An excess of <t>DraI</t> and <t>AlwnI</t> enzymes that digest this dsDNA into two fragments (1800 and 1500 bp) was added. The DraI-AlwnI digestion products of the plasmid DNA are shown as a control on the left. When the HIV-1 central DNA flap is fully synthesized (i. e. with NCp7), the 1800 bp fragment is shifted to a slower migrating band. Magnification is identical for panels A,B,C. The scale bars correspond to 250 nm.
    Drai, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 426 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    New England Biolabs alwni
    Extrusion of dsDNA produced by RT from ssDNA-NCp7 co-aggregates. Progression of DNA synthesis analyzed by TEM after 2 min. (A), 10 min. (B) and 40 min. (C) from reactions with ssDNA (5 nM), RT (50 nM) and NCp7 (3.4 µM). Disaggregation after 10 min. (B) appeared both at the periphery and within the aggregates. A few individual molecules were visible close to the aggregate after 40 min. (C). (D) A typical DNA product visualized by TEM after 40 min. of DNA synthesis with subsequent incubation for 15 min. at 70°C in the presence of 0.4 M NaCl. (E) Band shift analysis of DNA flap synthesis within the dsDNA produced by RT after 40 min. at 37°C with 50 nM RT, with or without 3.4 µM NCp7. An excess of <t>DraI</t> and <t>AlwnI</t> enzymes that digest this dsDNA into two fragments (1800 and 1500 bp) was added. The DraI-AlwnI digestion products of the plasmid DNA are shown as a control on the left. When the HIV-1 central DNA flap is fully synthesized (i. e. with NCp7), the 1800 bp fragment is shifted to a slower migrating band. Magnification is identical for panels A,B,C. The scale bars correspond to 250 nm.
    Alwni, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 170 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs t4 kinase
    Extrusion of dsDNA produced by RT from ssDNA-NCp7 co-aggregates. Progression of DNA synthesis analyzed by TEM after 2 min. (A), 10 min. (B) and 40 min. (C) from reactions with ssDNA (5 nM), RT (50 nM) and NCp7 (3.4 µM). Disaggregation after 10 min. (B) appeared both at the periphery and within the aggregates. A few individual molecules were visible close to the aggregate after 40 min. (C). (D) A typical DNA product visualized by TEM after 40 min. of DNA synthesis with subsequent incubation for 15 min. at 70°C in the presence of 0.4 M NaCl. (E) Band shift analysis of DNA flap synthesis within the dsDNA produced by RT after 40 min. at 37°C with 50 nM RT, with or without 3.4 µM NCp7. An excess of <t>DraI</t> and <t>AlwnI</t> enzymes that digest this dsDNA into two fragments (1800 and 1500 bp) was added. The DraI-AlwnI digestion products of the plasmid DNA are shown as a control on the left. When the HIV-1 central DNA flap is fully synthesized (i. e. with NCp7), the 1800 bp fragment is shifted to a slower migrating band. Magnification is identical for panels A,B,C. The scale bars correspond to 250 nm.
    T4 Kinase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 684 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs rnase inhibitor
    Gel shift assay of cRNA fragmented with <t>RNase</t> <t>III</t> and direct-labeled with pCpB. After RNase III fragmentation and labeling (RN), 93% of the cRNA fragments are shifted when incubated with streptavidin (+SA).
    Rnase Inhibitor, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2198 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs kpni
    In vitro selection process. ( A ) RNA <t>aptamer</t> library format, random region and tetraloop highlighted in black. ( B ) Fraction of RNA recovered from selections against BamHI (blue circles), <t>KpnI</t> (green triangles) and PacI (red squares), as a function of selection round.
    Kpni, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 3744 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs pmal c5x
    In vitro selection process. ( A ) RNA <t>aptamer</t> library format, random region and tetraloop highlighted in black. ( B ) Fraction of RNA recovered from selections against BamHI (blue circles), <t>KpnI</t> (green triangles) and PacI (red squares), as a function of selection round.
    Pmal C5x, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 569 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    New England Biolabs xmni
    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.
    Xmni, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 398 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs nt bspqi
    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 <t>XmnI,</t> which cuts p[ lacO x16] once, or <t>Nt.BspQI,</t> 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.
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    New England Biolabs pngasef kit
    Immunoblots showing that COX-1 and COX-2 are present in protein extractions from DRG (L4–L6) and dorsal and ventral spinal cord (segments L1–L6) of untreated rats. COX-1 and COX-2 isoforms ran to ∼72 kDa in 10% Bis-Tris gels. A second band was present at 74 kDa in spinal cord and DRG samples that were labeled with the COX-2 antibody. After deglycosidation by <t>PNGaseF</t> treatment, COX-2 displayed an electrophoretic mobility shift to 65 kDa, suggesting that N-linked carbohydrates had been removed.
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    New England Biolabs cutsmart buffer
    Immunoblots showing that COX-1 and COX-2 are present in protein extractions from DRG (L4–L6) and dorsal and ventral spinal cord (segments L1–L6) of untreated rats. COX-1 and COX-2 isoforms ran to ∼72 kDa in 10% Bis-Tris gels. A second band was present at 74 kDa in spinal cord and DRG samples that were labeled with the COX-2 antibody. After deglycosidation by <t>PNGaseF</t> treatment, COX-2 displayed an electrophoretic mobility shift to 65 kDa, suggesting that N-linked carbohydrates had been removed.
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    New England Biolabs bamhi
    Immunoblots showing that COX-1 and COX-2 are present in protein extractions from DRG (L4–L6) and dorsal and ventral spinal cord (segments L1–L6) of untreated rats. COX-1 and COX-2 isoforms ran to ∼72 kDa in 10% Bis-Tris gels. A second band was present at 74 kDa in spinal cord and DRG samples that were labeled with the COX-2 antibody. After deglycosidation by <t>PNGaseF</t> treatment, COX-2 displayed an electrophoretic mobility shift to 65 kDa, suggesting that N-linked carbohydrates had been removed.
    Bamhi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 11236 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs endonuclease n bst nbi
    Gel retardation assay with the nicking enzyme N. <t>Bst</t> <t>NBI</t> and its variants. Lane 1, three DNA fragments, 1067, 794 and 584 bp, generated by digestion of plasmid pNB1, a plasmid derived from pUC19 containing a single GAGTC site (16), with Bss SI and Bsr FI. Only the middle 794 bp fragment contains the GAGTC recognition sequence. The digested pNB1 substrate (0.25 pmol) was incubated with 0.75 pmol of purified N. Bst NBI (lane 2), and 1 µl of the crude cell extract containing ∼9.5 pmol of N. Bst NBI (lane 3), N. Bst NBI-D456A (lane 4), N. Bst NBI-E418A (lane 5), N. Bst NBI-E469A (lane 6), N. Bst NBI-E482A (lane 7). Lane 8 is the control plasmid only. Lane 9 is the size standard of λ DNA digested by Hin dIII and φ174 DNA digested by Hae III (New England Biolabs).
    Endonuclease N Bst Nbi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs recj fusion
    <t>RecJ</t> binds monomerically to DNA. Electrophoretic mobility shift assays with 5 nM oligonucleotide A treated with 100 nM RecJ alone, RecJf alone or with a mixture of the two ( A ). An identical experiment, except using the 10 nt 5′ tailed substrate ( B ). Two distinct shifts are noted, owing to the ∼40 <t>kDa</t> difference in molecular weight between RecJ and RecJf.
    Recj Fusion, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs calf intestinal phosphatase cip
    Activation of conventional PKCs causes a phosphorylation-dependent mobility shift of Rnd3. ( A ) NIH 3T3 cells expressing HA-Rnd3 were pretreated for 3 h with either DMSO vehicle, Y-27632 (10 μM) or Gö-6976 (2.5 μM). Cells were then treated with PMA (100 nM) for 10 min and cell lysates were resolved on SDS-PAGE. The slower migrating Rnd3 band (arrow) was seen in both the vehicle and the Y-27632 pretreated cells, but not in cells pretreated with the conventional PKC inhibitor Gö-6976. ( B ) Calf intestinal phosphatase <t>(CIP)</t> treatment causes disappearance of the slower migrating band of Rnd3 (arrow). NIH 3T3 cells transiently expressing HA-Rnd3 expression vector were treated with PMA (100 nM) + <t>ionomycin</t> (500 μg/mL). CIP was added to the cell lysate to reverse phosphorylation. Cell lysates were resolved on SDS-PAGE and blotted with anti-HA antibody. ( C ) A CAAX mutant of Rnd3 does not shift after activation of PKCs. NIH 3T3 cells expressing HA-tagged WT Rnd3 and a SAAX mutant were treated with PMA (100 nM) for 10 min and cell lysates were resolved on SDS-PAGE.
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    Image Search Results


    Asn86 is the N-glycosylation site of Panx2. ( A ) Based on sequence analysis, Panx2 (Uniprot ID: Q6IMP4-1) is predicted to contain four transmembrane domains, one intracellular (IL) and two extracellular loops (EL). The predicted N-glycosylation site is located at Asn86 in the first extracellular loop (EL1) (red residue). ( B ) Western blot (WB) comparing wildtype Panx2 and mutant N86Q, the latter shows a faster migrating band than the wildtype counterpart, indicative of decreased molecular weight. ( C ) Cell lysates of HEK293T transiently expressing Panx2 and N86Q mutant were subjected to enzymatic digestions with PNGase F and EndoH N-glycosidases. WB analysis confirmed that N86 is the only glycosylation site for Panx2 since only the wildtype protein exhibited a band shift after treatment with both glycosidases, and the de-glycosylated Panx2 band ran to the same position as the N86Q mutant. GAPDH was used as loading control. Molecular weights are noted in kDa.

    Journal: International Journal of Molecular Sciences

    Article Title: N-Glycosylation Regulates Pannexin 2 Localization but Is Not Required for Interacting with Pannexin 1

    doi: 10.3390/ijms19071837

    Figure Lengend Snippet: Asn86 is the N-glycosylation site of Panx2. ( A ) Based on sequence analysis, Panx2 (Uniprot ID: Q6IMP4-1) is predicted to contain four transmembrane domains, one intracellular (IL) and two extracellular loops (EL). The predicted N-glycosylation site is located at Asn86 in the first extracellular loop (EL1) (red residue). ( B ) Western blot (WB) comparing wildtype Panx2 and mutant N86Q, the latter shows a faster migrating band than the wildtype counterpart, indicative of decreased molecular weight. ( C ) Cell lysates of HEK293T transiently expressing Panx2 and N86Q mutant were subjected to enzymatic digestions with PNGase F and EndoH N-glycosidases. WB analysis confirmed that N86 is the only glycosylation site for Panx2 since only the wildtype protein exhibited a band shift after treatment with both glycosidases, and the de-glycosylated Panx2 band ran to the same position as the N86Q mutant. GAPDH was used as loading control. Molecular weights are noted in kDa.

    Article Snippet: PNGase F (Roche, Indianapolis, IN, USA) and EndoH (New England Biolabs Ipswich, MA, USA) digestions were performed according to their manufacturer’s instructions.

    Techniques: Sequencing, Western Blot, Mutagenesis, Molecular Weight, Expressing, Electrophoretic Mobility Shift Assay

    Y288C protein has altered glycosylation and localizes at endoplasmic reticulum (ER). a Protein lysate of MCF10A expressing PDGFRA WT or Y288C were digested with or without Endo H or PNGase F and the band shift of PDGFRA was analyzed by SDS-PAGE. b Cell surface expression of PDGFRA in MCF10A parental (gray), PDGFRA WT (blue) or Y288C (green) expressing cells was assessed by flow cytometry. c MCF10A cells expressing PDGFRA WT, Y288C, V561D, or D842V were fixed and stained with anti-PDGFRA (green), anti-GM130 (Golgi marker; red), or anti-calnexin (ER marker; red), and DAPI (nucleus; blue) to visualize the subcellular colocalization of PDGFRA with Golgi and ER. Bar, 25 μm. d An OptiPrep gradient ultracentrifugation was used to resolve the membrane fractions (100k) of MCF10A cells stably expressing PDGFRA WT (upper panel) or Y288C (lower panel). 11 fractions were collected. Equal volume of lysate of each fraction were separated by SDS-PAGE and immunoblotted with antibodies against PDGFRA, calnexin (ER marker), GM130 (cis-Golgi marker), and syntaxin 6 (trans-Golgi marker). Heat maps showing the distribution and the relative levels of indicated proteins across different factions were generated. The fraction with the highest abundance of indicated protein was defined as 1. CG, complex glycosylated; HM, high mannose. e MCF10A cells stably expressing PDGFRA WT or Y288C were treated with or without cycloheximide (CHX; 20 μg ml -1 ) for 2–6 h. The level of PDGFRA was analyzed by western blot and ERK2 was used as loading control. The band intensities of complex (solid line) or high mannose (dash line) glycosylated PDGFRA WT (blue) and Y288C (green) in three independent experiments were quantified with densitometry, normalized against ERK2, and presented as mean ± SD. Two-way ANOVA; ** p

    Journal: Nature Communications

    Article Title: Neomorphic PDGFRA extracellular domain driver mutations are resistant to PDGFRA targeted therapies

    doi: 10.1038/s41467-018-06949-w

    Figure Lengend Snippet: Y288C protein has altered glycosylation and localizes at endoplasmic reticulum (ER). a Protein lysate of MCF10A expressing PDGFRA WT or Y288C were digested with or without Endo H or PNGase F and the band shift of PDGFRA was analyzed by SDS-PAGE. b Cell surface expression of PDGFRA in MCF10A parental (gray), PDGFRA WT (blue) or Y288C (green) expressing cells was assessed by flow cytometry. c MCF10A cells expressing PDGFRA WT, Y288C, V561D, or D842V were fixed and stained with anti-PDGFRA (green), anti-GM130 (Golgi marker; red), or anti-calnexin (ER marker; red), and DAPI (nucleus; blue) to visualize the subcellular colocalization of PDGFRA with Golgi and ER. Bar, 25 μm. d An OptiPrep gradient ultracentrifugation was used to resolve the membrane fractions (100k) of MCF10A cells stably expressing PDGFRA WT (upper panel) or Y288C (lower panel). 11 fractions were collected. Equal volume of lysate of each fraction were separated by SDS-PAGE and immunoblotted with antibodies against PDGFRA, calnexin (ER marker), GM130 (cis-Golgi marker), and syntaxin 6 (trans-Golgi marker). Heat maps showing the distribution and the relative levels of indicated proteins across different factions were generated. The fraction with the highest abundance of indicated protein was defined as 1. CG, complex glycosylated; HM, high mannose. e MCF10A cells stably expressing PDGFRA WT or Y288C were treated with or without cycloheximide (CHX; 20 μg ml -1 ) for 2–6 h. The level of PDGFRA was analyzed by western blot and ERK2 was used as loading control. The band intensities of complex (solid line) or high mannose (dash line) glycosylated PDGFRA WT (blue) and Y288C (green) in three independent experiments were quantified with densitometry, normalized against ERK2, and presented as mean ± SD. Two-way ANOVA; ** p

    Article Snippet: Protein deglycosylation with PNGase F or Endo H (New England BioLabs) was carried out according to the manufacturer’s instructions.

    Techniques: Expressing, Electrophoretic Mobility Shift Assay, SDS Page, Flow Cytometry, Cytometry, Staining, Marker, Stable Transfection, Generated, Western Blot

    Characterization of phage T4 DNA modification. (A) Phage T4(glc-HMC), T4(HMC), and T4(C) DNA left untreated (−) or treated with (+) restriction enzymes AluI (top), which cleaves unmodified DNA; MspJI (middle), which cleaves HMC-containing DNA; or T4 glucosyltransferase (bottom), which increases the mobility of HMC-containing DNA by the addition of glucose groups. The arrows indicate the mobility shift due to glucose attachment. (B) Analysis of phage T4 DNA modification by single-molecule sequencing. Results are summarized for each genome by mapping IPD ratios at each base for each of the T4 strains studied. The coloration of each base is shown by the key at the bottom left. The T4 nucleotide sequence runs from top to bottom for each of the four genomes. The distance each colored point is displaced from the center indicates the IPD ratio (scale at bottom; leftward for the reverse strand, rightward for the forward strand). Examples of interpulse distances (indicative of modification) are shown to the right for a short segment of the T4 genome. Bars indicate the magnitude of the IPD ratio (upward for the forward strand and downward for the reverse strand). A 5′ GATC 3′ site of DAM methylation is highlighted in yellow. (C) Violin plot showing IPD ratios of A residues at 5′ GATC 3′ sequences.

    Journal: mBio

    Article Title: Covalent Modification of Bacteriophage T4 DNA Inhibits CRISPR-Cas9

    doi: 10.1128/mBio.00648-15

    Figure Lengend Snippet: Characterization of phage T4 DNA modification. (A) Phage T4(glc-HMC), T4(HMC), and T4(C) DNA left untreated (−) or treated with (+) restriction enzymes AluI (top), which cleaves unmodified DNA; MspJI (middle), which cleaves HMC-containing DNA; or T4 glucosyltransferase (bottom), which increases the mobility of HMC-containing DNA by the addition of glucose groups. The arrows indicate the mobility shift due to glucose attachment. (B) Analysis of phage T4 DNA modification by single-molecule sequencing. Results are summarized for each genome by mapping IPD ratios at each base for each of the T4 strains studied. The coloration of each base is shown by the key at the bottom left. The T4 nucleotide sequence runs from top to bottom for each of the four genomes. The distance each colored point is displaced from the center indicates the IPD ratio (scale at bottom; leftward for the reverse strand, rightward for the forward strand). Examples of interpulse distances (indicative of modification) are shown to the right for a short segment of the T4 genome. Bars indicate the magnitude of the IPD ratio (upward for the forward strand and downward for the reverse strand). A 5′ GATC 3′ site of DAM methylation is highlighted in yellow. (C) Violin plot showing IPD ratios of A residues at 5′ GATC 3′ sequences.

    Article Snippet: One microgram of T4(C), T4(HMC), or T4(glc-HMC) was digested with AluI (R0137s; NEB), MspJI (R0661S; NEB), or T4 phage β-glucosyltransferase (M0357S; NEB) in accordance with NEB-specified protocols.

    Techniques: Modification, Mobility Shift, Sequencing, Methylation

    Confocal imaging of dsRNA reporter N. benthamiana infected with TBSV, PVX, TCV, TRV, TuMV, and GFLV. Virus infection resulted in a variety of patterns of intracellular relocation of B2:GFP. Profound modifications of B2:GFP localization to large cytoplasmic aggregates were observed upon TBSV and PVX infections. Smaller cytoplasmic aggregates were observed upon TCV and TRV-infections and almost no modification in B2:GFP localization occurred upon TuMV and GFLV infection except for the near depletion of B2:GFP from the nucleoli (arrows, compare with Figure 4 ). Scale bars: 50 μm (upper panels) and 10 μm (lower panels).

    Journal: Frontiers in Plant Science

    Article Title: Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein

    doi: 10.3389/fpls.2018.00070

    Figure Lengend Snippet: Confocal imaging of dsRNA reporter N. benthamiana infected with TBSV, PVX, TCV, TRV, TuMV, and GFLV. Virus infection resulted in a variety of patterns of intracellular relocation of B2:GFP. Profound modifications of B2:GFP localization to large cytoplasmic aggregates were observed upon TBSV and PVX infections. Smaller cytoplasmic aggregates were observed upon TCV and TRV-infections and almost no modification in B2:GFP localization occurred upon TuMV and GFLV infection except for the near depletion of B2:GFP from the nucleoli (arrows, compare with Figure 4 ). Scale bars: 50 μm (upper panels) and 10 μm (lower panels).

    Article Snippet: Binding reactions were performed by incubating 300 ng of nucleic acids with 100 pmoles of purified B2 proteins in 0.5X TBE buffer containing 100 mM NaCl at room temperature for 15 min. After incubation, the products of the binding reaction were resolved by native 1% agarose gel electrophoresis at 10 V.cm−1 at 4°C, except for EMSA with dsRNA ladder (1.5 μg dsRNA + 300 pmoles of B2 purified protein per lane) resolved through a 12% polyacrylamide 19:1 gel in 1X TBE buffer containing 100 mM NaCl and 5% glycerol.

    Techniques: Imaging, Infection, Modification

    Co-localization of dsRNA with components of PVX infection. B2:RFP (A–C) or B2:GFP (D–K) dsRNA reporter in PVX-infected cells. (A) dsRNA granules within an X-body surrounded by GFP-CP-decorated virus particles. (B) The dsRNA reporter remains associated with virus particles in the absence of an X-body during infection with PVX.ΔTGB1.GFP-CP (Tilsner et al., 2012 ). (C) dsRNA-containing granules between “whorls” of single-stranded vRNA labeled by Pumilio-BiFC (Tilsner et al., 2009 ). (D–G) Within the X-body, none of the ectopically expressed viral proteins, truncated replicase (RdRP) or Triple Gene Block 1-3 (TGB1-3) co-localize with the dsRNA marker, although RdRP and TGB3 show granular locations within the X-body as well. Note that dsRNA is sometimes found in “whorls” similar to those observed for viral ssRNA (E–G,I) , which are surrounding aggregates of TGB1 protein (E) as described for ssRNA (Tilsner et al., 2009 ). (H) Association and partial co-localization of the dsRNA reporter with peripheral membrane structures labeled by TGB3, which have been shown to be associated with plasmodesmata (Tilsner et al., 2013 ). (I,J) Association of dsRNA marker with TGB3 and RdRP in peripheral replication sites. (K) Association of dsRNA marker with mCherry-CP labeled plasmodesmata. Where nuclei are visible in the images they are marked by an asterisk ( * ). Images (A,B,D,J,K) are maximum projections of entire confocal z-stacks whereas images (C,E–I) are individual confocal sections. Scale bars: 10 μm.

    Journal: Frontiers in Plant Science

    Article Title: Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein

    doi: 10.3389/fpls.2018.00070

    Figure Lengend Snippet: Co-localization of dsRNA with components of PVX infection. B2:RFP (A–C) or B2:GFP (D–K) dsRNA reporter in PVX-infected cells. (A) dsRNA granules within an X-body surrounded by GFP-CP-decorated virus particles. (B) The dsRNA reporter remains associated with virus particles in the absence of an X-body during infection with PVX.ΔTGB1.GFP-CP (Tilsner et al., 2012 ). (C) dsRNA-containing granules between “whorls” of single-stranded vRNA labeled by Pumilio-BiFC (Tilsner et al., 2009 ). (D–G) Within the X-body, none of the ectopically expressed viral proteins, truncated replicase (RdRP) or Triple Gene Block 1-3 (TGB1-3) co-localize with the dsRNA marker, although RdRP and TGB3 show granular locations within the X-body as well. Note that dsRNA is sometimes found in “whorls” similar to those observed for viral ssRNA (E–G,I) , which are surrounding aggregates of TGB1 protein (E) as described for ssRNA (Tilsner et al., 2009 ). (H) Association and partial co-localization of the dsRNA reporter with peripheral membrane structures labeled by TGB3, which have been shown to be associated with plasmodesmata (Tilsner et al., 2013 ). (I,J) Association of dsRNA marker with TGB3 and RdRP in peripheral replication sites. (K) Association of dsRNA marker with mCherry-CP labeled plasmodesmata. Where nuclei are visible in the images they are marked by an asterisk ( * ). Images (A,B,D,J,K) are maximum projections of entire confocal z-stacks whereas images (C,E–I) are individual confocal sections. Scale bars: 10 μm.

    Article Snippet: Binding reactions were performed by incubating 300 ng of nucleic acids with 100 pmoles of purified B2 proteins in 0.5X TBE buffer containing 100 mM NaCl at room temperature for 15 min. After incubation, the products of the binding reaction were resolved by native 1% agarose gel electrophoresis at 10 V.cm−1 at 4°C, except for EMSA with dsRNA ladder (1.5 μg dsRNA + 300 pmoles of B2 purified protein per lane) resolved through a 12% polyacrylamide 19:1 gel in 1X TBE buffer containing 100 mM NaCl and 5% glycerol.

    Techniques: Infection, Labeling, Bimolecular Fluorescence Complementation Assay, Blocking Assay, Marker

    Time course of PVX infection. Progress of a PVX.mCherry-CP infection on a dsRNA reporter N. benthamiana leaf over 17 h. Images (A,B) give an overview of the infection front at 0 and 17 h post-infection, respectively. The boxed area is enlarged in images (C–F) , which were taken at the indicated time points. As the cell marked with an asterisk ( * cell outline delineated in C–F ) becomes infected, red fluorescence of mCherry-CP becomes detectable (D) and B2:GFP labels small peripheral replication sites at the side of the cell adjacent to an already infected cell (arrow tips). As the infection progresses, replication sites increase in size with one becoming dominant ( E , arrow), and a neighboring cell shows the first faint signal from the mCherry-CP infection marker. About 10 h after the cell has become visibly infected (F) , the dominant replication site has developed into an X-body. Note that the nucleus has moved toward the X-body. All images are maximum projections of entire confocal z-stacks. Scale bars: 100 μm.

    Journal: Frontiers in Plant Science

    Article Title: Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein

    doi: 10.3389/fpls.2018.00070

    Figure Lengend Snippet: Time course of PVX infection. Progress of a PVX.mCherry-CP infection on a dsRNA reporter N. benthamiana leaf over 17 h. Images (A,B) give an overview of the infection front at 0 and 17 h post-infection, respectively. The boxed area is enlarged in images (C–F) , which were taken at the indicated time points. As the cell marked with an asterisk ( * cell outline delineated in C–F ) becomes infected, red fluorescence of mCherry-CP becomes detectable (D) and B2:GFP labels small peripheral replication sites at the side of the cell adjacent to an already infected cell (arrow tips). As the infection progresses, replication sites increase in size with one becoming dominant ( E , arrow), and a neighboring cell shows the first faint signal from the mCherry-CP infection marker. About 10 h after the cell has become visibly infected (F) , the dominant replication site has developed into an X-body. Note that the nucleus has moved toward the X-body. All images are maximum projections of entire confocal z-stacks. Scale bars: 100 μm.

    Article Snippet: Binding reactions were performed by incubating 300 ng of nucleic acids with 100 pmoles of purified B2 proteins in 0.5X TBE buffer containing 100 mM NaCl at room temperature for 15 min. After incubation, the products of the binding reaction were resolved by native 1% agarose gel electrophoresis at 10 V.cm−1 at 4°C, except for EMSA with dsRNA ladder (1.5 μg dsRNA + 300 pmoles of B2 purified protein per lane) resolved through a 12% polyacrylamide 19:1 gel in 1X TBE buffer containing 100 mM NaCl and 5% glycerol.

    Techniques: Infection, Fluorescence, Marker

    Colocalization of dsRNA with 2A protein from GFLV and 6K2 protein from TuMV. (A) 2A:TagRFP and (B) B2:GFP labeled replication complexes in the cytoplasm of GFLV-TagRFP-infected leaf epidermal cell of dsRNA reporter plant. (C) Merged ( A + B ) images. (D) 6K2:mCherry and (E) B2:GFP labeled replication complexes in the cytoplasm of TuMV.6K2:mCherry-infected leaf epidermal cell of dsRNA reporter plant. The nuclei (N) are delineated. (F) Merged ( D + E ) images. Scale bars: 5 μm.

    Journal: Frontiers in Plant Science

    Article Title: Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein

    doi: 10.3389/fpls.2018.00070

    Figure Lengend Snippet: Colocalization of dsRNA with 2A protein from GFLV and 6K2 protein from TuMV. (A) 2A:TagRFP and (B) B2:GFP labeled replication complexes in the cytoplasm of GFLV-TagRFP-infected leaf epidermal cell of dsRNA reporter plant. (C) Merged ( A + B ) images. (D) 6K2:mCherry and (E) B2:GFP labeled replication complexes in the cytoplasm of TuMV.6K2:mCherry-infected leaf epidermal cell of dsRNA reporter plant. The nuclei (N) are delineated. (F) Merged ( D + E ) images. Scale bars: 5 μm.

    Article Snippet: Binding reactions were performed by incubating 300 ng of nucleic acids with 100 pmoles of purified B2 proteins in 0.5X TBE buffer containing 100 mM NaCl at room temperature for 15 min. After incubation, the products of the binding reaction were resolved by native 1% agarose gel electrophoresis at 10 V.cm−1 at 4°C, except for EMSA with dsRNA ladder (1.5 μg dsRNA + 300 pmoles of B2 purified protein per lane) resolved through a 12% polyacrylamide 19:1 gel in 1X TBE buffer containing 100 mM NaCl and 5% glycerol.

    Techniques: Labeling, Infection

    Specific detection of dsRNA by northwestern blotting. (A) B2 was tested by northwestern blotting (upper panel) for its capacity to specifically recognize dsRNA. For this decreasing amounts of dsRNA Φ6 (from 100 to 0.4 ng) in the presence of a constant concentration of total RNA from healthy N. benthamiana (5 μg per lane) were probed with B2. Note that up to 0.4 ng of dsRNA can be detected. (B) B2 was tested by northwestern blotting (right panel) for its capacity to specifically recognize small dsRNA species. For this, synthetic 21 bp dsRNA duplexes, dsRNA ladder or dsDNA ladder were probed with B2. Note that only dsRNA species with size superior to 50 bp could be detected. (C) B2 was tested by northwestern blotting (upper panel) for its capacity to specifically recognize viral dsRNA species. For this, 10 μg of total RNA extracted from systemically-infected TBSV- PVX- TCV- TRV- TuMV and GFLV-infected N. benthamiana leaves collected at 11–14 dpi were probed with B2. A short (1–2 s) and a long exposure (10 min) of the same membrane are presented side by side. Note dsRNA species of various sizes could be detected in all samples except in healthy (NI) and GFLV-infected ones. (D) Northwestern detection of dsRNA present in total RNA extracts from healthy or TCV and TRV systemically-infected N. benthamiana (Nb) and A. thaliana (At) leaves collected at 11–14 dpi. (E) Northwestern detection of dsRNA present in total RNA from healthy or DCV-infected S2 insect cells. Ethidium bromide-stained total RNA was used as loading control (lower panels except in B , left panel).

    Journal: Frontiers in Plant Science

    Article Title: Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein

    doi: 10.3389/fpls.2018.00070

    Figure Lengend Snippet: Specific detection of dsRNA by northwestern blotting. (A) B2 was tested by northwestern blotting (upper panel) for its capacity to specifically recognize dsRNA. For this decreasing amounts of dsRNA Φ6 (from 100 to 0.4 ng) in the presence of a constant concentration of total RNA from healthy N. benthamiana (5 μg per lane) were probed with B2. Note that up to 0.4 ng of dsRNA can be detected. (B) B2 was tested by northwestern blotting (right panel) for its capacity to specifically recognize small dsRNA species. For this, synthetic 21 bp dsRNA duplexes, dsRNA ladder or dsDNA ladder were probed with B2. Note that only dsRNA species with size superior to 50 bp could be detected. (C) B2 was tested by northwestern blotting (upper panel) for its capacity to specifically recognize viral dsRNA species. For this, 10 μg of total RNA extracted from systemically-infected TBSV- PVX- TCV- TRV- TuMV and GFLV-infected N. benthamiana leaves collected at 11–14 dpi were probed with B2. A short (1–2 s) and a long exposure (10 min) of the same membrane are presented side by side. Note dsRNA species of various sizes could be detected in all samples except in healthy (NI) and GFLV-infected ones. (D) Northwestern detection of dsRNA present in total RNA extracts from healthy or TCV and TRV systemically-infected N. benthamiana (Nb) and A. thaliana (At) leaves collected at 11–14 dpi. (E) Northwestern detection of dsRNA present in total RNA from healthy or DCV-infected S2 insect cells. Ethidium bromide-stained total RNA was used as loading control (lower panels except in B , left panel).

    Article Snippet: Binding reactions were performed by incubating 300 ng of nucleic acids with 100 pmoles of purified B2 proteins in 0.5X TBE buffer containing 100 mM NaCl at room temperature for 15 min. After incubation, the products of the binding reaction were resolved by native 1% agarose gel electrophoresis at 10 V.cm−1 at 4°C, except for EMSA with dsRNA ladder (1.5 μg dsRNA + 300 pmoles of B2 purified protein per lane) resolved through a 12% polyacrylamide 19:1 gel in 1X TBE buffer containing 100 mM NaCl and 5% glycerol.

    Techniques: Concentration Assay, Infection, Staining

    Specific detection of viral dsRNA-species by fluorescence labeling in situ . B2:RFP purified from E. coli and J2 mAb were used as fluorescent probes to detected viral dsRNA species in plant protoplasts (A–L) or in insect cells (M–T) . To this extent, GFLV infected (A–H) or healthy Arabidopsis protoplasts (I–L) as well as DCV-infected (M–P) or healthy insect cells (Q–T) were fluorescently labeled. Note that fluorescent labeling was restricted to infected samples where B2:RFP-labeling colocalized with J2-labeling. Arrowhead point to an autofluorescent structure seen upon fixation of protoplasts with glutaraldehyde. Scale bars: 5 μm (E–H) , 10 μm ( A–D, I–L ) and 50 μm (M–T) .

    Journal: Frontiers in Plant Science

    Article Title: Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein

    doi: 10.3389/fpls.2018.00070

    Figure Lengend Snippet: Specific detection of viral dsRNA-species by fluorescence labeling in situ . B2:RFP purified from E. coli and J2 mAb were used as fluorescent probes to detected viral dsRNA species in plant protoplasts (A–L) or in insect cells (M–T) . To this extent, GFLV infected (A–H) or healthy Arabidopsis protoplasts (I–L) as well as DCV-infected (M–P) or healthy insect cells (Q–T) were fluorescently labeled. Note that fluorescent labeling was restricted to infected samples where B2:RFP-labeling colocalized with J2-labeling. Arrowhead point to an autofluorescent structure seen upon fixation of protoplasts with glutaraldehyde. Scale bars: 5 μm (E–H) , 10 μm ( A–D, I–L ) and 50 μm (M–T) .

    Article Snippet: Binding reactions were performed by incubating 300 ng of nucleic acids with 100 pmoles of purified B2 proteins in 0.5X TBE buffer containing 100 mM NaCl at room temperature for 15 min. After incubation, the products of the binding reaction were resolved by native 1% agarose gel electrophoresis at 10 V.cm−1 at 4°C, except for EMSA with dsRNA ladder (1.5 μg dsRNA + 300 pmoles of B2 purified protein per lane) resolved through a 12% polyacrylamide 19:1 gel in 1X TBE buffer containing 100 mM NaCl and 5% glycerol.

    Techniques: Fluorescence, Labeling, In Situ, Purification, Infection

    Confocal imaging of healthy dsRNA reporter N. benthamiana . Leaves from healthy dsRNA reporter N. benthamiana were observed at low (A) and high magnification (B,C) . Note the typical nucleo-cytoplasmic localization of B2:GFP in the leaf epidermal cells. At higher magnification, nuclear localization of B2:GFP appeared speckled and clearly enriched in the nucleoli (arrows in B,C ). Scale bars: 50 μm (A) and 10 μm (B,C) .

    Journal: Frontiers in Plant Science

    Article Title: Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein

    doi: 10.3389/fpls.2018.00070

    Figure Lengend Snippet: Confocal imaging of healthy dsRNA reporter N. benthamiana . Leaves from healthy dsRNA reporter N. benthamiana were observed at low (A) and high magnification (B,C) . Note the typical nucleo-cytoplasmic localization of B2:GFP in the leaf epidermal cells. At higher magnification, nuclear localization of B2:GFP appeared speckled and clearly enriched in the nucleoli (arrows in B,C ). Scale bars: 50 μm (A) and 10 μm (B,C) .

    Article Snippet: Binding reactions were performed by incubating 300 ng of nucleic acids with 100 pmoles of purified B2 proteins in 0.5X TBE buffer containing 100 mM NaCl at room temperature for 15 min. After incubation, the products of the binding reaction were resolved by native 1% agarose gel electrophoresis at 10 V.cm−1 at 4°C, except for EMSA with dsRNA ladder (1.5 μg dsRNA + 300 pmoles of B2 purified protein per lane) resolved through a 12% polyacrylamide 19:1 gel in 1X TBE buffer containing 100 mM NaCl and 5% glycerol.

    Techniques: Imaging

    Confocal imaging of B2:GFP in roots from TRV- and GFLV-infected dsRNA reporter N. benthamiana and in TRV-infected leaves coexpressing B2:GFP and the mitochondrial marker F 0 -ATPase:RFP. (A,D) TRV-infected, (B,E) GFLV-infected and (C,F) healthy root cells constitutively expressing B2:GFP. Note the intensely and moderately labeled punctate replication complexes found in the cytoplasm of TRV-and GFLV-infected root cells, respectively. In healthy root cells no cytoplasmic aggregates can be observed. Intracellular localization of the mitochondrial marker F 0 -ATPase:RFP (G,J) and B2:GFP (H,K) in leaf epidermal cell of dsRNA reporter plants. (I,L) Corresponding merged images. (G–I) correspond to TRV-infected and (J–L) to healthy reporter plants. Scale bars: 50 μm (A–C) and 10 μm (D–L) .

    Journal: Frontiers in Plant Science

    Article Title: Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein

    doi: 10.3389/fpls.2018.00070

    Figure Lengend Snippet: Confocal imaging of B2:GFP in roots from TRV- and GFLV-infected dsRNA reporter N. benthamiana and in TRV-infected leaves coexpressing B2:GFP and the mitochondrial marker F 0 -ATPase:RFP. (A,D) TRV-infected, (B,E) GFLV-infected and (C,F) healthy root cells constitutively expressing B2:GFP. Note the intensely and moderately labeled punctate replication complexes found in the cytoplasm of TRV-and GFLV-infected root cells, respectively. In healthy root cells no cytoplasmic aggregates can be observed. Intracellular localization of the mitochondrial marker F 0 -ATPase:RFP (G,J) and B2:GFP (H,K) in leaf epidermal cell of dsRNA reporter plants. (I,L) Corresponding merged images. (G–I) correspond to TRV-infected and (J–L) to healthy reporter plants. Scale bars: 50 μm (A–C) and 10 μm (D–L) .

    Article Snippet: Binding reactions were performed by incubating 300 ng of nucleic acids with 100 pmoles of purified B2 proteins in 0.5X TBE buffer containing 100 mM NaCl at room temperature for 15 min. After incubation, the products of the binding reaction were resolved by native 1% agarose gel electrophoresis at 10 V.cm−1 at 4°C, except for EMSA with dsRNA ladder (1.5 μg dsRNA + 300 pmoles of B2 purified protein per lane) resolved through a 12% polyacrylamide 19:1 gel in 1X TBE buffer containing 100 mM NaCl and 5% glycerol.

    Techniques: Imaging, Infection, Marker, Expressing, Labeling

    Binding specificity of B2 and B2:GFP in vitro . B2 (A,F) , B2m (B,F) , and B2:GFP (C–F) were tested by EMSA for their capacity to bind single-stranded (ss) and double-stranded (ds) DNA and RNA. Nucleic acid mobility shift occurred only with dsRNA in the presence of B2 and B2:GFP as indicated by white arrows (A,C,E) but not in the presence of B2m (B,F) . dsRNA used for EMSA was of bacteriophage Phi6 (Φ6) or synthetic origin except in (F) where low molecular weight dsRNA ladder was used for EMSA. In the latter case, clear mobility shift was restricted to dsRNA species longer than 30 bp (F) . Numbers below Φ6 lanes correspond to the amount (in ng) of B2, B2m and B2:GFP added in each sample. Acquisitions were performed under UV excitation for nucleic acid visualization (A–C) or at 488 nm for B2:GFP visualization (D) . (E,F) are composite images showing nucleic acids (white) and B2:GFP (Green). Ladders are indicated by L and corresponding sizes are given in kbp on the left sides except in (F) (bp).

    Journal: Frontiers in Plant Science

    Article Title: Efficient Detection of Long dsRNA in Vitro and in Vivo Using the dsRNA Binding Domain from FHV B2 Protein

    doi: 10.3389/fpls.2018.00070

    Figure Lengend Snippet: Binding specificity of B2 and B2:GFP in vitro . B2 (A,F) , B2m (B,F) , and B2:GFP (C–F) were tested by EMSA for their capacity to bind single-stranded (ss) and double-stranded (ds) DNA and RNA. Nucleic acid mobility shift occurred only with dsRNA in the presence of B2 and B2:GFP as indicated by white arrows (A,C,E) but not in the presence of B2m (B,F) . dsRNA used for EMSA was of bacteriophage Phi6 (Φ6) or synthetic origin except in (F) where low molecular weight dsRNA ladder was used for EMSA. In the latter case, clear mobility shift was restricted to dsRNA species longer than 30 bp (F) . Numbers below Φ6 lanes correspond to the amount (in ng) of B2, B2m and B2:GFP added in each sample. Acquisitions were performed under UV excitation for nucleic acid visualization (A–C) or at 488 nm for B2:GFP visualization (D) . (E,F) are composite images showing nucleic acids (white) and B2:GFP (Green). Ladders are indicated by L and corresponding sizes are given in kbp on the left sides except in (F) (bp).

    Article Snippet: Binding reactions were performed by incubating 300 ng of nucleic acids with 100 pmoles of purified B2 proteins in 0.5X TBE buffer containing 100 mM NaCl at room temperature for 15 min. After incubation, the products of the binding reaction were resolved by native 1% agarose gel electrophoresis at 10 V.cm−1 at 4°C, except for EMSA with dsRNA ladder (1.5 μg dsRNA + 300 pmoles of B2 purified protein per lane) resolved through a 12% polyacrylamide 19:1 gel in 1X TBE buffer containing 100 mM NaCl and 5% glycerol.

    Techniques: Binding Assay, In Vitro, Mobility Shift, Molecular Weight

    Characterization of phage T4 DNA modification. (A) Phage T4(glc-HMC), T4(HMC), and T4(C) DNA left untreated (−) or treated with (+) restriction enzymes AluI (top), which cleaves unmodified DNA; MspJI (middle), which cleaves HMC-containing DNA; or T4 glucosyltransferase (bottom), which increases the mobility of HMC-containing DNA by the addition of glucose groups. The arrows indicate the mobility shift due to glucose attachment. (B) Analysis of phage T4 DNA modification by single-molecule sequencing. Results are summarized for each genome by mapping IPD ratios at each base for each of the T4 strains studied. The coloration of each base is shown by the key at the bottom left. The T4 nucleotide sequence runs from top to bottom for each of the four genomes. The distance each colored point is displaced from the center indicates the IPD ratio (scale at bottom; leftward for the reverse strand, rightward for the forward strand). Examples of interpulse distances (indicative of modification) are shown to the right for a short segment of the T4 genome. Bars indicate the magnitude of the IPD ratio (upward for the forward strand and downward for the reverse strand). A 5′ GATC 3′ site of DAM methylation is highlighted in yellow. (C) Violin plot showing IPD ratios of A residues at 5′ GATC 3′ sequences.

    Journal: mBio

    Article Title: Covalent Modification of Bacteriophage T4 DNA Inhibits CRISPR-Cas9

    doi: 10.1128/mBio.00648-15

    Figure Lengend Snippet: Characterization of phage T4 DNA modification. (A) Phage T4(glc-HMC), T4(HMC), and T4(C) DNA left untreated (−) or treated with (+) restriction enzymes AluI (top), which cleaves unmodified DNA; MspJI (middle), which cleaves HMC-containing DNA; or T4 glucosyltransferase (bottom), which increases the mobility of HMC-containing DNA by the addition of glucose groups. The arrows indicate the mobility shift due to glucose attachment. (B) Analysis of phage T4 DNA modification by single-molecule sequencing. Results are summarized for each genome by mapping IPD ratios at each base for each of the T4 strains studied. The coloration of each base is shown by the key at the bottom left. The T4 nucleotide sequence runs from top to bottom for each of the four genomes. The distance each colored point is displaced from the center indicates the IPD ratio (scale at bottom; leftward for the reverse strand, rightward for the forward strand). Examples of interpulse distances (indicative of modification) are shown to the right for a short segment of the T4 genome. Bars indicate the magnitude of the IPD ratio (upward for the forward strand and downward for the reverse strand). A 5′ GATC 3′ site of DAM methylation is highlighted in yellow. (C) Violin plot showing IPD ratios of A residues at 5′ GATC 3′ sequences.

    Article Snippet: One microgram of T4(C), T4(HMC), or T4(glc-HMC) was digested with AluI (R0137s; NEB), MspJI (R0661S; NEB), or T4 phage β-glucosyltransferase (M0357S; NEB) in accordance with NEB-specified protocols.

    Techniques: Modification, Mobility Shift, Sequencing, Methylation

    5′-adenylation of long RNA substrates. ( A ) Schematic diagram of the experimental strategy. The  > 100-mer RNA substrate is too long for 5′-AppRNA formation to induce a measurable gel shift relative to a 5′-monophosphate. Therefore, an appropriate 8–17 deoxyribozyme is used to cleave the 5′-portion of the RNA substrate, leaving a small fragment for which 5′-AppRNA formation does cause a gel shift. ( B ) The strategy in  A  applied to the 160-nt P4–P6 domain of the  Tetrahymena  group I intron RNA. Blocking oligos were uncapped. The three time points are at 0.5 min, 10 min, and 1 h (6% PAGE). The RNA substrate was internally radiolabeled by transcription incorporating α- 32 P-ATP; the 5′-monophosphate was provided by performing the transcription in the presence of excess GMP (see Materials and Methods). Although the side products have not been studied in great detail, the side product formed in the first experiment (P4–P6 with no DNA blocking oligo) is tentatively assigned as circularized P4–P6 on the basis of attempted 5′- 32 P-radiolabeling with T4 polynucleotide kinase and γ- 32 P-ATP; no reaction was observed alongside a positive control. Only the  lower  band (a mixture of 5′-monophosphate and 5′-AppRNA) was carried to the 8–17 deoxyribozyme cleavage experiment. std, P4–P6 standard RNA carried through all reactions with no blocking oligo, except that T4 RNA ligase was omitted. ( C ) The strategy in  A ).

    Journal: RNA

    Article Title: Practical and general synthesis of 5?-adenylated RNA (5?-AppRNA)

    doi: 10.1261/rna.5247704

    Figure Lengend Snippet: 5′-adenylation of long RNA substrates. ( A ) Schematic diagram of the experimental strategy. The > 100-mer RNA substrate is too long for 5′-AppRNA formation to induce a measurable gel shift relative to a 5′-monophosphate. Therefore, an appropriate 8–17 deoxyribozyme is used to cleave the 5′-portion of the RNA substrate, leaving a small fragment for which 5′-AppRNA formation does cause a gel shift. ( B ) The strategy in A applied to the 160-nt P4–P6 domain of the Tetrahymena group I intron RNA. Blocking oligos were uncapped. The three time points are at 0.5 min, 10 min, and 1 h (6% PAGE). The RNA substrate was internally radiolabeled by transcription incorporating α- 32 P-ATP; the 5′-monophosphate was provided by performing the transcription in the presence of excess GMP (see Materials and Methods). Although the side products have not been studied in great detail, the side product formed in the first experiment (P4–P6 with no DNA blocking oligo) is tentatively assigned as circularized P4–P6 on the basis of attempted 5′- 32 P-radiolabeling with T4 polynucleotide kinase and γ- 32 P-ATP; no reaction was observed alongside a positive control. Only the lower band (a mixture of 5′-monophosphate and 5′-AppRNA) was carried to the 8–17 deoxyribozyme cleavage experiment. std, P4–P6 standard RNA carried through all reactions with no blocking oligo, except that T4 RNA ligase was omitted. ( C ) The strategy in A ).

    Article Snippet: Radiolabeled RNAs were prepared with γ-32 P-ATP (PerkinElmer) and T4 PNK (New England Biolabs) and purified by 20% denaturing PAGE followed by ethanol precipitation.

    Techniques: Electrophoretic Mobility Shift Assay, Blocking Assay, Polyacrylamide Gel Electrophoresis, Radioactivity, Positive Control

    Assessment of the removal of rotationally and translationally positioned uracils by UDG and APE1. A, NCPs containing a single uracil at different sites were incubated with UDG and APE1. Open symbols represent in uracils as follows: red square , NCP-UI

    Journal: The Journal of Biological Chemistry

    Article Title: The Structural Location of DNA Lesions in Nucleosome Core Particles Determines Accessibility by Base Excision Repair Enzymes *

    doi: 10.1074/jbc.M112.441444

    Figure Lengend Snippet: Assessment of the removal of rotationally and translationally positioned uracils by UDG and APE1. A, NCPs containing a single uracil at different sites were incubated with UDG and APE1. Open symbols represent in uracils as follows: red square , NCP-UI

    Article Snippet: After confirmation of formaldehyde cross-linking efficiency by electrophoretic mobility shift assays (EMSA) (data not shown), cross-linked and noncross-linked NCPs were incubated with UDG and APE1 for different times (0–40 min).

    Techniques: Incubation

    UDG and APE1 Digestion

    Journal: The Journal of Biological Chemistry

    Article Title: The Structural Location of DNA Lesions in Nucleosome Core Particles Determines Accessibility by Base Excision Repair Enzymes *

    doi: 10.1074/jbc.M112.441444

    Figure Lengend Snippet: UDG and APE1 Digestion

    Article Snippet: After confirmation of formaldehyde cross-linking efficiency by electrophoretic mobility shift assays (EMSA) (data not shown), cross-linked and noncross-linked NCPs were incubated with UDG and APE1 for different times (0–40 min).

    Techniques:

    Polymerase β extension activity in NCPs near the dyad. A, representative gels for NCP-gO (+10) and NCP-gI (+4) pol β (100 n m ) extension in the absence of APE1. B, NCP-gO (+10) and NCP-gI (+4) were incubated with pol β and APE1

    Journal: The Journal of Biological Chemistry

    Article Title: The Structural Location of DNA Lesions in Nucleosome Core Particles Determines Accessibility by Base Excision Repair Enzymes *

    doi: 10.1074/jbc.M112.441444

    Figure Lengend Snippet: Polymerase β extension activity in NCPs near the dyad. A, representative gels for NCP-gO (+10) and NCP-gI (+4) pol β (100 n m ) extension in the absence of APE1. B, NCP-gO (+10) and NCP-gI (+4) were incubated with pol β and APE1

    Article Snippet: After confirmation of formaldehyde cross-linking efficiency by electrophoretic mobility shift assays (EMSA) (data not shown), cross-linked and noncross-linked NCPs were incubated with UDG and APE1 for different times (0–40 min).

    Techniques: Activity Assay, Incubation

    Polymerase β extension activity in NCPs near DNA ends. A, representative gels for NCP-gO (−35) and NCP-gI (−49) pol β (100 n m ) extension in the absence of APE1. B, NCP-gO (−35) and NCP-gI (−49) were incubated

    Journal: The Journal of Biological Chemistry

    Article Title: The Structural Location of DNA Lesions in Nucleosome Core Particles Determines Accessibility by Base Excision Repair Enzymes *

    doi: 10.1074/jbc.M112.441444

    Figure Lengend Snippet: Polymerase β extension activity in NCPs near DNA ends. A, representative gels for NCP-gO (−35) and NCP-gI (−49) pol β (100 n m ) extension in the absence of APE1. B, NCP-gO (−35) and NCP-gI (−49) were incubated

    Article Snippet: After confirmation of formaldehyde cross-linking efficiency by electrophoretic mobility shift assays (EMSA) (data not shown), cross-linked and noncross-linked NCPs were incubated with UDG and APE1 for different times (0–40 min).

    Techniques: Activity Assay, Incubation

    UDG and APE1 Digestion

    Journal: The Journal of Biological Chemistry

    Article Title: The Structural Location of DNA Lesions in Nucleosome Core Particles Determines Accessibility by Base Excision Repair Enzymes *

    doi: 10.1074/jbc.M112.441444

    Figure Lengend Snippet: UDG and APE1 Digestion

    Article Snippet: After confirmation of formaldehyde cross-linking efficiency by electrophoretic mobility shift assays (EMSA) (data not shown), cross-linked and noncross-linked NCPs were incubated with UDG and APE1 for different times (0–40 min).

    Techniques:

    Identification of a Hyperactive Phosphomutant ATOH1 (A) Diagram depicting the location of proline-directed kinase motifs (serine-proline [SP] or threonine-proline [TP]) in Atoh1 protein and mutations of these sites into alanine in ATOH1 phosphomutants. (B) In vitro -translated Atoh1 protein band-shift following incubation with different cyclin-dependent kinases (CDKs). Ngn3 was used as a positive control. (C) WT ATOH1 bands (arrows) collapse following λ phosphatase treatment, demonstrating phosphorylation. (D) DLD-1 cell proliferation following doxycycline (Dox)-induced expression of WT or phosphomutant Atoh1 (n = 3 biological replicates, 2 technical replicates, mean ± SEM). (E) Cell cycle profile of uninduced and Dox-treated cells showing increased G1 and decreased S/M populations upon induction of 9S/T-A Atoh1 . (F) Gene expression of Atoh1 and its target and secretory differentiation genes 72 hr after Dox induction of DLD-1 cells (n = 3 biological replicates, 2 technical replicates; Gapdh-normalized, mean ± SEM). Two-way ANOVA was used for statistical analysis; ∗ p

    Journal: Cell Stem Cell

    Article Title: Phospho-regulation of ATOH1 Is Required for Plasticity of Secretory Progenitors and Tissue Regeneration

    doi: 10.1016/j.stem.2018.07.002

    Figure Lengend Snippet: Identification of a Hyperactive Phosphomutant ATOH1 (A) Diagram depicting the location of proline-directed kinase motifs (serine-proline [SP] or threonine-proline [TP]) in Atoh1 protein and mutations of these sites into alanine in ATOH1 phosphomutants. (B) In vitro -translated Atoh1 protein band-shift following incubation with different cyclin-dependent kinases (CDKs). Ngn3 was used as a positive control. (C) WT ATOH1 bands (arrows) collapse following λ phosphatase treatment, demonstrating phosphorylation. (D) DLD-1 cell proliferation following doxycycline (Dox)-induced expression of WT or phosphomutant Atoh1 (n = 3 biological replicates, 2 technical replicates, mean ± SEM). (E) Cell cycle profile of uninduced and Dox-treated cells showing increased G1 and decreased S/M populations upon induction of 9S/T-A Atoh1 . (F) Gene expression of Atoh1 and its target and secretory differentiation genes 72 hr after Dox induction of DLD-1 cells (n = 3 biological replicates, 2 technical replicates; Gapdh-normalized, mean ± SEM). Two-way ANOVA was used for statistical analysis; ∗ p

    Article Snippet: For some experiments, protein extracts were incubated with λ phosphatase (New England Biolabs) prior to western blotting, according to the manufacturer’s instructions.

    Techniques: In Vitro, Electrophoretic Mobility Shift Assay, Incubation, Positive Control, Expressing

    Extrusion of dsDNA produced by RT from ssDNA-NCp7 co-aggregates. Progression of DNA synthesis analyzed by TEM after 2 min. (A), 10 min. (B) and 40 min. (C) from reactions with ssDNA (5 nM), RT (50 nM) and NCp7 (3.4 µM). Disaggregation after 10 min. (B) appeared both at the periphery and within the aggregates. A few individual molecules were visible close to the aggregate after 40 min. (C). (D) A typical DNA product visualized by TEM after 40 min. of DNA synthesis with subsequent incubation for 15 min. at 70°C in the presence of 0.4 M NaCl. (E) Band shift analysis of DNA flap synthesis within the dsDNA produced by RT after 40 min. at 37°C with 50 nM RT, with or without 3.4 µM NCp7. An excess of DraI and AlwnI enzymes that digest this dsDNA into two fragments (1800 and 1500 bp) was added. The DraI-AlwnI digestion products of the plasmid DNA are shown as a control on the left. When the HIV-1 central DNA flap is fully synthesized (i. e. with NCp7), the 1800 bp fragment is shifted to a slower migrating band. Magnification is identical for panels A,B,C. The scale bars correspond to 250 nm.

    Journal: PLoS ONE

    Article Title: HIV-1 Protease and Reverse Transcriptase Control the Architecture of Their Nucleocapsid Partner

    doi: 10.1371/journal.pone.0000669

    Figure Lengend Snippet: Extrusion of dsDNA produced by RT from ssDNA-NCp7 co-aggregates. Progression of DNA synthesis analyzed by TEM after 2 min. (A), 10 min. (B) and 40 min. (C) from reactions with ssDNA (5 nM), RT (50 nM) and NCp7 (3.4 µM). Disaggregation after 10 min. (B) appeared both at the periphery and within the aggregates. A few individual molecules were visible close to the aggregate after 40 min. (C). (D) A typical DNA product visualized by TEM after 40 min. of DNA synthesis with subsequent incubation for 15 min. at 70°C in the presence of 0.4 M NaCl. (E) Band shift analysis of DNA flap synthesis within the dsDNA produced by RT after 40 min. at 37°C with 50 nM RT, with or without 3.4 µM NCp7. An excess of DraI and AlwnI enzymes that digest this dsDNA into two fragments (1800 and 1500 bp) was added. The DraI-AlwnI digestion products of the plasmid DNA are shown as a control on the left. When the HIV-1 central DNA flap is fully synthesized (i. e. with NCp7), the 1800 bp fragment is shifted to a slower migrating band. Magnification is identical for panels A,B,C. The scale bars correspond to 250 nm.

    Article Snippet: AlwNI , DraI and Mo-MuLV RT enzymes were purchased from New England Biolabs (Ipswich, MA).

    Techniques: Produced, DNA Synthesis, Transmission Electron Microscopy, Incubation, Electrophoretic Mobility Shift Assay, Plasmid Preparation, Synthesized

    Extrusion of dsDNA produced by RT from ssDNA-NCp7 co-aggregates. Progression of DNA synthesis analyzed by TEM after 2 min. (A), 10 min. (B) and 40 min. (C) from reactions with ssDNA (5 nM), RT (50 nM) and NCp7 (3.4 µM). Disaggregation after 10 min. (B) appeared both at the periphery and within the aggregates. A few individual molecules were visible close to the aggregate after 40 min. (C). (D) A typical DNA product visualized by TEM after 40 min. of DNA synthesis with subsequent incubation for 15 min. at 70°C in the presence of 0.4 M NaCl. (E) Band shift analysis of DNA flap synthesis within the dsDNA produced by RT after 40 min. at 37°C with 50 nM RT, with or without 3.4 µM NCp7. An excess of DraI and AlwnI enzymes that digest this dsDNA into two fragments (1800 and 1500 bp) was added. The DraI-AlwnI digestion products of the plasmid DNA are shown as a control on the left. When the HIV-1 central DNA flap is fully synthesized (i. e. with NCp7), the 1800 bp fragment is shifted to a slower migrating band. Magnification is identical for panels A,B,C. The scale bars correspond to 250 nm.

    Journal: PLoS ONE

    Article Title: HIV-1 Protease and Reverse Transcriptase Control the Architecture of Their Nucleocapsid Partner

    doi: 10.1371/journal.pone.0000669

    Figure Lengend Snippet: Extrusion of dsDNA produced by RT from ssDNA-NCp7 co-aggregates. Progression of DNA synthesis analyzed by TEM after 2 min. (A), 10 min. (B) and 40 min. (C) from reactions with ssDNA (5 nM), RT (50 nM) and NCp7 (3.4 µM). Disaggregation after 10 min. (B) appeared both at the periphery and within the aggregates. A few individual molecules were visible close to the aggregate after 40 min. (C). (D) A typical DNA product visualized by TEM after 40 min. of DNA synthesis with subsequent incubation for 15 min. at 70°C in the presence of 0.4 M NaCl. (E) Band shift analysis of DNA flap synthesis within the dsDNA produced by RT after 40 min. at 37°C with 50 nM RT, with or without 3.4 µM NCp7. An excess of DraI and AlwnI enzymes that digest this dsDNA into two fragments (1800 and 1500 bp) was added. The DraI-AlwnI digestion products of the plasmid DNA are shown as a control on the left. When the HIV-1 central DNA flap is fully synthesized (i. e. with NCp7), the 1800 bp fragment is shifted to a slower migrating band. Magnification is identical for panels A,B,C. The scale bars correspond to 250 nm.

    Article Snippet: AlwNI , DraI and Mo-MuLV RT enzymes were purchased from New England Biolabs (Ipswich, MA).

    Techniques: Produced, DNA Synthesis, Transmission Electron Microscopy, Incubation, Electrophoretic Mobility Shift Assay, Plasmid Preparation, Synthesized

    Gel shift assay of cRNA fragmented with RNase III and direct-labeled with pCpB. After RNase III fragmentation and labeling (RN), 93% of the cRNA fragments are shifted when incubated with streptavidin (+SA).

    Journal: Nucleic Acids Research

    Article Title: Direct labeling of RNA with multiple biotins allows sensitive expression profiling of acute leukemia class predictor genes

    doi: 10.1093/nar/gnh085

    Figure Lengend Snippet: Gel shift assay of cRNA fragmented with RNase III and direct-labeled with pCpB. After RNase III fragmentation and labeling (RN), 93% of the cRNA fragments are shifted when incubated with streptavidin (+SA).

    Article Snippet: Both enzymes fragmented cRNA, however RNase III produced a fragment size range more similar to the standard method of magnesium hydrolysis (Figure ). cRNA, total RNA and poly(A) RNA were all fragmented by RNase III and the average fragment size ranged from ∼20 to 200 nt.

    Techniques: Electrophoretic Mobility Shift Assay, Labeling, Incubation

    Comparison of cRNA fragmented by RNase III and magnesium hydrolysis. Unlabeled cRNA fragmented with RNase III (RN) has a similar size distribution to cRNA fragmented by magnesium hydrolysis (Mg). Fragments 20–100 nt are ideal for array hybridization.

    Journal: Nucleic Acids Research

    Article Title: Direct labeling of RNA with multiple biotins allows sensitive expression profiling of acute leukemia class predictor genes

    doi: 10.1093/nar/gnh085

    Figure Lengend Snippet: Comparison of cRNA fragmented by RNase III and magnesium hydrolysis. Unlabeled cRNA fragmented with RNase III (RN) has a similar size distribution to cRNA fragmented by magnesium hydrolysis (Mg). Fragments 20–100 nt are ideal for array hybridization.

    Article Snippet: Both enzymes fragmented cRNA, however RNase III produced a fragment size range more similar to the standard method of magnesium hydrolysis (Figure ). cRNA, total RNA and poly(A) RNA were all fragmented by RNase III and the average fragment size ranged from ∼20 to 200 nt.

    Techniques: Hybridization

    In vitro selection process. ( A ) RNA aptamer library format, random region and tetraloop highlighted in black. ( B ) Fraction of RNA recovered from selections against BamHI (blue circles), KpnI (green triangles) and PacI (red squares), as a function of selection round.

    Journal: Nucleic Acids Research

    Article Title: RNA aptamer inhibitors of a restriction endonuclease

    doi: 10.1093/nar/gkv702

    Figure Lengend Snippet: In vitro selection process. ( A ) RNA aptamer library format, random region and tetraloop highlighted in black. ( B ) Fraction of RNA recovered from selections against BamHI (blue circles), KpnI (green triangles) and PacI (red squares), as a function of selection round.

    Article Snippet: Binding reactions contain radiolabeled aptamer (13 nM) and the indicated concentration of KpnI in New England Biolabs 1.1 buffer (10 mM Bis-Tris-Propane-HCl, pH 7, 10 mM MgCl2 , 100 μg/ml bovine serum albumin (BSA)) for 30 min at 37°C.

    Techniques: In Vitro, Selection

    Quantitation of KpnI binding affinity by electrophoretic gel mobility shift assay for ( A ) aptamer 20; ( B ) aptamer 24; ( C ) aptamer 29; and ( D ) aptamer 30. Replicate data are shown as black and gray points.

    Journal: Nucleic Acids Research

    Article Title: RNA aptamer inhibitors of a restriction endonuclease

    doi: 10.1093/nar/gkv702

    Figure Lengend Snippet: Quantitation of KpnI binding affinity by electrophoretic gel mobility shift assay for ( A ) aptamer 20; ( B ) aptamer 24; ( C ) aptamer 29; and ( D ) aptamer 30. Replicate data are shown as black and gray points.

    Article Snippet: Binding reactions contain radiolabeled aptamer (13 nM) and the indicated concentration of KpnI in New England Biolabs 1.1 buffer (10 mM Bis-Tris-Propane-HCl, pH 7, 10 mM MgCl2 , 100 μg/ml bovine serum albumin (BSA)) for 30 min at 37°C.

    Techniques: Quantitation Assay, Binding Assay, Mobility Shift

    Titration of 50 nM KpnI with high affinity radiolabeled aptamer 20 in the concentration range 100 pM–500 nM.

    Journal: Nucleic Acids Research

    Article Title: RNA aptamer inhibitors of a restriction endonuclease

    doi: 10.1093/nar/gkv702

    Figure Lengend Snippet: Titration of 50 nM KpnI with high affinity radiolabeled aptamer 20 in the concentration range 100 pM–500 nM.

    Article Snippet: Binding reactions contain radiolabeled aptamer (13 nM) and the indicated concentration of KpnI in New England Biolabs 1.1 buffer (10 mM Bis-Tris-Propane-HCl, pH 7, 10 mM MgCl2 , 100 μg/ml bovine serum albumin (BSA)) for 30 min at 37°C.

    Techniques: Titration, Concentration Assay

    Example of anti-KpnI aptamer 20 binding to KpnI as measured by quantitative electrophoretic gel mobility shift assay. A total of 13 nM radiolabeled RNA aptamer titrated with increasing concentrations of KpnI as indicated.

    Journal: Nucleic Acids Research

    Article Title: RNA aptamer inhibitors of a restriction endonuclease

    doi: 10.1093/nar/gkv702

    Figure Lengend Snippet: Example of anti-KpnI aptamer 20 binding to KpnI as measured by quantitative electrophoretic gel mobility shift assay. A total of 13 nM radiolabeled RNA aptamer titrated with increasing concentrations of KpnI as indicated.

    Article Snippet: Binding reactions contain radiolabeled aptamer (13 nM) and the indicated concentration of KpnI in New England Biolabs 1.1 buffer (10 mM Bis-Tris-Propane-HCl, pH 7, 10 mM MgCl2 , 100 μg/ml bovine serum albumin (BSA)) for 30 min at 37°C.

    Techniques: Binding Assay, Mobility Shift

    Example gels showing results of qualitative screens for aptamer activity. ( A ) Electrophoretic gel mobility shift binding screen. Radiolabeled aptamer candidates (4 nM) were exposed to their corresponding protein targets, in this case KpnI (20 nM; even lanes). ( B ) Restriction inhibition screen. Fluorescent DNA probe (20 nM, see panel C ) was exposed to REase that had been incubated with a high concentration of the indicated RNA aptamers (40 μM). The examples shown are RNA aptamers against KpnI.

    Journal: Nucleic Acids Research

    Article Title: RNA aptamer inhibitors of a restriction endonuclease

    doi: 10.1093/nar/gkv702

    Figure Lengend Snippet: Example gels showing results of qualitative screens for aptamer activity. ( A ) Electrophoretic gel mobility shift binding screen. Radiolabeled aptamer candidates (4 nM) were exposed to their corresponding protein targets, in this case KpnI (20 nM; even lanes). ( B ) Restriction inhibition screen. Fluorescent DNA probe (20 nM, see panel C ) was exposed to REase that had been incubated with a high concentration of the indicated RNA aptamers (40 μM). The examples shown are RNA aptamers against KpnI.

    Article Snippet: Binding reactions contain radiolabeled aptamer (13 nM) and the indicated concentration of KpnI in New England Biolabs 1.1 buffer (10 mM Bis-Tris-Propane-HCl, pH 7, 10 mM MgCl2 , 100 μg/ml bovine serum albumin (BSA)) for 30 min at 37°C.

    Techniques: Activity Assay, Mobility Shift, Binding Assay, Inhibition, Incubation, Concentration Assay

    Competition gel shi ft assay showing inhibition of KpnI binding to fluorescent DNA probe in the presence of 100 pM–10 μM anti-KpnI RNA aptamers. ( A ) Aptamer 20, ( B ) aptamer 24.

    Journal: Nucleic Acids Research

    Article Title: RNA aptamer inhibitors of a restriction endonuclease

    doi: 10.1093/nar/gkv702

    Figure Lengend Snippet: Competition gel shi ft assay showing inhibition of KpnI binding to fluorescent DNA probe in the presence of 100 pM–10 μM anti-KpnI RNA aptamers. ( A ) Aptamer 20, ( B ) aptamer 24.

    Article Snippet: Binding reactions contain radiolabeled aptamer (13 nM) and the indicated concentration of KpnI in New England Biolabs 1.1 buffer (10 mM Bis-Tris-Propane-HCl, pH 7, 10 mM MgCl2 , 100 μg/ml bovine serum albumin (BSA)) for 30 min at 37°C.

    Techniques: Inhibition, Binding Assay

    ( A ) In-line probing ( 36 ) of high-affinity anti-KpnI aptamers 20, 24, 29, 30. (UN: unmodified samples (lane 1, 6, 11 and 16); T1: RNAse T1 digestions (lane 2, 7, 12 and 17); OH: alkaline hydrolysis reactions (lane 3, 8, 13 and 18); IN: In-line attack reactions after 24 h (lane 4, 9, 14 and 19) and 48 h (lane 5, 10, 15 and 20). ( B ) Predicted secondary structure of aptamer 20 and validation by in-line probing shown as color-coded heat map (red, high in-line attack rate; white, intermediate in-line attack rate; blue, low in-line attack rate). Black box highlights the nucleotides that were randomized in the original library.

    Journal: Nucleic Acids Research

    Article Title: RNA aptamer inhibitors of a restriction endonuclease

    doi: 10.1093/nar/gkv702

    Figure Lengend Snippet: ( A ) In-line probing ( 36 ) of high-affinity anti-KpnI aptamers 20, 24, 29, 30. (UN: unmodified samples (lane 1, 6, 11 and 16); T1: RNAse T1 digestions (lane 2, 7, 12 and 17); OH: alkaline hydrolysis reactions (lane 3, 8, 13 and 18); IN: In-line attack reactions after 24 h (lane 4, 9, 14 and 19) and 48 h (lane 5, 10, 15 and 20). ( B ) Predicted secondary structure of aptamer 20 and validation by in-line probing shown as color-coded heat map (red, high in-line attack rate; white, intermediate in-line attack rate; blue, low in-line attack rate). Black box highlights the nucleotides that were randomized in the original library.

    Article Snippet: Binding reactions contain radiolabeled aptamer (13 nM) and the indicated concentration of KpnI in New England Biolabs 1.1 buffer (10 mM Bis-Tris-Propane-HCl, pH 7, 10 mM MgCl2 , 100 μg/ml bovine serum albumin (BSA)) for 30 min at 37°C.

    Techniques:

    KpnI inhibition by anti-KpnI RNA aptamers. KpnI in the presence of 20 nM fluorescent DNA probe was incubated with anti-KpnI aptamers in the concentration range 10 pM–100 μM. ( A ) Aptamer 20; ( B ) aptamer 24; ( C ) aptamer 29; ( D ) aptamer 30.

    Journal: Nucleic Acids Research

    Article Title: RNA aptamer inhibitors of a restriction endonuclease

    doi: 10.1093/nar/gkv702

    Figure Lengend Snippet: KpnI inhibition by anti-KpnI RNA aptamers. KpnI in the presence of 20 nM fluorescent DNA probe was incubated with anti-KpnI aptamers in the concentration range 10 pM–100 μM. ( A ) Aptamer 20; ( B ) aptamer 24; ( C ) aptamer 29; ( D ) aptamer 30.

    Article Snippet: Binding reactions contain radiolabeled aptamer (13 nM) and the indicated concentration of KpnI in New England Biolabs 1.1 buffer (10 mM Bis-Tris-Propane-HCl, pH 7, 10 mM MgCl2 , 100 μg/ml bovine serum albumin (BSA)) for 30 min at 37°C.

    Techniques: Inhibition, Incubation, Concentration Assay

    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

    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: To analyze topoisomers ( ) 0.25 ng/µl of radiolabelled DNA was incubated in 1X Buffer A and 1X Buffer B (Topogen) with 0.2 U/µl Human Topo II-α (Topogen) at 37°C for 15 minutes, or in CutSmart Buffer with 0.4 U/µl XmnI or 0.04 U/µl Nt.BspQI (New England Biolabs) for 1 hour.

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

    Immunoblots showing that COX-1 and COX-2 are present in protein extractions from DRG (L4–L6) and dorsal and ventral spinal cord (segments L1–L6) of untreated rats. COX-1 and COX-2 isoforms ran to ∼72 kDa in 10% Bis-Tris gels. A second band was present at 74 kDa in spinal cord and DRG samples that were labeled with the COX-2 antibody. After deglycosidation by PNGaseF treatment, COX-2 displayed an electrophoretic mobility shift to 65 kDa, suggesting that N-linked carbohydrates had been removed.

    Journal: The Journal of Neuroscience

    Article Title: The Acute Antihyperalgesic Action of Nonsteroidal, Anti-Inflammatory Drugs and Release of Spinal Prostaglandin E2 Is Mediated by the Inhibition of Constitutive Spinal Cyclooxygenase-2 (COX-2) but not COX-1

    doi: 10.1523/JNEUROSCI.21-16-05847.2001

    Figure Lengend Snippet: Immunoblots showing that COX-1 and COX-2 are present in protein extractions from DRG (L4–L6) and dorsal and ventral spinal cord (segments L1–L6) of untreated rats. COX-1 and COX-2 isoforms ran to ∼72 kDa in 10% Bis-Tris gels. A second band was present at 74 kDa in spinal cord and DRG samples that were labeled with the COX-2 antibody. After deglycosidation by PNGaseF treatment, COX-2 displayed an electrophoretic mobility shift to 65 kDa, suggesting that N-linked carbohydrates had been removed.

    Article Snippet: The PNGaseF kit was purchased from New England Biolabs (Beverly, MA).

    Techniques: Western Blot, Labeling, Electrophoretic Mobility Shift Assay

    Gel retardation assay with the nicking enzyme N. Bst NBI and its variants. Lane 1, three DNA fragments, 1067, 794 and 584 bp, generated by digestion of plasmid pNB1, a plasmid derived from pUC19 containing a single GAGTC site (16), with Bss SI and Bsr FI. Only the middle 794 bp fragment contains the GAGTC recognition sequence. The digested pNB1 substrate (0.25 pmol) was incubated with 0.75 pmol of purified N. Bst NBI (lane 2), and 1 µl of the crude cell extract containing ∼9.5 pmol of N. Bst NBI (lane 3), N. Bst NBI-D456A (lane 4), N. Bst NBI-E418A (lane 5), N. Bst NBI-E469A (lane 6), N. Bst NBI-E482A (lane 7). Lane 8 is the control plasmid only. Lane 9 is the size standard of λ DNA digested by Hin dIII and φ174 DNA digested by Hae III (New England Biolabs).

    Journal: Nucleic Acids Research

    Article Title: The nicking endonuclease N.BstNBI is closely related to Type IIs restriction endonucleases MlyI and PleI

    doi:

    Figure Lengend Snippet: Gel retardation assay with the nicking enzyme N. Bst NBI and its variants. Lane 1, three DNA fragments, 1067, 794 and 584 bp, generated by digestion of plasmid pNB1, a plasmid derived from pUC19 containing a single GAGTC site (16), with Bss SI and Bsr FI. Only the middle 794 bp fragment contains the GAGTC recognition sequence. The digested pNB1 substrate (0.25 pmol) was incubated with 0.75 pmol of purified N. Bst NBI (lane 2), and 1 µl of the crude cell extract containing ∼9.5 pmol of N. Bst NBI (lane 3), N. Bst NBI-D456A (lane 4), N. Bst NBI-E418A (lane 5), N. Bst NBI-E469A (lane 6), N. Bst NBI-E482A (lane 7). Lane 8 is the control plasmid only. Lane 9 is the size standard of λ DNA digested by Hin dIII and φ174 DNA digested by Hae III (New England Biolabs).

    Article Snippet: The nicking endonuclease N. Bst NBI was originally isolated from the thermophilic bacterium B.stearothermophilus (NEB 928).

    Techniques: Electrophoretic Mobility Shift Assay, Generated, Plasmid Preparation, Derivative Assay, Sequencing, Incubation, Purification

    Digestion reactions of plasmid pNB1 with Mly I, Ple I and N. Bst NBI. Plasmid pNB1 (7.8 nM) was incubated with ( A ) Mly I (2 nM), ( B ) Ple I (14.2 nM) and ( C ) N. Bst NBI (0.8 nM). Aliquots were removed at different time intervals. M, DNA size marker.

    Journal: Nucleic Acids Research

    Article Title: The nicking endonuclease N.BstNBI is closely related to Type IIs restriction endonucleases MlyI and PleI

    doi:

    Figure Lengend Snippet: Digestion reactions of plasmid pNB1 with Mly I, Ple I and N. Bst NBI. Plasmid pNB1 (7.8 nM) was incubated with ( A ) Mly I (2 nM), ( B ) Ple I (14.2 nM) and ( C ) N. Bst NBI (0.8 nM). Aliquots were removed at different time intervals. M, DNA size marker.

    Article Snippet: The nicking endonuclease N. Bst NBI was originally isolated from the thermophilic bacterium B.stearothermophilus (NEB 928).

    Techniques: Plasmid Preparation, Incubation, Marker

    Schematic diagram showing the hypothesized DNA cleavage models of Type IIs restriction endonucleases Fok I ( A ), Mly I and Ple I ( B ) and the N. Bst NBI nicking enzyme ( C ). Ellipses indicate the DNA binding domain and the gray circles represent DNA cleavage domains of the Type IIs restriction endonucleases. Double strand DNA is shown as two solid lines and the GAGTC recognition sequence is indicated by the black-filled box between the two lines.

    Journal: Nucleic Acids Research

    Article Title: The nicking endonuclease N.BstNBI is closely related to Type IIs restriction endonucleases MlyI and PleI

    doi:

    Figure Lengend Snippet: Schematic diagram showing the hypothesized DNA cleavage models of Type IIs restriction endonucleases Fok I ( A ), Mly I and Ple I ( B ) and the N. Bst NBI nicking enzyme ( C ). Ellipses indicate the DNA binding domain and the gray circles represent DNA cleavage domains of the Type IIs restriction endonucleases. Double strand DNA is shown as two solid lines and the GAGTC recognition sequence is indicated by the black-filled box between the two lines.

    Article Snippet: The nicking endonuclease N. Bst NBI was originally isolated from the thermophilic bacterium B.stearothermophilus (NEB 928).

    Techniques: Binding Assay, Sequencing

    Recognition sequences and gene organizations of N. Bst NBI, Ple I and Mly I R–M systems. The recognition sequence, GAGTC, is highlighted in bold, and cleavage sites are indicated by small arrows. Open boxes represent the ORFs and their directions are indicated by arrows. Endonuclease genes are shown in black and methyltransferase genes are shown in white. The size of each gene is indicated under the gene.

    Journal: Nucleic Acids Research

    Article Title: The nicking endonuclease N.BstNBI is closely related to Type IIs restriction endonucleases MlyI and PleI

    doi:

    Figure Lengend Snippet: Recognition sequences and gene organizations of N. Bst NBI, Ple I and Mly I R–M systems. The recognition sequence, GAGTC, is highlighted in bold, and cleavage sites are indicated by small arrows. Open boxes represent the ORFs and their directions are indicated by arrows. Endonuclease genes are shown in black and methyltransferase genes are shown in white. The size of each gene is indicated under the gene.

    Article Snippet: The nicking endonuclease N. Bst NBI was originally isolated from the thermophilic bacterium B.stearothermophilus (NEB 928).

    Techniques: Sequencing

    RecJ binds monomerically to DNA. Electrophoretic mobility shift assays with 5 nM oligonucleotide A treated with 100 nM RecJ alone, RecJf alone or with a mixture of the two ( A ). An identical experiment, except using the 10 nt 5′ tailed substrate ( B ). Two distinct shifts are noted, owing to the ∼40 kDa difference in molecular weight between RecJ and RecJf.

    Journal: Nucleic Acids Research

    Article Title: RecJ exonuclease: substrates, products and interaction with SSB

    doi: 10.1093/nar/gkj503

    Figure Lengend Snippet: RecJ binds monomerically to DNA. Electrophoretic mobility shift assays with 5 nM oligonucleotide A treated with 100 nM RecJ alone, RecJf alone or with a mixture of the two ( A ). An identical experiment, except using the 10 nt 5′ tailed substrate ( B ). Two distinct shifts are noted, owing to the ∼40 kDa difference in molecular weight between RecJ and RecJf.

    Article Snippet: We tested binding of purified native wild-type RecJ (∼60 kDa in molecular weight) and a RecJ fusion to maltose-binding protein (RecJf, New England Biolabs; 100 kDa) to oligonucleotide A or a 10 nt 5′ single-stranded tailed substrate.

    Techniques: Electrophoretic Mobility Shift Assay, Molecular Weight

    Activation of conventional PKCs causes a phosphorylation-dependent mobility shift of Rnd3. ( A ) NIH 3T3 cells expressing HA-Rnd3 were pretreated for 3 h with either DMSO vehicle, Y-27632 (10 μM) or Gö-6976 (2.5 μM). Cells were then treated with PMA (100 nM) for 10 min and cell lysates were resolved on SDS-PAGE. The slower migrating Rnd3 band (arrow) was seen in both the vehicle and the Y-27632 pretreated cells, but not in cells pretreated with the conventional PKC inhibitor Gö-6976. ( B ) Calf intestinal phosphatase (CIP) treatment causes disappearance of the slower migrating band of Rnd3 (arrow). NIH 3T3 cells transiently expressing HA-Rnd3 expression vector were treated with PMA (100 nM) + ionomycin (500 μg/mL). CIP was added to the cell lysate to reverse phosphorylation. Cell lysates were resolved on SDS-PAGE and blotted with anti-HA antibody. ( C ) A CAAX mutant of Rnd3 does not shift after activation of PKCs. NIH 3T3 cells expressing HA-tagged WT Rnd3 and a SAAX mutant were treated with PMA (100 nM) for 10 min and cell lysates were resolved on SDS-PAGE.

    Journal: The Biochemical journal

    Article Title: Regulation of Rnd3 Localization and Function By PKC?-Mediated Phosphorylation

    doi: 10.1042/BJ20082377

    Figure Lengend Snippet: Activation of conventional PKCs causes a phosphorylation-dependent mobility shift of Rnd3. ( A ) NIH 3T3 cells expressing HA-Rnd3 were pretreated for 3 h with either DMSO vehicle, Y-27632 (10 μM) or Gö-6976 (2.5 μM). Cells were then treated with PMA (100 nM) for 10 min and cell lysates were resolved on SDS-PAGE. The slower migrating Rnd3 band (arrow) was seen in both the vehicle and the Y-27632 pretreated cells, but not in cells pretreated with the conventional PKC inhibitor Gö-6976. ( B ) Calf intestinal phosphatase (CIP) treatment causes disappearance of the slower migrating band of Rnd3 (arrow). NIH 3T3 cells transiently expressing HA-Rnd3 expression vector were treated with PMA (100 nM) + ionomycin (500 μg/mL). CIP was added to the cell lysate to reverse phosphorylation. Cell lysates were resolved on SDS-PAGE and blotted with anti-HA antibody. ( C ) A CAAX mutant of Rnd3 does not shift after activation of PKCs. NIH 3T3 cells expressing HA-tagged WT Rnd3 and a SAAX mutant were treated with PMA (100 nM) for 10 min and cell lysates were resolved on SDS-PAGE.

    Article Snippet: Other reagents included ionomycin and Y-27632 [Calbiochem], Bryostatin-1 and Gö-6976 [BIOMOL Research Laboratories] and calf intestinal phosphatase (CIP) [New England Biolabs].

    Techniques: Activation Assay, Mobility Shift, Expressing, SDS Page, Plasmid Preparation, Mutagenesis