e coli dna polymerase  (New England Biolabs)


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    Structured Review

    New England Biolabs e coli dna polymerase
    Proposed model for RNAP-collision-driven bidirectional transcription termination. The RNA hairpin structure formed at the overlapping region of the bidirectional terminator induces pausing of the EC from either direction. The opposite RNAP runs into the paused RNAP, forming a stable head-on collided complex that does not efficiently dissociate by itself ( Step 1 ). When a trailing EC newly initiated from the promoter runs into the head-on collided complex, the RNAP that transcribed past the overlapping region is dislodged from the <t>DNA</t> along with its nascent RNA due to its lower stability induced by the RNA hairpin ( Step 2 ). The trailing EC may either transcribes the overlapping region and forms a new head-on collided complex with the remaining EC ( Step 3 , a ), or pushes its leading EC across the overlapping region and dislodges it ( Step 3 , b ). Note that the new EC illustrated here travels in the forward direction. A reciprocal scenario where the new EC travels in the reverse direction is omitted for simplicity. This model explains the predominantly uniform 3’ ends of nascent transcripts observed at the bidirectional terminators (e.g., Figure S3A and S7).
    E Coli Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Head-on and co-directional RNA polymerase collisions orchestrate bidirectional transcription termination"

    Article Title: Head-on and co-directional RNA polymerase collisions orchestrate bidirectional transcription termination

    Journal: bioRxiv

    doi: 10.1101/2022.10.23.513370

    Proposed model for RNAP-collision-driven bidirectional transcription termination. The RNA hairpin structure formed at the overlapping region of the bidirectional terminator induces pausing of the EC from either direction. The opposite RNAP runs into the paused RNAP, forming a stable head-on collided complex that does not efficiently dissociate by itself ( Step 1 ). When a trailing EC newly initiated from the promoter runs into the head-on collided complex, the RNAP that transcribed past the overlapping region is dislodged from the DNA along with its nascent RNA due to its lower stability induced by the RNA hairpin ( Step 2 ). The trailing EC may either transcribes the overlapping region and forms a new head-on collided complex with the remaining EC ( Step 3 , a ), or pushes its leading EC across the overlapping region and dislodges it ( Step 3 , b ). Note that the new EC illustrated here travels in the forward direction. A reciprocal scenario where the new EC travels in the reverse direction is omitted for simplicity. This model explains the predominantly uniform 3’ ends of nascent transcripts observed at the bidirectional terminators (e.g., Figure S3A and S7).
    Figure Legend Snippet: Proposed model for RNAP-collision-driven bidirectional transcription termination. The RNA hairpin structure formed at the overlapping region of the bidirectional terminator induces pausing of the EC from either direction. The opposite RNAP runs into the paused RNAP, forming a stable head-on collided complex that does not efficiently dissociate by itself ( Step 1 ). When a trailing EC newly initiated from the promoter runs into the head-on collided complex, the RNAP that transcribed past the overlapping region is dislodged from the DNA along with its nascent RNA due to its lower stability induced by the RNA hairpin ( Step 2 ). The trailing EC may either transcribes the overlapping region and forms a new head-on collided complex with the remaining EC ( Step 3 , a ), or pushes its leading EC across the overlapping region and dislodges it ( Step 3 , b ). Note that the new EC illustrated here travels in the forward direction. A reciprocal scenario where the new EC travels in the reverse direction is omitted for simplicity. This model explains the predominantly uniform 3’ ends of nascent transcripts observed at the bidirectional terminators (e.g., Figure S3A and S7).

    Techniques Used:

    Screening for factors that facilitate the dissociation of head-on collided complexes. ( A ) Experimental workflow. Briefly, in vitro transcription reactions were performed on biotinylated DNA templates in the presence of fluorescently labeled probes to detect nascent RNA. After reaction, DNA substrates were pulled down by streptavidin magnetic beads. After thoroughly washing out the released RNAP and RNA products upon termination, the amount of RNA retained on the DNA template was assessed by the probe intensity from the gel. ( B ) Gel results for the assay described in (A) using DNA substrates derived from Template1 (with an intrinsic terminator) and Template2 (with a bidirectional terminator). AlexaFluor488-labeled forward probe intensities were normalized to the Template1 value. ( C ) Gel results showing the effects of a panel of E. coli transcription factors on the amount of RNA products retained on Template2 . AlexaFluor488-labeled forward probe and Cy5-labeled reverse probe intensities were normalized to the no-factor condition. The +GreB condition (outlined in red) exhibited reduced probe intensities. ( D ) Gel results showing the effect of GreB and/or GreA on the amount of retained RNA probes on Template2 under the single-round or multi-round transcription condition. Probe intensities were normalized to the no-factor condition. ( E ) Pie chart showing the fraction of different outcomes for head-on collisions observed on Template2 in the presence of GreB in the single-molecule assay. GreB was supplied in channel 4 at a final concentration of 800 nM. N denotes the total number of collision events. ( F ) Fraction of arrested forward and reverse ECs observed on Template2 with or without GreB. Data are presented as mean ± SEM from three independent experiments. Significance was determined using two-sided unpaired Student’s t -tests (* P
    Figure Legend Snippet: Screening for factors that facilitate the dissociation of head-on collided complexes. ( A ) Experimental workflow. Briefly, in vitro transcription reactions were performed on biotinylated DNA templates in the presence of fluorescently labeled probes to detect nascent RNA. After reaction, DNA substrates were pulled down by streptavidin magnetic beads. After thoroughly washing out the released RNAP and RNA products upon termination, the amount of RNA retained on the DNA template was assessed by the probe intensity from the gel. ( B ) Gel results for the assay described in (A) using DNA substrates derived from Template1 (with an intrinsic terminator) and Template2 (with a bidirectional terminator). AlexaFluor488-labeled forward probe intensities were normalized to the Template1 value. ( C ) Gel results showing the effects of a panel of E. coli transcription factors on the amount of RNA products retained on Template2 . AlexaFluor488-labeled forward probe and Cy5-labeled reverse probe intensities were normalized to the no-factor condition. The +GreB condition (outlined in red) exhibited reduced probe intensities. ( D ) Gel results showing the effect of GreB and/or GreA on the amount of retained RNA probes on Template2 under the single-round or multi-round transcription condition. Probe intensities were normalized to the no-factor condition. ( E ) Pie chart showing the fraction of different outcomes for head-on collisions observed on Template2 in the presence of GreB in the single-molecule assay. GreB was supplied in channel 4 at a final concentration of 800 nM. N denotes the total number of collision events. ( F ) Fraction of arrested forward and reverse ECs observed on Template2 with or without GreB. Data are presented as mean ± SEM from three independent experiments. Significance was determined using two-sided unpaired Student’s t -tests (* P

    Techniques Used: In Vitro, Labeling, Magnetic Beads, Derivative Assay, Concentration Assay

    Direct visualization of convergent transcription. ( A ) ( Top ) A representative kymograph showing convergent transcription and head-on collision of two RNAPs. ( Bottom ) Extracted positions of the two RNAPs as a function of time for the same kymograph. Raw data and smoothed trajectories (± 10-s moving average of tracked points) are shown in green dots and black lines, respectively. Gray region indicates the bidirectional terminator position (2250 ± 400 bp). ( B ) Multiple overlaid trajectories of convergent RNAP pairs on Template2 show collisions occur uniformly within the termination zone. ( C ) Frequency of RNAP reading through the ynaJ - uspE terminator in the forward or reverse direction observed on DNA templates that only allow unidirectional transcription ( Template3 and Template4 ; see Figure 2 ) versus on Template2 that allows convergent transcription. Data are presented as mean ± SEM from three independent experiments. Significance was determined using two-sided unpaired Student’s t -tests (* P
    Figure Legend Snippet: Direct visualization of convergent transcription. ( A ) ( Top ) A representative kymograph showing convergent transcription and head-on collision of two RNAPs. ( Bottom ) Extracted positions of the two RNAPs as a function of time for the same kymograph. Raw data and smoothed trajectories (± 10-s moving average of tracked points) are shown in green dots and black lines, respectively. Gray region indicates the bidirectional terminator position (2250 ± 400 bp). ( B ) Multiple overlaid trajectories of convergent RNAP pairs on Template2 show collisions occur uniformly within the termination zone. ( C ) Frequency of RNAP reading through the ynaJ - uspE terminator in the forward or reverse direction observed on DNA templates that only allow unidirectional transcription ( Template3 and Template4 ; see Figure 2 ) versus on Template2 that allows convergent transcription. Data are presented as mean ± SEM from three independent experiments. Significance was determined using two-sided unpaired Student’s t -tests (* P

    Techniques Used:

    Outcome of head-on collisions at the bidirectional terminator. ( A ) Three different outcomes of head-on collision between two convergent RNAPs at the bidirectional terminator on Template2 (depicted in Figure 2A ). ( Top ) Representative kymographs showing both RNAPs remaining on DNA ( Left ), one of the RNAPs dissociated ( Middle ), or both RNAPs dissociated ( Right ) after collision. ( Bottom ) Cy3 intensity profiles for selected regions from the kymograph on top (colored boxes). The summed pixel intensities of individual frames were displayed as gray dots. The filtered values (± 10-s average of frames) were shown as colored lines corresponding to the colored boxes. The number of RNAPs within the selected regions were assigned based on the photon count: under 1 as zero RNAP; between 1 and 3.5 as one RNAP (yellow shade); between 3.5 and 6.5 as two RNAPs (green shade). ( B ) Pie chart showing the fraction of each outcome illustrated in (A). ( C ) Visualizing head-on collisions by three-color imaging (Cy3 for RNAP, AlexaFluor488 for forward RNA, Cy5 for reverse RNA). Representative kymographs showing both ECs remaining on DNA ( Left ), one of the ECs dissociated ( Middle ; forward EC dissociated in this example), or both ECs dissociated ( Right ) after collision. ( D ) Pie chart showing the fraction of each outcome illustrated in (C). N denotes the total number of collision events.
    Figure Legend Snippet: Outcome of head-on collisions at the bidirectional terminator. ( A ) Three different outcomes of head-on collision between two convergent RNAPs at the bidirectional terminator on Template2 (depicted in Figure 2A ). ( Top ) Representative kymographs showing both RNAPs remaining on DNA ( Left ), one of the RNAPs dissociated ( Middle ), or both RNAPs dissociated ( Right ) after collision. ( Bottom ) Cy3 intensity profiles for selected regions from the kymograph on top (colored boxes). The summed pixel intensities of individual frames were displayed as gray dots. The filtered values (± 10-s average of frames) were shown as colored lines corresponding to the colored boxes. The number of RNAPs within the selected regions were assigned based on the photon count: under 1 as zero RNAP; between 1 and 3.5 as one RNAP (yellow shade); between 3.5 and 6.5 as two RNAPs (green shade). ( B ) Pie chart showing the fraction of each outcome illustrated in (A). ( C ) Visualizing head-on collisions by three-color imaging (Cy3 for RNAP, AlexaFluor488 for forward RNA, Cy5 for reverse RNA). Representative kymographs showing both ECs remaining on DNA ( Left ), one of the ECs dissociated ( Middle ; forward EC dissociated in this example), or both ECs dissociated ( Right ) after collision. ( D ) Pie chart showing the fraction of each outcome illustrated in (C). N denotes the total number of collision events.

    Techniques Used: Imaging

    Single-molecule platform for visualizing transcription elongation and termination. ( A ) Schematic of Template1 that contains a λ tR’ intrinsic terminator. See main text for a detailed description of the transcription unit. ( B ) Single-molecule experimental setup. Streptavidin-coated beads, stalled elongation complexes (ECs) and imaging buffer were flown into channels 1—3, respectively. A single DNA tether loaded with a stalled EC (visualized by Cy3-RNAP fluorescence) was moved to channel 4 to resume transcription. ( C ) A representative kymograph showing unidirectional translocation of a restarted EC along Template1 . ( D ) Position of the Cy3-RNAP on DNA as a function of time extracted from the kymograph in (C). Raw data and smoothed trajectory ((± 10-s moving average of tracked points) are shown in green dots and black lines, respectively. Gray region indicates the intrinsic terminator position (2150 ± 200 bp). ( E ) Multiple overlaid individual RNAP trajectories on Template1 show RNAP release within the termination zone. ( Inset ) Distribution of the average elongation speed for 57 individual RNAPs. Error bars represent SD. ( F ) A representative two-color kymograph showing the concomitant translocation and release (white arrows) of both RNAP (green) and nascent RNA (red). ( G ) Pie chart showing the fraction of EC release, retention and readthrough events observed on Template1 . N denotes the total number of ECs. See also Figures S1 and S2.
    Figure Legend Snippet: Single-molecule platform for visualizing transcription elongation and termination. ( A ) Schematic of Template1 that contains a λ tR’ intrinsic terminator. See main text for a detailed description of the transcription unit. ( B ) Single-molecule experimental setup. Streptavidin-coated beads, stalled elongation complexes (ECs) and imaging buffer were flown into channels 1—3, respectively. A single DNA tether loaded with a stalled EC (visualized by Cy3-RNAP fluorescence) was moved to channel 4 to resume transcription. ( C ) A representative kymograph showing unidirectional translocation of a restarted EC along Template1 . ( D ) Position of the Cy3-RNAP on DNA as a function of time extracted from the kymograph in (C). Raw data and smoothed trajectory ((± 10-s moving average of tracked points) are shown in green dots and black lines, respectively. Gray region indicates the intrinsic terminator position (2150 ± 200 bp). ( E ) Multiple overlaid individual RNAP trajectories on Template1 show RNAP release within the termination zone. ( Inset ) Distribution of the average elongation speed for 57 individual RNAPs. Error bars represent SD. ( F ) A representative two-color kymograph showing the concomitant translocation and release (white arrows) of both RNAP (green) and nascent RNA (red). ( G ) Pie chart showing the fraction of EC release, retention and readthrough events observed on Template1 . N denotes the total number of ECs. See also Figures S1 and S2.

    Techniques Used: Imaging, Fluorescence, Translocation Assay

    2) Product Images from "Nucleosome-directed replication origin licensing independent of a consensus DNA sequence"

    Article Title: Nucleosome-directed replication origin licensing independent of a consensus DNA sequence

    Journal: Nature Communications

    doi: 10.1038/s41467-022-32657-7

    MCMs are recruited by ORC to nucleosomes independently of ARS DNA. a Cartoon (top), an example kymograph (middle) and the corresponding fluorescence intensities (bottom) of the pre-RC assembly experiment using Cy3-labeled X.l. nucleosomes (green), LD650-labeled MCM (red), unlabeled ORC, Cdc6 and Cdt1. Yellow arrowhead in the kymograph indicates the time when the MCM fluorescence signal appeared at the nucleosomal site. b Cartoon (top), an example kymograph (middle) and the corresponding fluorescence intensities (bottom) of the pre-RC assembly experiment using A488-labeled S.c. nucleosomes (blue), LD650-labeled MCM (red), unlabeled ORC, Cdc6 and Cdt1. In both examples in a , b the nucleosomes were at non-ARS1 positions on the DNA. c Fraction of nucleosomes (X.l. or S.c.) that were observed to have colocalized MCM signals in the presence or absence of ORC. n indicates the number of nucleosomes analyzed for each condition. d Fraction of MCM complexes on a nucleosome-loaded (X.l. or S.c.) tether that colocalized with a nucleosome vs. with nucleosome-free DNA. n indicates the number of MCM complexes analyzed. e Fraction of MCM-nucleosome (X.l. or S.c.) colocalization events observed at ARS1 vs. non-ARS1 positions. n indicates the number of events analyzed. f Cartoon (top) and an example kymograph (bottom) of the three-color experiment using A488-labeled S.c. nucleosomes (blue), both LD650-labeled MCM (red) and Cy3-labeled MCM (green), unlabeled ORC, Cdc6 and Cdt1. The colocalization of a dual-color MCM with a nucleosome indicates MCM DH recruitment to the nucleosomal site. Individual lasers were occasionally turned off to confirm the fluorescence signals from the other channels. Source data are provided as a Source Data file.
    Figure Legend Snippet: MCMs are recruited by ORC to nucleosomes independently of ARS DNA. a Cartoon (top), an example kymograph (middle) and the corresponding fluorescence intensities (bottom) of the pre-RC assembly experiment using Cy3-labeled X.l. nucleosomes (green), LD650-labeled MCM (red), unlabeled ORC, Cdc6 and Cdt1. Yellow arrowhead in the kymograph indicates the time when the MCM fluorescence signal appeared at the nucleosomal site. b Cartoon (top), an example kymograph (middle) and the corresponding fluorescence intensities (bottom) of the pre-RC assembly experiment using A488-labeled S.c. nucleosomes (blue), LD650-labeled MCM (red), unlabeled ORC, Cdc6 and Cdt1. In both examples in a , b the nucleosomes were at non-ARS1 positions on the DNA. c Fraction of nucleosomes (X.l. or S.c.) that were observed to have colocalized MCM signals in the presence or absence of ORC. n indicates the number of nucleosomes analyzed for each condition. d Fraction of MCM complexes on a nucleosome-loaded (X.l. or S.c.) tether that colocalized with a nucleosome vs. with nucleosome-free DNA. n indicates the number of MCM complexes analyzed. e Fraction of MCM-nucleosome (X.l. or S.c.) colocalization events observed at ARS1 vs. non-ARS1 positions. n indicates the number of events analyzed. f Cartoon (top) and an example kymograph (bottom) of the three-color experiment using A488-labeled S.c. nucleosomes (blue), both LD650-labeled MCM (red) and Cy3-labeled MCM (green), unlabeled ORC, Cdc6 and Cdt1. The colocalization of a dual-color MCM with a nucleosome indicates MCM DH recruitment to the nucleosomal site. Individual lasers were occasionally turned off to confirm the fluorescence signals from the other channels. Source data are provided as a Source Data file.

    Techniques Used: Fluorescence, Labeling

    ORC-mediated MCM loading occurs at nucleosomal sites. a Cartoon illustrating the experimental assay that evaluates MCM loading at nucleosomal sites via high-salt wash. b A representative kymograph showing that MCM complexes (red) formed on a DNA tether in the presence of unlabeled ORC, Cdc6 and Cdt1. Upon moving to a high-salt buffer (0.5 M NaCl), a fraction of the MCMs displayed diffusive behavior without dissociation, demonstrating their successful loading onto DNA. MCM diffusion could occur from both bare DNA and nucleosome sites (white arrowheads in the zoomed-in view). Blue arrowheads indicate nucleosome positions, all of which were at non-ARS1 sites in this example. MCM and nucleosome fluorescence signals are also separately shown in gray scale at the bottom. c Fraction of nucleosome-colocalized MCM complexes that underwent diffusion without dissociation (red), remained stably bound to the nucleosome (black), or dissociated into solution (white) upon high-salt wash. n indicates the number of MCM complexes analyzed. Source data are provided as a Source Data file.
    Figure Legend Snippet: ORC-mediated MCM loading occurs at nucleosomal sites. a Cartoon illustrating the experimental assay that evaluates MCM loading at nucleosomal sites via high-salt wash. b A representative kymograph showing that MCM complexes (red) formed on a DNA tether in the presence of unlabeled ORC, Cdc6 and Cdt1. Upon moving to a high-salt buffer (0.5 M NaCl), a fraction of the MCMs displayed diffusive behavior without dissociation, demonstrating their successful loading onto DNA. MCM diffusion could occur from both bare DNA and nucleosome sites (white arrowheads in the zoomed-in view). Blue arrowheads indicate nucleosome positions, all of which were at non-ARS1 sites in this example. MCM and nucleosome fluorescence signals are also separately shown in gray scale at the bottom. c Fraction of nucleosome-colocalized MCM complexes that underwent diffusion without dissociation (red), remained stably bound to the nucleosome (black), or dissociated into solution (white) upon high-salt wash. n indicates the number of MCM complexes analyzed. Source data are provided as a Source Data file.

    Techniques Used: Diffusion-based Assay, Fluorescence, Stable Transfection

    A single-molecule platform to study eukaryotic replication initiation. a Cartoon of the λ ARS1 DNA template. The inserted ARS1 element is illustrated in the inset box. b Schematic of the single-molecule experimental setup. Channels 1–3 are separated by laminar flow. Beads are optically trapped in channel 1, moved to channel 2 to tether DNA, then moved to channel 3 to characterize the force-extension curve of the tether. Once a correct tether is confirmed, the beads-DNA assembly is moved to channel 4 or 5 containing the proteins. The zoom-in box illustrates the final assembly in the imaging channel (not drawn to scale). c A representative kymograph showing the behavior of Cy3-labeled ORC on λ ARS1 DNA in the presence of Cdc6. The engineered ARS1 position is indicated. d Fraction of λ ARS1 DNA tethers that were observed to have at least one ORC bound in the presence or absence of Cdc6. The protein concentrations used in this experiment are: 2 nM for ORC, and 5 nM for Cdc6. The number of tethers analyzed for each condition is indicated. Data are presented as mean values ± SD from three independent experiments. Significance was obtained using an unpaired two-tailed t- test (* p
    Figure Legend Snippet: A single-molecule platform to study eukaryotic replication initiation. a Cartoon of the λ ARS1 DNA template. The inserted ARS1 element is illustrated in the inset box. b Schematic of the single-molecule experimental setup. Channels 1–3 are separated by laminar flow. Beads are optically trapped in channel 1, moved to channel 2 to tether DNA, then moved to channel 3 to characterize the force-extension curve of the tether. Once a correct tether is confirmed, the beads-DNA assembly is moved to channel 4 or 5 containing the proteins. The zoom-in box illustrates the final assembly in the imaging channel (not drawn to scale). c A representative kymograph showing the behavior of Cy3-labeled ORC on λ ARS1 DNA in the presence of Cdc6. The engineered ARS1 position is indicated. d Fraction of λ ARS1 DNA tethers that were observed to have at least one ORC bound in the presence or absence of Cdc6. The protein concentrations used in this experiment are: 2 nM for ORC, and 5 nM for Cdc6. The number of tethers analyzed for each condition is indicated. Data are presented as mean values ± SD from three independent experiments. Significance was obtained using an unpaired two-tailed t- test (* p

    Techniques Used: Imaging, Labeling, Two Tailed Test

    ORC predominantly binds to nucleosomes over bare DNA. a Cartoon of the λ ARS1 DNA sparsely loaded with Cy3-labeled S.c. nucleosomes (green) and incubated with LD650-labeled ORC (red) and Cdc6. b A representative kymograph showing a λ ARS1 DNA tether loaded with multiple nucleosomes (positions indicated by green arrowheads), all of which were located at non-ARS1 sites except one. Each nucleosome was observed to be stably bound by ORC (red). The presence of ORC on the nucleosomes is confirmed by turning off the green laser, which showed only the red fluorescence from ORC; alternatively, turning off the red laser showed only the green fluorescence from the nucleosomes. c Fraction of ORC stably bound to nucleosomes (X.l. or S.c.) located at either the ARS1 site or non-ARS1 sites. n indicates the number of ORC molecules analyzed for each condition. d Fraction of nucleosomes (X.l. or S.c.) within a given DNA tether that were observed to be ORC-bound in the presence of 2 nM ORC and 5 nM Cdc6. The number of tethers analyzed for each condition is indicated. Data are presented as mean values ± SD. Significance was obtained using an unpaired two-tailed t -test (ns, p = 0.41). Source data are provided as a Source Data file.
    Figure Legend Snippet: ORC predominantly binds to nucleosomes over bare DNA. a Cartoon of the λ ARS1 DNA sparsely loaded with Cy3-labeled S.c. nucleosomes (green) and incubated with LD650-labeled ORC (red) and Cdc6. b A representative kymograph showing a λ ARS1 DNA tether loaded with multiple nucleosomes (positions indicated by green arrowheads), all of which were located at non-ARS1 sites except one. Each nucleosome was observed to be stably bound by ORC (red). The presence of ORC on the nucleosomes is confirmed by turning off the green laser, which showed only the red fluorescence from ORC; alternatively, turning off the red laser showed only the green fluorescence from the nucleosomes. c Fraction of ORC stably bound to nucleosomes (X.l. or S.c.) located at either the ARS1 site or non-ARS1 sites. n indicates the number of ORC molecules analyzed for each condition. d Fraction of nucleosomes (X.l. or S.c.) within a given DNA tether that were observed to be ORC-bound in the presence of 2 nM ORC and 5 nM Cdc6. The number of tethers analyzed for each condition is indicated. Data are presented as mean values ± SD. Significance was obtained using an unpaired two-tailed t -test (ns, p = 0.41). Source data are provided as a Source Data file.

    Techniques Used: Labeling, Incubation, Stable Transfection, Fluorescence, Two Tailed Test

    ORC-dependent MCM loading occurs frequently at non-ARS DNA sites. a Cartoon of the single-molecule pre-RC assembly experiment using λ ARS1 DNA, unlabeled ORC, Cdc6, Cdt1, and LD650-labeled MCM (red). b An example kymograph showing that the MCM fluorescence signal appeared at a non-ARS1 position on DNA. c Fraction of stably bound MCM complexes that were observed at ARS1 vs. non-ARS1 positions. n indicates the number of MCM complexes analyzed. d Example kymographs showing the photobleaching steps (white arrows) of MCM fluorescence at ARS1 and non-ARS1 positions on the DNA tether. e Distribution of the number of photobleaching steps observed in each MCM fluorescence trajectory. n indicates the number of trajectories analyzed. f Fraction of DNA tethers that were observed to harbor at least one fluorescent MCM complex in the presence or absence of ORC. The protein concentrations used in this experiment are: 10 nM for MCM, 2 nM for ORC, and 5 nM for Cdc6. The number of tethers analyzed for each condition is indicated. g Cartoon of the high-salt wash experiment to demonstrate MCM loading on DNA using a mixture of LD650-MCM and Cy3-MCM, unlabeled ORC, Cdc6 and Cdt1. h A representative kymograph showing large-scale mobility of an MCM DH (indicated by the dual-color complex which appeared as yellow) loaded at a non-ARS1 position traversing the entire length of the tethered DNA upon high-salt wash (yellow arrowhead). The other MCM complex dissociated at high salt (white arrowhead). i Fraction of MCM complexes on nucleosome-free DNA that underwent sliding on DNA without dissociation (red), remained stably bound to the DNA position (black), or dissociated into solution (white) upon high-salt wash. n indicates the number of MCM complexes analyzed. Source data are provided as a Source Data file.
    Figure Legend Snippet: ORC-dependent MCM loading occurs frequently at non-ARS DNA sites. a Cartoon of the single-molecule pre-RC assembly experiment using λ ARS1 DNA, unlabeled ORC, Cdc6, Cdt1, and LD650-labeled MCM (red). b An example kymograph showing that the MCM fluorescence signal appeared at a non-ARS1 position on DNA. c Fraction of stably bound MCM complexes that were observed at ARS1 vs. non-ARS1 positions. n indicates the number of MCM complexes analyzed. d Example kymographs showing the photobleaching steps (white arrows) of MCM fluorescence at ARS1 and non-ARS1 positions on the DNA tether. e Distribution of the number of photobleaching steps observed in each MCM fluorescence trajectory. n indicates the number of trajectories analyzed. f Fraction of DNA tethers that were observed to harbor at least one fluorescent MCM complex in the presence or absence of ORC. The protein concentrations used in this experiment are: 10 nM for MCM, 2 nM for ORC, and 5 nM for Cdc6. The number of tethers analyzed for each condition is indicated. g Cartoon of the high-salt wash experiment to demonstrate MCM loading on DNA using a mixture of LD650-MCM and Cy3-MCM, unlabeled ORC, Cdc6 and Cdt1. h A representative kymograph showing large-scale mobility of an MCM DH (indicated by the dual-color complex which appeared as yellow) loaded at a non-ARS1 position traversing the entire length of the tethered DNA upon high-salt wash (yellow arrowhead). The other MCM complex dissociated at high salt (white arrowhead). i Fraction of MCM complexes on nucleosome-free DNA that underwent sliding on DNA without dissociation (red), remained stably bound to the DNA position (black), or dissociated into solution (white) upon high-salt wash. n indicates the number of MCM complexes analyzed. Source data are provided as a Source Data file.

    Techniques Used: Labeling, Fluorescence, Stable Transfection

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    New England Biolabs dna polymerase i large klenow fragment
    Experimental procedure, time, and cost of multiple genetic abnormalities sequencing (MGA-Seq). (A) Flowchart of MGA-Seq. Nuclei were cross-linked with 0.5% formaldehyde and then digested with HindIII. 5’ <t>DNA</t> overhangs of digested chromatin fragments were filled in by <t>DNA</t> <t>polymerase</t> and then proximity ligated by T4 DNA ligase. The proximity ligation products were fragmented and then subjected to high-throughput sequencing library construction. After sequencing, all the reads were used to generate chromatin contact matrix for genome structural variation calling. In the sequencing library, the reads without ligation junction “AAGCTAGCTT” were used for the detection of CNV, SNP, small indels (
    Dna Polymerase I Large Klenow Fragment, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Experimental procedure, time, and cost of multiple genetic abnormalities sequencing (MGA-Seq). (A) Flowchart of MGA-Seq. Nuclei were cross-linked with 0.5% formaldehyde and then digested with HindIII. 5’ <t>DNA</t> overhangs of digested chromatin fragments were filled in by <t>DNA</t> <t>polymerase</t> and then proximity ligated by T4 DNA ligase. The proximity ligation products were fragmented and then subjected to high-throughput sequencing library construction. After sequencing, all the reads were used to generate chromatin contact matrix for genome structural variation calling. In the sequencing library, the reads without ligation junction “AAGCTAGCTT” were used for the detection of CNV, SNP, small indels (
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    Experimental procedure, time, and cost of multiple genetic abnormalities sequencing (MGA-Seq). (A) Flowchart of MGA-Seq. Nuclei were cross-linked with 0.5% formaldehyde and then digested with HindIII. 5’ DNA overhangs of digested chromatin fragments were filled in by DNA polymerase and then proximity ligated by T4 DNA ligase. The proximity ligation products were fragmented and then subjected to high-throughput sequencing library construction. After sequencing, all the reads were used to generate chromatin contact matrix for genome structural variation calling. In the sequencing library, the reads without ligation junction “AAGCTAGCTT” were used for the detection of CNV, SNP, small indels (

    Journal: bioRxiv

    Article Title: Robust identification of extrachromosomal DNA and genetic variants using multiple genetic abnormality sequencing (MGA-Seq)

    doi: 10.1101/2022.11.18.517160

    Figure Lengend Snippet: Experimental procedure, time, and cost of multiple genetic abnormalities sequencing (MGA-Seq). (A) Flowchart of MGA-Seq. Nuclei were cross-linked with 0.5% formaldehyde and then digested with HindIII. 5’ DNA overhangs of digested chromatin fragments were filled in by DNA polymerase and then proximity ligated by T4 DNA ligase. The proximity ligation products were fragmented and then subjected to high-throughput sequencing library construction. After sequencing, all the reads were used to generate chromatin contact matrix for genome structural variation calling. In the sequencing library, the reads without ligation junction “AAGCTAGCTT” were used for the detection of CNV, SNP, small indels (

    Article Snippet: End filling-inAdd 5 μl of dNTP mix (10 mM each) and 5 μl of DNA polymerase I Klenow fragment (NEB, M0210) to the reaction system, place the sample in thermomixer with rotation at 37 °C at 1000 r.p.m for 30 mins.

    Techniques: Sequencing, Ligation, Next-Generation Sequencing