dna pol  (New England Biolabs)


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    Structured Review

    New England Biolabs dna pol
    Pol α and PCNA are not directly down-regulated by the replication arrest pathway. (A) EC C and EC D were prepared as in Fig. 3 . 2,000/μl sperm chromatin was added (to EC C in EC C + s.c. lane, and to EC D in EC D + s.c. lane) and the reactions were incubated for 30 min. NPE was added and incubation was continued for 30 min. The chromatin was isolated and probed for the presence of pol δ (50- and 66-kD subunit), pol ε (60-kD subunit), and PCNA. To control for the specificity of the chromatin isolation procedure, a sample containing EC C but lacking sperm chromatin was also processed and analyzed (EC C − s.c. lane). (B) Schematic renditions of the <t>DNA</t> structures used to assess PCNA DNA-binding activity. The circled “5′” refers to the position of the biotin group on the 5′ end of the top strand in each structure. See text and methods for details on construction of these structures. (C) The indicated DNA structure was incubated in the indicated extract for 30 min (EC, EC C , and EC D refer to the extracts described in Fig. 3 ). The structures were recovered on a magnetic stand, the beads were washed, and bound proteins were eluted with SDS-PAGE sample buffer. The eluted proteins were probed for PCNA by immunoblotting. (D) M13 ssDNA was added, along with radio-labeled <t>dATP</t> and the indicated supplements, to either EC C or EC D . After a 30-min incubation, replication of the M13 ssDNA was assessed by agarose gel electrophoresis as described for sperm chromatin in Fig. 3 B. (E) EC D extract was treated with immobilized antibodies against pol α (Δ pol α) or with immobilized nonspecific antibodies (mock), and the depleted extracts were probed by immunoblotting for pol α (p60 subunit). (F) M13 ssDNA replication was measured in the extracts described in E. The sample labeled “Δ pol α + primer” received M13 ssDNA to which the M13 universal primer had been preannealed.
    Dna Pol, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Price from $9.99 to $1999.99
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    1) Product Images from "DNA damage-induced replication arrest in Xenopus egg extracts"

    Article Title: DNA damage-induced replication arrest in Xenopus egg extracts

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200306006

    Pol α and PCNA are not directly down-regulated by the replication arrest pathway. (A) EC C and EC D were prepared as in Fig. 3 . 2,000/μl sperm chromatin was added (to EC C in EC C + s.c. lane, and to EC D in EC D + s.c. lane) and the reactions were incubated for 30 min. NPE was added and incubation was continued for 30 min. The chromatin was isolated and probed for the presence of pol δ (50- and 66-kD subunit), pol ε (60-kD subunit), and PCNA. To control for the specificity of the chromatin isolation procedure, a sample containing EC C but lacking sperm chromatin was also processed and analyzed (EC C − s.c. lane). (B) Schematic renditions of the DNA structures used to assess PCNA DNA-binding activity. The circled “5′” refers to the position of the biotin group on the 5′ end of the top strand in each structure. See text and methods for details on construction of these structures. (C) The indicated DNA structure was incubated in the indicated extract for 30 min (EC, EC C , and EC D refer to the extracts described in Fig. 3 ). The structures were recovered on a magnetic stand, the beads were washed, and bound proteins were eluted with SDS-PAGE sample buffer. The eluted proteins were probed for PCNA by immunoblotting. (D) M13 ssDNA was added, along with radio-labeled dATP and the indicated supplements, to either EC C or EC D . After a 30-min incubation, replication of the M13 ssDNA was assessed by agarose gel electrophoresis as described for sperm chromatin in Fig. 3 B. (E) EC D extract was treated with immobilized antibodies against pol α (Δ pol α) or with immobilized nonspecific antibodies (mock), and the depleted extracts were probed by immunoblotting for pol α (p60 subunit). (F) M13 ssDNA replication was measured in the extracts described in E. The sample labeled “Δ pol α + primer” received M13 ssDNA to which the M13 universal primer had been preannealed.
    Figure Legend Snippet: Pol α and PCNA are not directly down-regulated by the replication arrest pathway. (A) EC C and EC D were prepared as in Fig. 3 . 2,000/μl sperm chromatin was added (to EC C in EC C + s.c. lane, and to EC D in EC D + s.c. lane) and the reactions were incubated for 30 min. NPE was added and incubation was continued for 30 min. The chromatin was isolated and probed for the presence of pol δ (50- and 66-kD subunit), pol ε (60-kD subunit), and PCNA. To control for the specificity of the chromatin isolation procedure, a sample containing EC C but lacking sperm chromatin was also processed and analyzed (EC C − s.c. lane). (B) Schematic renditions of the DNA structures used to assess PCNA DNA-binding activity. The circled “5′” refers to the position of the biotin group on the 5′ end of the top strand in each structure. See text and methods for details on construction of these structures. (C) The indicated DNA structure was incubated in the indicated extract for 30 min (EC, EC C , and EC D refer to the extracts described in Fig. 3 ). The structures were recovered on a magnetic stand, the beads were washed, and bound proteins were eluted with SDS-PAGE sample buffer. The eluted proteins were probed for PCNA by immunoblotting. (D) M13 ssDNA was added, along with radio-labeled dATP and the indicated supplements, to either EC C or EC D . After a 30-min incubation, replication of the M13 ssDNA was assessed by agarose gel electrophoresis as described for sperm chromatin in Fig. 3 B. (E) EC D extract was treated with immobilized antibodies against pol α (Δ pol α) or with immobilized nonspecific antibodies (mock), and the depleted extracts were probed by immunoblotting for pol α (p60 subunit). (F) M13 ssDNA replication was measured in the extracts described in E. The sample labeled “Δ pol α + primer” received M13 ssDNA to which the M13 universal primer had been preannealed.

    Techniques Used: Incubation, Isolation, Binding Assay, Activity Assay, SDS Page, Labeling, Agarose Gel Electrophoresis

    MMS-treated DNA generates a diffusible inhibitor of chromosomal replication. (A) Experimental strategy. Egg cytosol (EC) was mixed with either damaged or undamaged plasmid DNAs that had been immobilized on magnetic beads. After a 30-min incubation, the beads were separated from the extract by collection on a magnetic stand, and the extract was recovered. Extracts that had been exposed to undamaged, control plasmid are defined as EC C , whereas extracts that had been exposed to MMS-treated plasmid are defined as EC D . (B) Either EC C or EC D was mixed with 2,000/μl sperm chromatin for 30 min, followed by addition of NPE containing α- 32 P–labeled dATP. Replication of the sperm chromatin was assessed after every 30 min of additional incubation by agarose gel electrophoresis as described previously ( Walter and Newport, 1999 ). The dried gels were exposed to a PhosphorImager screen, and the amount of radioactivity incorporated into the DNA was determined by PhosphorImager analysis. The amount of DNA synthesis observed in the last time point for the EC C extract was arbitrarily set to 100, and all other values were adjusted accordingly. (C) EC was mixed with three parts buffer (sperm dilution buffer; Murray, 1991 ), or three parts EC D , and incubated with sperm chromatin for 30 min. NPE was added, and replication was assessed 30 min later. Replication in the diluted extracts was compared with that observed with undiluted EC, where the value was arbitrarily set to 100. All reactions contained a total of 12,000 sperm nuclei. Replication was analyzed as in B. The experiment was performed three times, and the bars represent the mean averages for each experiment. The error bars refer to one SD from that mean.
    Figure Legend Snippet: MMS-treated DNA generates a diffusible inhibitor of chromosomal replication. (A) Experimental strategy. Egg cytosol (EC) was mixed with either damaged or undamaged plasmid DNAs that had been immobilized on magnetic beads. After a 30-min incubation, the beads were separated from the extract by collection on a magnetic stand, and the extract was recovered. Extracts that had been exposed to undamaged, control plasmid are defined as EC C , whereas extracts that had been exposed to MMS-treated plasmid are defined as EC D . (B) Either EC C or EC D was mixed with 2,000/μl sperm chromatin for 30 min, followed by addition of NPE containing α- 32 P–labeled dATP. Replication of the sperm chromatin was assessed after every 30 min of additional incubation by agarose gel electrophoresis as described previously ( Walter and Newport, 1999 ). The dried gels were exposed to a PhosphorImager screen, and the amount of radioactivity incorporated into the DNA was determined by PhosphorImager analysis. The amount of DNA synthesis observed in the last time point for the EC C extract was arbitrarily set to 100, and all other values were adjusted accordingly. (C) EC was mixed with three parts buffer (sperm dilution buffer; Murray, 1991 ), or three parts EC D , and incubated with sperm chromatin for 30 min. NPE was added, and replication was assessed 30 min later. Replication in the diluted extracts was compared with that observed with undiluted EC, where the value was arbitrarily set to 100. All reactions contained a total of 12,000 sperm nuclei. Replication was analyzed as in B. The experiment was performed three times, and the bars represent the mean averages for each experiment. The error bars refer to one SD from that mean.

    Techniques Used: Plasmid Preparation, Magnetic Beads, Incubation, Labeling, Agarose Gel Electrophoresis, Radioactivity, DNA Synthesis

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    New England Biolabs dna polymerase i
    Pol I removes the 5′-dRP-H4 DPC following strand-displacement <t>DNA</t> synthesis . A , top, the nucleotide sequence of duplex DNA containing an AP-H4 DPC prepared via reductive amination. A Cy5 is at the 5′-terminus, and a FdT is at the second position from the 3′-terminus. The numbers above the nucleotide sequence indicate the lengths from the 3′-terminus. Bottom, a scheme showing the procedures of investigating the 5′-dRP-H4 DPC removal by Pol I. The reaction samples were finally treated by proteinase K for urea-PAGE analysis of the unexcised 5′-dRP-H4 DPC. B , a representative 20% urea-PAGE gel showing the strand-displacement DNA synthesis ( top , Cy5) and removal of 5′-dRP-H4 DPC (20 nM, bottom, FdT) by Pol I (4 nM) in the presence of different dNTPs (2 μM) at 37 ° C for 30 min. C , a scatter plot with the mean and standard deviation showing the efficiency of 5′-dRP-H4 DPC removal by Pol I from the experiments in B (bottom). The data are from three independent experiments. AP site, apurinic/apyrimidinic or abasic site; DPC, DNA–protein cross-link; FdT, fluorescein dT; Pol I, DNA polymerase I.
    Dna Polymerase I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Pol I removes the 5′-dRP-H4 DPC following strand-displacement DNA synthesis . A , top, the nucleotide sequence of duplex DNA containing an AP-H4 DPC prepared via reductive amination. A Cy5 is at the 5′-terminus, and a FdT is at the second position from the 3′-terminus. The numbers above the nucleotide sequence indicate the lengths from the 3′-terminus. Bottom, a scheme showing the procedures of investigating the 5′-dRP-H4 DPC removal by Pol I. The reaction samples were finally treated by proteinase K for urea-PAGE analysis of the unexcised 5′-dRP-H4 DPC. B , a representative 20% urea-PAGE gel showing the strand-displacement DNA synthesis ( top , Cy5) and removal of 5′-dRP-H4 DPC (20 nM, bottom, FdT) by Pol I (4 nM) in the presence of different dNTPs (2 μM) at 37 ° C for 30 min. C , a scatter plot with the mean and standard deviation showing the efficiency of 5′-dRP-H4 DPC removal by Pol I from the experiments in B (bottom). The data are from three independent experiments. AP site, apurinic/apyrimidinic or abasic site; DPC, DNA–protein cross-link; FdT, fluorescein dT; Pol I, DNA polymerase I.

    Journal: The Journal of Biological Chemistry

    Article Title: In vitro eradication of abasic site-mediated DNA–peptide/protein cross-links by Escherichia coli long-patch base excision repair

    doi: 10.1016/j.jbc.2022.102055

    Figure Lengend Snippet: Pol I removes the 5′-dRP-H4 DPC following strand-displacement DNA synthesis . A , top, the nucleotide sequence of duplex DNA containing an AP-H4 DPC prepared via reductive amination. A Cy5 is at the 5′-terminus, and a FdT is at the second position from the 3′-terminus. The numbers above the nucleotide sequence indicate the lengths from the 3′-terminus. Bottom, a scheme showing the procedures of investigating the 5′-dRP-H4 DPC removal by Pol I. The reaction samples were finally treated by proteinase K for urea-PAGE analysis of the unexcised 5′-dRP-H4 DPC. B , a representative 20% urea-PAGE gel showing the strand-displacement DNA synthesis ( top , Cy5) and removal of 5′-dRP-H4 DPC (20 nM, bottom, FdT) by Pol I (4 nM) in the presence of different dNTPs (2 μM) at 37 ° C for 30 min. C , a scatter plot with the mean and standard deviation showing the efficiency of 5′-dRP-H4 DPC removal by Pol I from the experiments in B (bottom). The data are from three independent experiments. AP site, apurinic/apyrimidinic or abasic site; DPC, DNA–protein cross-link; FdT, fluorescein dT; Pol I, DNA polymerase I.

    Article Snippet: Proteinase K (Cat. #: P8107S), Endo IV (Cat. #: M0304S), Exo III (Cat. #: M0206S), Pol I (Cat. #: M0209S), ligase (Cat. #: M0205S), human APE1 (Cat. #: M0282S), uracil-DNA glycosylase (UDG, Cat. #: M0280S), and Nt.BstNBI (Cat. #: R0607S) were purchased from the New England Biolabs.

    Techniques: DNA Synthesis, Sequencing, Polyacrylamide Gel Electrophoresis, Standard Deviation

    Pol I removes 5′-dRP-peptide 10mer following strand-displacement DNA synthesis . A , the nucleotide sequence of duplex DNA containing AP-peptide 10mer . A Cy5 is at the 5′-terminus, and a FdT is at the second position from the 3′-terminus. The numbers above the nucleotide sequence indicate the lengths from the 3′-terminus. B , a scheme showing the procedures of investigating the strand-displacement DNA synthesis and 5′-dRP-peptide 10mer removal by Pol I following the Endo IV-induced strand incision. C , a representative 20% urea-PAGE gel showing the removal of 5′-dRP-peptide 10mer (20 nM, top, FdT) and strand-displacement DNA synthesis (bottom, Cy5) by Pol I (4 nM) in the presence of different dNTPs (2 μM). The reactions were carried out at 37 ° C for 30 min. The asterisks (top) indicate the predominant excision products. D , a scatter plot with the mean and standard deviation showing the efficiency of 5′-dRP-peptide 10mer removal by Pol I from reactions in C (top). The data are from three independent experiments. E , the predominant excision sites of Pol I following the strand-displacement DNA synthesis that were determined from the results in C (top, Lanes 4–6). AP, apurinic/apyrimidinic or abasic; Endo IV, endonuclease IV; FdT, fluorescein dT; Pol I, DNA polymerase I.

    Journal: The Journal of Biological Chemistry

    Article Title: In vitro eradication of abasic site-mediated DNA–peptide/protein cross-links by Escherichia coli long-patch base excision repair

    doi: 10.1016/j.jbc.2022.102055

    Figure Lengend Snippet: Pol I removes 5′-dRP-peptide 10mer following strand-displacement DNA synthesis . A , the nucleotide sequence of duplex DNA containing AP-peptide 10mer . A Cy5 is at the 5′-terminus, and a FdT is at the second position from the 3′-terminus. The numbers above the nucleotide sequence indicate the lengths from the 3′-terminus. B , a scheme showing the procedures of investigating the strand-displacement DNA synthesis and 5′-dRP-peptide 10mer removal by Pol I following the Endo IV-induced strand incision. C , a representative 20% urea-PAGE gel showing the removal of 5′-dRP-peptide 10mer (20 nM, top, FdT) and strand-displacement DNA synthesis (bottom, Cy5) by Pol I (4 nM) in the presence of different dNTPs (2 μM). The reactions were carried out at 37 ° C for 30 min. The asterisks (top) indicate the predominant excision products. D , a scatter plot with the mean and standard deviation showing the efficiency of 5′-dRP-peptide 10mer removal by Pol I from reactions in C (top). The data are from three independent experiments. E , the predominant excision sites of Pol I following the strand-displacement DNA synthesis that were determined from the results in C (top, Lanes 4–6). AP, apurinic/apyrimidinic or abasic; Endo IV, endonuclease IV; FdT, fluorescein dT; Pol I, DNA polymerase I.

    Article Snippet: Proteinase K (Cat. #: P8107S), Endo IV (Cat. #: M0304S), Exo III (Cat. #: M0206S), Pol I (Cat. #: M0209S), ligase (Cat. #: M0205S), human APE1 (Cat. #: M0282S), uracil-DNA glycosylase (UDG, Cat. #: M0280S), and Nt.BstNBI (Cat. #: R0607S) were purchased from the New England Biolabs.

    Techniques: DNA Synthesis, Sequencing, Polyacrylamide Gel Electrophoresis, Standard Deviation

    Removal of independently generated 5′-dRP-peptide 10mer -containing flaps by Pol I . A , nucleotide sequences of nicked DNA containing independently generated 5′-dRP-peptide 10mer within different lengths of flaps. The arrows indicate the predominant incision sites by Pol I that were determined from the results in B. B , top, a representative 20% urea-PAGE gel showing the removal of 5′-dRP-peptide 10mer (20 nM) by Pol I (0–20 nM) at 37 ° C for 30 min. The oligos and DNA–peptide adducts were visualized by using the fluorescence of 6-FAM. Bottom, a scatter plot with the mean and standard deviation showing the efficiency of 5′-dRP-peptide 10mer removal by Pol I as a function of Pol I concentration. The data are from three independent experiments. Pol I, DNA polymerase I.

    Journal: The Journal of Biological Chemistry

    Article Title: In vitro eradication of abasic site-mediated DNA–peptide/protein cross-links by Escherichia coli long-patch base excision repair

    doi: 10.1016/j.jbc.2022.102055

    Figure Lengend Snippet: Removal of independently generated 5′-dRP-peptide 10mer -containing flaps by Pol I . A , nucleotide sequences of nicked DNA containing independently generated 5′-dRP-peptide 10mer within different lengths of flaps. The arrows indicate the predominant incision sites by Pol I that were determined from the results in B. B , top, a representative 20% urea-PAGE gel showing the removal of 5′-dRP-peptide 10mer (20 nM) by Pol I (0–20 nM) at 37 ° C for 30 min. The oligos and DNA–peptide adducts were visualized by using the fluorescence of 6-FAM. Bottom, a scatter plot with the mean and standard deviation showing the efficiency of 5′-dRP-peptide 10mer removal by Pol I as a function of Pol I concentration. The data are from three independent experiments. Pol I, DNA polymerase I.

    Article Snippet: Proteinase K (Cat. #: P8107S), Endo IV (Cat. #: M0304S), Exo III (Cat. #: M0206S), Pol I (Cat. #: M0209S), ligase (Cat. #: M0205S), human APE1 (Cat. #: M0282S), uracil-DNA glycosylase (UDG, Cat. #: M0280S), and Nt.BstNBI (Cat. #: R0607S) were purchased from the New England Biolabs.

    Techniques: Generated, Polyacrylamide Gel Electrophoresis, Fluorescence, Standard Deviation, Concentration Assay

    DPE-PCR enables sensitive and quantitative detection of Gram-negative and Gram-positive bacteria via measurement of DPE activity in crude lysates. ( A ) Decreasing amounts of E. coli colony forming unit were spiked into bead lysis-coupled DPE-PCR. NIC were also included to monitor reagent background levels. All colony forming unit spikes and NICs were performed in triplicate. A representative DPE-PCR curve is shown below for each level of bacterial input. Colony count plating and gsPCR were performed in an effort to obtain a better estimate of the actual colony forming unit placed into each reaction and is presented in Supplementary Figure S5 ( B ) A plot of E. coli DNA polymerase activity and linear regression analysis is presented. Graphs were generated using the average C t values obtained from triplicate reactions of bacterial spikes ranging from 1 × 10 5 to 1 × 10 1 input colony forming unit. ( C and D ) Colony forming unit titration experiments were performed for S. aureus exactly as described above for E. coli . Colony count plating and gsPCR were performed in an effort to obtain a better estimate of the actual colony forming unit placed into each reaction and is presented in Supplementary Figure S6 .

    Journal: Nucleic Acids Research

    Article Title: Characterization of a novel DNA polymerase activity assay enabling sensitive, quantitative and universal detection of viable microbes

    doi: 10.1093/nar/gks316

    Figure Lengend Snippet: DPE-PCR enables sensitive and quantitative detection of Gram-negative and Gram-positive bacteria via measurement of DPE activity in crude lysates. ( A ) Decreasing amounts of E. coli colony forming unit were spiked into bead lysis-coupled DPE-PCR. NIC were also included to monitor reagent background levels. All colony forming unit spikes and NICs were performed in triplicate. A representative DPE-PCR curve is shown below for each level of bacterial input. Colony count plating and gsPCR were performed in an effort to obtain a better estimate of the actual colony forming unit placed into each reaction and is presented in Supplementary Figure S5 ( B ) A plot of E. coli DNA polymerase activity and linear regression analysis is presented. Graphs were generated using the average C t values obtained from triplicate reactions of bacterial spikes ranging from 1 × 10 5 to 1 × 10 1 input colony forming unit. ( C and D ) Colony forming unit titration experiments were performed for S. aureus exactly as described above for E. coli . Colony count plating and gsPCR were performed in an effort to obtain a better estimate of the actual colony forming unit placed into each reaction and is presented in Supplementary Figure S6 .

    Article Snippet: DPE reaction conditions DNA Pol I (NEB cat# M0209L), Klenow (NEB cat# M0210S) and Klenow exo(−) (NEB cat# M0212S) were diluted to the indicated units per microliter stock in sterile Tris–EDTA, pH 8.0.

    Techniques: Polymerase Chain Reaction, Activity Assay, Lysis, Generated, Titration

    Sensitive detection of purified DNA polymerase using DPE-PCR. ( A ) A commercial source of DNA polymerase I was assayed in duplicate at 10-fold increments starting at 2 × 10 −5 U down to 2 × 10 −11 U per reaction. A representative DPE-PCR curve is shown for each polymerase input level and NIC. ( B ) A plot was constructed from n = 4 data points per polymerase input level, taken from two independent experiments and linear regression analysis was performed. ( C ) Triplicate reactions containing 2 × 10 −7 U of DNA polymerase I, Klenow, Klenow (exo−) and E. coli DNA Ligase were assayed in comparison to an NIC. A representative DPE-PCR curve is presented for each of the assayed enzymes and NIC. ( D ) Triplicate DPE-PCR curves are shown from corresponding DPE reactions containing a 50 -µM (dATP, dGTP, dTTP) mixture supplemented with 50 µM of either dCTP or ddCTP. A schematic representing some of the first available sites for dCTP or ddCTP incorporation within the DNA substrate is presented adjacent to the DPE-PCR curves.

    Journal: Nucleic Acids Research

    Article Title: Characterization of a novel DNA polymerase activity assay enabling sensitive, quantitative and universal detection of viable microbes

    doi: 10.1093/nar/gks316

    Figure Lengend Snippet: Sensitive detection of purified DNA polymerase using DPE-PCR. ( A ) A commercial source of DNA polymerase I was assayed in duplicate at 10-fold increments starting at 2 × 10 −5 U down to 2 × 10 −11 U per reaction. A representative DPE-PCR curve is shown for each polymerase input level and NIC. ( B ) A plot was constructed from n = 4 data points per polymerase input level, taken from two independent experiments and linear regression analysis was performed. ( C ) Triplicate reactions containing 2 × 10 −7 U of DNA polymerase I, Klenow, Klenow (exo−) and E. coli DNA Ligase were assayed in comparison to an NIC. A representative DPE-PCR curve is presented for each of the assayed enzymes and NIC. ( D ) Triplicate DPE-PCR curves are shown from corresponding DPE reactions containing a 50 -µM (dATP, dGTP, dTTP) mixture supplemented with 50 µM of either dCTP or ddCTP. A schematic representing some of the first available sites for dCTP or ddCTP incorporation within the DNA substrate is presented adjacent to the DPE-PCR curves.

    Article Snippet: DPE reaction conditions DNA Pol I (NEB cat# M0209L), Klenow (NEB cat# M0210S) and Klenow exo(−) (NEB cat# M0212S) were diluted to the indicated units per microliter stock in sterile Tris–EDTA, pH 8.0.

    Techniques: Purification, Polymerase Chain Reaction, Construct

    Detection of bacteria by DPE-PCR is blocked by ddCTP and rescued with dCTP. ( A ) E. coli suspensions were added to bead lysis-coupled DNA polymerase assays composed of a 50 µM (dATP, dGTP, dTTP) mixture supplemented with either 50 µM dCTP or 50 µM ddCTP. DPE-PCR curves representing E. coli -derived DNA polymerase activity is presented. Approximate colony forming unit input as determined by plating is presented in the upper left region of the qPCR graph ( B ) E. coli suspensions were added to bead lysis tubes containing 50 µl reaction buffer with 50-µM (dATP, dGTP, dTTP, ddCTP). Prior to lysis, 1 µl of dCTP (2.5, 0.25, 0.025 and 0.0025 mM) was added to selected ddCTP-containing reactions. Reactions containing 50 µM (dATP, dGTP, dTTP, dCTP) alone or 50 µM (dATP, dGTP, dTTP, ddCTP) alone were run in parallel as ‘non-terminated’ and ‘terminated’ comparators. The resultant DPE-PCR curves representing E. coli -derived DNA polymerase activity is presented. Approximate colony forming unit input as determined by plating is presented in the lower left region of the qPCR graph. ( C ) Escherichia coli gene-specific PCR was also performed on the same lysates used for DNA polymerase detection presented in Figure 2 B. Linear plots of dCTP-dependent rescue of bacterial DNA polymerase detection versus gsPCR of genomic DNA are shown. Plots were generated using the average qPCR C t values from triplicate reactions at the indicated conditions. ( D–F ) ddCTP termination and dCTP rescue experiments were performed for S. aureus exactly as described above for E. coli .

    Journal: Nucleic Acids Research

    Article Title: Characterization of a novel DNA polymerase activity assay enabling sensitive, quantitative and universal detection of viable microbes

    doi: 10.1093/nar/gks316

    Figure Lengend Snippet: Detection of bacteria by DPE-PCR is blocked by ddCTP and rescued with dCTP. ( A ) E. coli suspensions were added to bead lysis-coupled DNA polymerase assays composed of a 50 µM (dATP, dGTP, dTTP) mixture supplemented with either 50 µM dCTP or 50 µM ddCTP. DPE-PCR curves representing E. coli -derived DNA polymerase activity is presented. Approximate colony forming unit input as determined by plating is presented in the upper left region of the qPCR graph ( B ) E. coli suspensions were added to bead lysis tubes containing 50 µl reaction buffer with 50-µM (dATP, dGTP, dTTP, ddCTP). Prior to lysis, 1 µl of dCTP (2.5, 0.25, 0.025 and 0.0025 mM) was added to selected ddCTP-containing reactions. Reactions containing 50 µM (dATP, dGTP, dTTP, dCTP) alone or 50 µM (dATP, dGTP, dTTP, ddCTP) alone were run in parallel as ‘non-terminated’ and ‘terminated’ comparators. The resultant DPE-PCR curves representing E. coli -derived DNA polymerase activity is presented. Approximate colony forming unit input as determined by plating is presented in the lower left region of the qPCR graph. ( C ) Escherichia coli gene-specific PCR was also performed on the same lysates used for DNA polymerase detection presented in Figure 2 B. Linear plots of dCTP-dependent rescue of bacterial DNA polymerase detection versus gsPCR of genomic DNA are shown. Plots were generated using the average qPCR C t values from triplicate reactions at the indicated conditions. ( D–F ) ddCTP termination and dCTP rescue experiments were performed for S. aureus exactly as described above for E. coli .

    Article Snippet: DPE reaction conditions DNA Pol I (NEB cat# M0209L), Klenow (NEB cat# M0210S) and Klenow exo(−) (NEB cat# M0212S) were diluted to the indicated units per microliter stock in sterile Tris–EDTA, pH 8.0.

    Techniques: Polymerase Chain Reaction, Lysis, Derivative Assay, Activity Assay, Real-time Polymerase Chain Reaction, Generated

    DPE-PCR as an indicator of E. coli viability in response to heat treatment. ( A ) Aliquots of an E. coli suspension (∼2000 cfu/µl) were incubated at 25°C, 45°C, 65°C, 85°C and 105°C for 20 min. After heating, each bacterial stock was cooled to room temperature and 5 µl were transferred to the bead lysis-coupled DPE-PCR assay. DPE-PCR curves representing E. coli -derived DNA polymerase activity following each of the indicated temperature treatments are presented. ( B ) Plots were generated from triplicate DPE-PCRs and gsPCR of genomic DNA (from the same lysates) after the indicated temperature treatments of E. coli suspensions. Parallel plating was also performed in triplicate for each of the treated E. coli suspensions. Representative colony forming unit monitoring plates are presented below the graph, revealing bacterial viability status after treatment at each temperature. ( C ) DPE-PCR is compared with gsPCR of genomic DNA in response to the various temperature treatments. ‘Fold Reduction of qPCR Signal’ was calculated using the indicated equation and the values obtained were used to generate comparative bar graphs.

    Journal: Nucleic Acids Research

    Article Title: Characterization of a novel DNA polymerase activity assay enabling sensitive, quantitative and universal detection of viable microbes

    doi: 10.1093/nar/gks316

    Figure Lengend Snippet: DPE-PCR as an indicator of E. coli viability in response to heat treatment. ( A ) Aliquots of an E. coli suspension (∼2000 cfu/µl) were incubated at 25°C, 45°C, 65°C, 85°C and 105°C for 20 min. After heating, each bacterial stock was cooled to room temperature and 5 µl were transferred to the bead lysis-coupled DPE-PCR assay. DPE-PCR curves representing E. coli -derived DNA polymerase activity following each of the indicated temperature treatments are presented. ( B ) Plots were generated from triplicate DPE-PCRs and gsPCR of genomic DNA (from the same lysates) after the indicated temperature treatments of E. coli suspensions. Parallel plating was also performed in triplicate for each of the treated E. coli suspensions. Representative colony forming unit monitoring plates are presented below the graph, revealing bacterial viability status after treatment at each temperature. ( C ) DPE-PCR is compared with gsPCR of genomic DNA in response to the various temperature treatments. ‘Fold Reduction of qPCR Signal’ was calculated using the indicated equation and the values obtained were used to generate comparative bar graphs.

    Article Snippet: DPE reaction conditions DNA Pol I (NEB cat# M0209L), Klenow (NEB cat# M0210S) and Klenow exo(−) (NEB cat# M0212S) were diluted to the indicated units per microliter stock in sterile Tris–EDTA, pH 8.0.

    Techniques: Polymerase Chain Reaction, Incubation, Lysis, Derivative Assay, Activity Assay, Generated, Real-time Polymerase Chain Reaction

    Pol α and PCNA are not directly down-regulated by the replication arrest pathway. (A) EC C and EC D were prepared as in Fig. 3 . 2,000/μl sperm chromatin was added (to EC C in EC C + s.c. lane, and to EC D in EC D + s.c. lane) and the reactions were incubated for 30 min. NPE was added and incubation was continued for 30 min. The chromatin was isolated and probed for the presence of pol δ (50- and 66-kD subunit), pol ε (60-kD subunit), and PCNA. To control for the specificity of the chromatin isolation procedure, a sample containing EC C but lacking sperm chromatin was also processed and analyzed (EC C − s.c. lane). (B) Schematic renditions of the DNA structures used to assess PCNA DNA-binding activity. The circled “5′” refers to the position of the biotin group on the 5′ end of the top strand in each structure. See text and methods for details on construction of these structures. (C) The indicated DNA structure was incubated in the indicated extract for 30 min (EC, EC C , and EC D refer to the extracts described in Fig. 3 ). The structures were recovered on a magnetic stand, the beads were washed, and bound proteins were eluted with SDS-PAGE sample buffer. The eluted proteins were probed for PCNA by immunoblotting. (D) M13 ssDNA was added, along with radio-labeled dATP and the indicated supplements, to either EC C or EC D . After a 30-min incubation, replication of the M13 ssDNA was assessed by agarose gel electrophoresis as described for sperm chromatin in Fig. 3 B. (E) EC D extract was treated with immobilized antibodies against pol α (Δ pol α) or with immobilized nonspecific antibodies (mock), and the depleted extracts were probed by immunoblotting for pol α (p60 subunit). (F) M13 ssDNA replication was measured in the extracts described in E. The sample labeled “Δ pol α + primer” received M13 ssDNA to which the M13 universal primer had been preannealed.

    Journal: The Journal of Cell Biology

    Article Title: DNA damage-induced replication arrest in Xenopus egg extracts

    doi: 10.1083/jcb.200306006

    Figure Lengend Snippet: Pol α and PCNA are not directly down-regulated by the replication arrest pathway. (A) EC C and EC D were prepared as in Fig. 3 . 2,000/μl sperm chromatin was added (to EC C in EC C + s.c. lane, and to EC D in EC D + s.c. lane) and the reactions were incubated for 30 min. NPE was added and incubation was continued for 30 min. The chromatin was isolated and probed for the presence of pol δ (50- and 66-kD subunit), pol ε (60-kD subunit), and PCNA. To control for the specificity of the chromatin isolation procedure, a sample containing EC C but lacking sperm chromatin was also processed and analyzed (EC C − s.c. lane). (B) Schematic renditions of the DNA structures used to assess PCNA DNA-binding activity. The circled “5′” refers to the position of the biotin group on the 5′ end of the top strand in each structure. See text and methods for details on construction of these structures. (C) The indicated DNA structure was incubated in the indicated extract for 30 min (EC, EC C , and EC D refer to the extracts described in Fig. 3 ). The structures were recovered on a magnetic stand, the beads were washed, and bound proteins were eluted with SDS-PAGE sample buffer. The eluted proteins were probed for PCNA by immunoblotting. (D) M13 ssDNA was added, along with radio-labeled dATP and the indicated supplements, to either EC C or EC D . After a 30-min incubation, replication of the M13 ssDNA was assessed by agarose gel electrophoresis as described for sperm chromatin in Fig. 3 B. (E) EC D extract was treated with immobilized antibodies against pol α (Δ pol α) or with immobilized nonspecific antibodies (mock), and the depleted extracts were probed by immunoblotting for pol α (p60 subunit). (F) M13 ssDNA replication was measured in the extracts described in E. The sample labeled “Δ pol α + primer” received M13 ssDNA to which the M13 universal primer had been preannealed.

    Article Snippet: Linear pSP72 was labeled with biotin 14-dATP (GIBCO BRL) in a reaction containing 33 μM biotin-14-dATP, and 10 U DNA pol I Klenow fragment (New England Biolabs, Inc.) per microgram of DNA, for 30 min at room temperature.

    Techniques: Incubation, Isolation, Binding Assay, Activity Assay, SDS Page, Labeling, Agarose Gel Electrophoresis

    MMS-treated DNA generates a diffusible inhibitor of chromosomal replication. (A) Experimental strategy. Egg cytosol (EC) was mixed with either damaged or undamaged plasmid DNAs that had been immobilized on magnetic beads. After a 30-min incubation, the beads were separated from the extract by collection on a magnetic stand, and the extract was recovered. Extracts that had been exposed to undamaged, control plasmid are defined as EC C , whereas extracts that had been exposed to MMS-treated plasmid are defined as EC D . (B) Either EC C or EC D was mixed with 2,000/μl sperm chromatin for 30 min, followed by addition of NPE containing α- 32 P–labeled dATP. Replication of the sperm chromatin was assessed after every 30 min of additional incubation by agarose gel electrophoresis as described previously ( Walter and Newport, 1999 ). The dried gels were exposed to a PhosphorImager screen, and the amount of radioactivity incorporated into the DNA was determined by PhosphorImager analysis. The amount of DNA synthesis observed in the last time point for the EC C extract was arbitrarily set to 100, and all other values were adjusted accordingly. (C) EC was mixed with three parts buffer (sperm dilution buffer; Murray, 1991 ), or three parts EC D , and incubated with sperm chromatin for 30 min. NPE was added, and replication was assessed 30 min later. Replication in the diluted extracts was compared with that observed with undiluted EC, where the value was arbitrarily set to 100. All reactions contained a total of 12,000 sperm nuclei. Replication was analyzed as in B. The experiment was performed three times, and the bars represent the mean averages for each experiment. The error bars refer to one SD from that mean.

    Journal: The Journal of Cell Biology

    Article Title: DNA damage-induced replication arrest in Xenopus egg extracts

    doi: 10.1083/jcb.200306006

    Figure Lengend Snippet: MMS-treated DNA generates a diffusible inhibitor of chromosomal replication. (A) Experimental strategy. Egg cytosol (EC) was mixed with either damaged or undamaged plasmid DNAs that had been immobilized on magnetic beads. After a 30-min incubation, the beads were separated from the extract by collection on a magnetic stand, and the extract was recovered. Extracts that had been exposed to undamaged, control plasmid are defined as EC C , whereas extracts that had been exposed to MMS-treated plasmid are defined as EC D . (B) Either EC C or EC D was mixed with 2,000/μl sperm chromatin for 30 min, followed by addition of NPE containing α- 32 P–labeled dATP. Replication of the sperm chromatin was assessed after every 30 min of additional incubation by agarose gel electrophoresis as described previously ( Walter and Newport, 1999 ). The dried gels were exposed to a PhosphorImager screen, and the amount of radioactivity incorporated into the DNA was determined by PhosphorImager analysis. The amount of DNA synthesis observed in the last time point for the EC C extract was arbitrarily set to 100, and all other values were adjusted accordingly. (C) EC was mixed with three parts buffer (sperm dilution buffer; Murray, 1991 ), or three parts EC D , and incubated with sperm chromatin for 30 min. NPE was added, and replication was assessed 30 min later. Replication in the diluted extracts was compared with that observed with undiluted EC, where the value was arbitrarily set to 100. All reactions contained a total of 12,000 sperm nuclei. Replication was analyzed as in B. The experiment was performed three times, and the bars represent the mean averages for each experiment. The error bars refer to one SD from that mean.

    Article Snippet: Linear pSP72 was labeled with biotin 14-dATP (GIBCO BRL) in a reaction containing 33 μM biotin-14-dATP, and 10 U DNA pol I Klenow fragment (New England Biolabs, Inc.) per microgram of DNA, for 30 min at room temperature.

    Techniques: Plasmid Preparation, Magnetic Beads, Incubation, Labeling, Agarose Gel Electrophoresis, Radioactivity, DNA Synthesis