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Effects of DNA3'pp5'G capping on 3' end repair reactions and of an embedded pyrophosphate-linked guanylate on ribonucleotide surveillance.

Chauleau M, Das U, Shuman S - Nucleic Acids Res. (2015)

Bottom Line: Human DNA polymerase β (X family) extends the cap primer predominantly by a single templated addition step.Cap-primed synthesis by templated polymerases embeds a pyrophosphate-linked ribonucleotide in DNA.We find that the embedded ppG is refractory to surveillance and incision by RNase H2.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA.

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The cap pyrophosphate prevents nick sealing by DNA ligases. Left panel: Chlorella virus DNA ligase reaction mixtures (10 μl) containing 50 mM Tris–HCl, pH 7.5, 5 mM DTT, 10 mM MgCl2, 1 mM ATP, 0.4 pmol 5′ 32P-labeled pDNAp(dG)OH or pDNApp(rG)OH nicked duplex (shown below with the 32P-label denoted by •) and either no enzyme (−) or 1 pmol ChVLig (+) were incubated at 37°C for 30 min. Right panel: Escherichia coli DNA ligase reaction mixtures (10 μl) containing 50 mM Tris–HCl, pH 7.5, 5 mM DTT, 5 mM MgCl2, 1 mM NAD+, 10 mM (NH4)2SO4, 0.4 pmol pDNAp(dG)OH or pDNApp(rG)OH nicked duplex, and either no enzyme (−) or 1 pmol EcoLigA (+) were incubated at 37°C for 30 min. The reactions were quenched with formamide, EDTA. The products were analyzed by urea-PAGE and visualized by autoradiography.
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Figure 8: The cap pyrophosphate prevents nick sealing by DNA ligases. Left panel: Chlorella virus DNA ligase reaction mixtures (10 μl) containing 50 mM Tris–HCl, pH 7.5, 5 mM DTT, 10 mM MgCl2, 1 mM ATP, 0.4 pmol 5′ 32P-labeled pDNAp(dG)OH or pDNApp(rG)OH nicked duplex (shown below with the 32P-label denoted by •) and either no enzyme (−) or 1 pmol ChVLig (+) were incubated at 37°C for 30 min. Right panel: Escherichia coli DNA ligase reaction mixtures (10 μl) containing 50 mM Tris–HCl, pH 7.5, 5 mM DTT, 5 mM MgCl2, 1 mM NAD+, 10 mM (NH4)2SO4, 0.4 pmol pDNAp(dG)OH or pDNApp(rG)OH nicked duplex, and either no enzyme (−) or 1 pmol EcoLigA (+) were incubated at 37°C for 30 min. The reactions were quenched with formamide, EDTA. The products were analyzed by urea-PAGE and visualized by autoradiography.

Mentions: Chlorella virus DNA ligase (ChVLig) and E. coli DNA ligase (EcoLigA) are structurally and biochemically well-characterized exemplars of the ATP-dependent and NAD+-dependent clades of nick-sealing DNA ligases (20,21). We reacted ChVLig and EcoLigA with singly nicked DNAs prepared by annealing 21-mer 5′ 32P-labeled pDNAp(dG)OH or pDNApp(rG)OH strands and an unlabeled 10-mer pDNAOH strand to a complementary 31-mer DNA strand to form otherwise identical substrates with either a phosphodiester or pyrophosphate linkage preceding the nick 3′-OH nucleoside (Figure 8). Whereas 83% and 81% of the input 5′ 32P-labeled pDNAp(dG)OH strand were ligated by ChVLig and EcoLigA, respectively, there was no detectable sealing of the 32P-labeled pDNApp(rG)OH strand (Figure 8). Because it is well established that ChVLig and EcoLigA are adept at sealing nicks containing a 3′-OH ribonucleotide (21), we considered it unlikely that the general inability to ligate a DNAppGOH strand to a 5′-PO4 strand had to do with the cap being a ribonucleotide. However, to consolidate this point, we prepared otherwise identical singly nicked duplexes with 21-mer 5′ 32P-labeled pDNAp(dG)OH or pDNApp(dG)OH strands on the 3′-OH side of the nick. We found that ChVLig and EcoLig sealed 88% of the input 32P-labeled pDNAp(dG)OH strand, but there was no detectable sealing of the 32P-labeled pDNApp(dG)OH strand by either ligase (not shown). We conclude that the pyrophosphate bridge of the 3′ cap precludes 3′-OH/5′-PO4 nick sealing by canonical ATP-dependent and NAD+-dependent DNA ligases.


Effects of DNA3'pp5'G capping on 3' end repair reactions and of an embedded pyrophosphate-linked guanylate on ribonucleotide surveillance.

Chauleau M, Das U, Shuman S - Nucleic Acids Res. (2015)

The cap pyrophosphate prevents nick sealing by DNA ligases. Left panel: Chlorella virus DNA ligase reaction mixtures (10 μl) containing 50 mM Tris–HCl, pH 7.5, 5 mM DTT, 10 mM MgCl2, 1 mM ATP, 0.4 pmol 5′ 32P-labeled pDNAp(dG)OH or pDNApp(rG)OH nicked duplex (shown below with the 32P-label denoted by •) and either no enzyme (−) or 1 pmol ChVLig (+) were incubated at 37°C for 30 min. Right panel: Escherichia coli DNA ligase reaction mixtures (10 μl) containing 50 mM Tris–HCl, pH 7.5, 5 mM DTT, 5 mM MgCl2, 1 mM NAD+, 10 mM (NH4)2SO4, 0.4 pmol pDNAp(dG)OH or pDNApp(rG)OH nicked duplex, and either no enzyme (−) or 1 pmol EcoLigA (+) were incubated at 37°C for 30 min. The reactions were quenched with formamide, EDTA. The products were analyzed by urea-PAGE and visualized by autoradiography.
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Related In: Results  -  Collection

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Show All Figures
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Figure 8: The cap pyrophosphate prevents nick sealing by DNA ligases. Left panel: Chlorella virus DNA ligase reaction mixtures (10 μl) containing 50 mM Tris–HCl, pH 7.5, 5 mM DTT, 10 mM MgCl2, 1 mM ATP, 0.4 pmol 5′ 32P-labeled pDNAp(dG)OH or pDNApp(rG)OH nicked duplex (shown below with the 32P-label denoted by •) and either no enzyme (−) or 1 pmol ChVLig (+) were incubated at 37°C for 30 min. Right panel: Escherichia coli DNA ligase reaction mixtures (10 μl) containing 50 mM Tris–HCl, pH 7.5, 5 mM DTT, 5 mM MgCl2, 1 mM NAD+, 10 mM (NH4)2SO4, 0.4 pmol pDNAp(dG)OH or pDNApp(rG)OH nicked duplex, and either no enzyme (−) or 1 pmol EcoLigA (+) were incubated at 37°C for 30 min. The reactions were quenched with formamide, EDTA. The products were analyzed by urea-PAGE and visualized by autoradiography.
Mentions: Chlorella virus DNA ligase (ChVLig) and E. coli DNA ligase (EcoLigA) are structurally and biochemically well-characterized exemplars of the ATP-dependent and NAD+-dependent clades of nick-sealing DNA ligases (20,21). We reacted ChVLig and EcoLigA with singly nicked DNAs prepared by annealing 21-mer 5′ 32P-labeled pDNAp(dG)OH or pDNApp(rG)OH strands and an unlabeled 10-mer pDNAOH strand to a complementary 31-mer DNA strand to form otherwise identical substrates with either a phosphodiester or pyrophosphate linkage preceding the nick 3′-OH nucleoside (Figure 8). Whereas 83% and 81% of the input 5′ 32P-labeled pDNAp(dG)OH strand were ligated by ChVLig and EcoLigA, respectively, there was no detectable sealing of the 32P-labeled pDNApp(rG)OH strand (Figure 8). Because it is well established that ChVLig and EcoLigA are adept at sealing nicks containing a 3′-OH ribonucleotide (21), we considered it unlikely that the general inability to ligate a DNAppGOH strand to a 5′-PO4 strand had to do with the cap being a ribonucleotide. However, to consolidate this point, we prepared otherwise identical singly nicked duplexes with 21-mer 5′ 32P-labeled pDNAp(dG)OH or pDNApp(dG)OH strands on the 3′-OH side of the nick. We found that ChVLig and EcoLig sealed 88% of the input 32P-labeled pDNAp(dG)OH strand, but there was no detectable sealing of the 32P-labeled pDNApp(dG)OH strand by either ligase (not shown). We conclude that the pyrophosphate bridge of the 3′ cap precludes 3′-OH/5′-PO4 nick sealing by canonical ATP-dependent and NAD+-dependent DNA ligases.

Bottom Line: Human DNA polymerase β (X family) extends the cap primer predominantly by a single templated addition step.Cap-primed synthesis by templated polymerases embeds a pyrophosphate-linked ribonucleotide in DNA.We find that the embedded ppG is refractory to surveillance and incision by RNase H2.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA.

Show MeSH
Related in: MedlinePlus