Replisome-mediated translesion synthesis and leading strand template lesion skipping are competing bypass mechanisms

Abstract

A number of different enzymatic pathways have evolved to ensure that DNA replication can proceed past template base damage. These pathways include lesion skipping by the replisome, replication fork regression followed by either correction of the damage and origin-independent replication restart or homologous recombination-mediated restart of replication downstream of the lesion, and bypass of the damage by a translesion synthesis DNA polymerase. We report here that of two translesion synthesis polymerases tested, only DNA polymerase IV, not DNA polymerase II, could engage productively with the Escherichia coli replisome to bypass leading strand template damage, despite the fact that both enzymes are shown to be interacting with the replicase. Inactivation of the 3' → 5' proofreading exonuclease of DNA polymerase II did not enable bypass. Bypass by DNA polymerase IV required its ability to interact with the β clamp and act as a translesion polymerase but did not require its "little finger" domain, a secondary region of interaction with the β clamp. Bypass by DNA polymerase IV came at the expense of the inherent leading strand lesion skipping activity of the replisome, indicating that they are competing reactions.

Keywords: DNA Damage; DNA Polymerase; DNA Repair; DNA Replication; Genomic Instability.

FIGURE 1.

TLS bypass of THF and CPD lesions by polymerases on oligonucleotide primer templates. The indicated concentrations of DNA polymerases were incubated with the CPD ([5′-³²P]CG18:CG17CPD), THF ([5′-³²P]CG18:CG17THF), and undamaged ([5′-³²P]CG18:CG16) primer templates for 10 min at 37 °C, and the DNA products were processed and analyzed as described under “Experimental Procedures.” The white asterisks correspond to the position of the CPD or THF lesion, accordingly, in the template strand. P denotes the position of the unextended primer. The extent of bypass (mean and standard deviation from three experiments) is given as the percentage of total radioactivity in extended primer species. Representative gels are shown. A, Pol IV bypasses both a THF and CPD lesion. B, Pol II bypasses a THF, but not a CPD, lesion. C, Pol III* does not bypass either a THF or CPD lesion. D, inactivation of the 3′ → 5′ proofreading exonuclease of Pol II stimulates TLS. E, Pol IV ΔC5, but not Pol IV F13V, bypasses a CPD lesion. F, Pol IV ΔC5, but not Pol IV F13V, bypasses a THF lesion. G, Pol IV F13V is active on an undamaged primer template. H, Pol IV L1 (LF domain variant) is active in TLS. Representative gels are shown.

FIGURE 2.

Increasing deoxynucleoside triphosphate concentration stimulates TLS bypass by Pol IV and II on oligonucleotide primer templates. Either Pol IV or Pol II were incubated with the CPD, THF, and undamaged primer templates for 10 min at 37 °C at the indicated concentration of dNTPs (increasing by a factor of 2 from left to right), and the DNA products were processed and analyzed as described under “Experimental Procedures.” The white asterisks correspond to the position of the CPD or THF lesion, accordingly, in the template strand. P denotes the position of the unextended primer. The extent of bypass (mean and standard deviation from three experiments) is given as the percentage of total radioactivity in extended primer species. A representative gel is shown.

FIGURE 3.

Pol IV, but not Pol II, catalyzes replisome-mediated TLS. A, DNA template. Panel i, map showing the positions of the leading strand template lesion relative to the origin of DNA replication, the Ter sites, and the relevant restriction sites used for analysis. Panel ii, DNA sequence about the site of the template lesion. B, replication reaction scheme showing the possible replicated DNA products. C, elevated nucleoside triphosphate concentration is required for Pol IV-catalyzed, replisome-mediated TLS on a THF template. Standard gyrase reactions containing the indicated TLS polymerase (100 nm Pol IV, 20 nm Pol II) and concentrations of dNTPS were incubated, processed, and analyzed as described under “Experimental Procedures.” D, Pol IV, but not Pol II, catalyzes replisome-mediated TLS. Standard gyrase replication reactions containing the THF template and the indicated concentrations of TLS polymerase, standard concentrations of dCTP and TTP, and 750 μm dATP and dGTP were incubated, processed, and analyzed as described under “Experimental Procedures.” E, Pol IV L1 (LF domain variant), but not Pol II D155A/E157A (the 3′ → 5′ exonuclease-defective variant), catalyzes replisome-mediated TLS. Standard gyrase reactions containing the indicated TLS polymerase (100 nm), standard concentrations of dCTP and TTP, and 750 μm dATP and dGTP were incubated, processed, and analyzed as described under “Experimental Procedures.” RC, greater than unit length, rolling circle DNA products that arise from nicks in the DNA template; FL, full-length nascent leading strand spanning the PvuI to EcoRI sites; stall, the nascent leading strand stall product spanning the distance from the PvuI site to the site of the template lesion; restart, nascent leading strands restarted downstream of the damage by leading strand lesion skipping; OF, Okazaki fragments. The extent of bypass in D, lanes 1–7, was (calculated as FL/FL + stall) 0.04 ± 0.01, 0.03 ± 0.01, 0.03 ± 0.01, 0.12 ± 0.04, 0.14 ± 0.03, 0.19 ± 0.04, and 0.03 ± 0.01, respectively (mean and standard deviation from four experiments). The extent of bypass in E, lanes 1 and 2, was 0.24 ± 0.1 and 0.1 ± 0.03, respectively (mean and standard deviation from three experiments). Representative gels are shown.

FIGURE 4.

Replisome-mediated Pol IV-catalyzed TLS bypass requires interaction with β and TLS activity. Standard gyrase replication reactions containing either the CPD (A) or THF template (B), the indicated TLS polymerase at 100 nm, standard concentrations of dCTP and TTP, and 750 μm dATP and dGTP were incubated, processed, and analyzed as described under “Experimental Procedures.” ΔC5, Pol IV ΔC5; F13V, Pol IV F13V. Other abbreviations are as defined in the legend to Fig. 3.

FIGURE 5.

Nucleotide requirements for Pol IV-catalyzed, replisome-mediated TLS bypass. A and B, standard gyrase replication reactions containing either the CPD template (A) or the THF template (B), and the indicated elevated concentrations (0.75 mm) of dATP, dGTP, or TTP were incubated in either the presence or absence of Pol IV (100 nm), processed, and analyzed as described under “Experimental Procedures.” C, quantification of TLS bypass for reactions containing Pol IV. Given are the means and standard deviations from three experiments. Representative gels are shown. Abbreviations are as defined in the legend to Fig. 3.

FIGURE 6.

Pol IV-catalyzed, replisome-mediated TLS bypass generates a −1 frameshift on the THF template. A, scheme for analyzing the nascent DNA products. B, a THF lesion inhibits digestion of DNA by the PsiI restriction endonuclease. Panel i, schematic of the two duplex DNA oligonucleotides used as substrates. The DNA sequence is identical to that about the site of the lesion in the template DNA used for replication. Panel ii, different combinations of oligonucleotides were treated with PsiI as indicated, and the DNA products were analyzed by denaturing polyacrylamide gel electrophoresis as described under “Experimental Procedures.” An asterisk on an oligonucleotide name denotes that it was 5′-[³²P] end-labeled. C, leading strand replication is contiguous across the site of the template lesion. DNA products generated by replication with either the undamaged, CPD, or THF template either in the presence or absence of Pol IV (100 nm) and the presence or absence of elevated concentrations (0.75 mm) of dATP and dGTP were digested with DrdI and Acc65I either with or without digestion with PsiI as indicated, processed, and analyzed by electrophoresis through a denaturing polyacrylamide gel as described under “Experimental Procedures.” RF, replicative form DNA. D, Pol IV-catalyzed, replisome-mediated TLS bypass generates a −1 frameshift on the THF template. DNA products generated as described for C in the presence of Pol IV (100 nm), and elevated nucleotide concentrations (0.75 mm) were digested with DrdI and Acc65I and analyzed by electrophoresis through a high resolution denaturing 6% polyacrylamide gel (19:1, acrylamide:bisacrylamide) as described under “Experimental Procedures.” T, C, G, and A show a DNA sequencing ladder prepared from undamaged DNA using a primer that has the same 5′ end as the DrdI-digested nascent leading strand DNA. For reasons that are unclear, migration of the digested nascent leading strand from the CPD template was somewhat variable compared with the product from the undamaged template; it could have the same mobility or be a bit slower. We think this could be because of differences in loading volumes necessitated by the differences in the extent of replication with the two templates. E, leading strand replication stalls just 5′ of the template lesion. DNA products generated using either the THF or CPD templates, either in the presence or absence of Pol IV (100 nm) and either in the presence or absence of elevated concentrations (0.75 mm) of dATP and dGTP were prepared and analyzed as in D except they were digested only with DrdI.

FIGURE 7.

Stalled nascent leading strand is chased into full-length product during Pol IV-catalyzed, replisome-mediated TLS bypass. A, scheme of the replication reaction. B and C, pulse-chase EcoRI replication reactions containing [α-³²P]dATP using either the THF (B) or CPD (C) template either in the presence or absence of Pol IV (100 nm) were incubated, processed, and analyzed as described under “Experimental Procedures.” Times are post chase. ERI, early replication intermediate; topo, topoisomerase. Other abbreviations are as defined in the legend to Fig. 3.

FIGURE 8.

Pol IV-catalyzed, replisome-mediated TLS bypass occurs at the expense of leading strand lesion skipping. A, EcoRI replication reactions using the THF template either in the presence or absence of elevated concentrations (0.75 mm) of dATP and dGTP and either in the presence or absence of Pol IV (100 nm), as indicated, were incubated, processed, and analyzed as described under “Experimental Procedures.” Times are post EcoRI addition. B and C, gel lane traces prepared by PhosphorImager analysis comparing the 6-min lanes in A for reactions either in the absence (B) or presence (C) of Pol IV either in the presence or absence of elevated concentrations of dATP and dGTP. PSL, photo stimulated luminescence; RC, greater than unit length, rolling circle DNA products that arise from nicks in the DNA template; FL, full-length nascent leading strand spanning the PvuI to EcoRI sites; stall, the nascent leading strand stall product spanning the distance from the PvuI site to the site of the template lesion; restart, nascent leading strands restarted downstream of the damage by leading strand lesion skipping.