Homologous recombination (HR) and nonhomologous end joining (NHEJ) play overlapping roles in repair of DNA double-strand breaks (DSBs) generated during the S phase of the cell cycle. Here, we characterized the involvement of HR and NHEJ in the rescue of DNA replication forks arrested or slowed by treatment of hamster cells with hydroxyurea or thymidine. We show that the arrest of replication with hydroxyurea generates DNA fragmentation as a consequence of the formation of DSBs at newly replicated DNA. Both HR and NHEJ protected cells from the lethal effects of hydroxyurea, and this agent also increased the frequency of recombination mediated by both homologous and nonhomologous exchanges. Thymidine induced a less stringent arrest of replication and did not generate detectable DSBs. HR alone rescued cells from the lethal effects of thymidine. Furthermore, thymidine increased the frequency of DNA exchange mediated solely by HR in the absence of detectable DSBs. Our data suggest that both NHEJ and HR are involved in repair of arrested replication forks that include a DSB, while HR alone is required for the repair of slowed replication forks in the absence of detectable DSBs.

Base excision repair (BER) represents the most important repair pathway of endogenous DNA lesions. Initially, a base damage is recognized, excised and a DNA single-strand break (SSB) intermediate forms. The SSB is then ligated, a process that employs proteins also involved in SSB repair, e.g. XRCC1, Ligase III and possibly PARP1. Here, we confirm the role of XRCC1 and PARP in direct SSB repair. Interestingly, we uncover a synthetic lethality between XRCC1 deficiency and PARP inhibition. We also treated cells with alkylating agent dimethyl sulfate (DMS) and monitored the SSB intermediates formed during BER. DMS-induced SSBs were quickly repaired in wild-type cells; while a rapid accumulation of SSBs was observed in cells where post-incision repair was blocked by a PARP inhibitor or by XRCC1 deficiency (EM9 cells). Interestingly, DMS-induced SSBs did not accumulate in PARP1 siRNA depleted cells, demonstrating that PARP1 is not required for efficient completion of BER. Based on these results we suggest no immediate role for PARP1 in BER, but that PARP inhibitors trap PARP on the SSB intermediate formed during BER. Unexpectedly, addition of PARP inhibitor 2 h after DMS treatment still increased SSB levels indicating ongoing repair even at this late time point.

Homologous recombination (HR) deficient cells are sensitive to methyl methanesulfonate (MMS). HR is usually involved in the repair of DNA double-strand breaks (DSBs) in Saccharomyces cerevisiae implying that MMS somehow induces DSBs in vivo. Indeed there is evidence, based on pulsed-field gel electrophoresis (PFGE), that MMS causes DNA fragmentation. However, the mechanism through which MMS induces DSBs has not been demonstrated. Here, we show that DNA fragmentation following MMS treatment, and detected by PFGE is not the consequence of production of cellular DSBs. Instead, DSBs seen following MMS treatment are produced during sample preparation where heat-labile methylated DNA is converted into DSBs. Furthermore, we show that the repair of MMS-induced heat-labile damage requires the base excision repair protein XRCC1, and is independent of HR in both S.cerevisiae and mammalian cells. We speculate that the reason for recombination-deficient cells being sensitive to MMS is due to the role of HR in repair of MMS-induced stalled replication forks, rather than for repair of cellular DSBs or heat-labile damage.

The Y family DNA polymerase Rev1 has been proposed to play a regulatory role in the replication of damaged templates. To elucidate the mechanism by which Rev1 promotes DNA damage bypass, we have analyzed the progression of replication on UV light-damaged DNA in mouse embryonic fibroblasts that contain a defined deletion in the N-terminal BRCT domain of Rev1 or that are deficient for Rev1. We provide evidence that Rev1 plays a coordinating role in two modes of DNA damage bypass, i.e., an early and a late pathway. The cells carrying the deletion in the BRCT domain are deficient for the early pathway, reflecting a role of the BRCT domain of Rev1 in mutagenic translesion synthesis. Rev1-deficient cells display a defect in both modes of DNA damage bypass. Despite the persistent defect in the late replicational bypass of fork-blocking (6-4)pyrimidine-pyrimidone photoproducts, overall replication is not strongly affected by Rev1 deficiency. This results in almost completely replicated templates that contain gaps encompassing the photoproducts. These gaps are inducers of DNA damage signaling leading to an irreversible G 2 arrest. Our results corroborate a model in which Rev1-mediated DNA damage bypass at postreplicative gaps quenches irreversible DNA damage responses.Unrepaired DNA damage usually leads to an arrest of replicative polymerases. Nonetheless, prokaryotic and eukaryotic cells display progression of replication on damaged templates, allowing replication to be completed and averting replication fork collapse (12). Direct bypass of the fork-blocking lesion, permitting replication to proceed with little delay, would be an attractive mechanism to release an arrested replicon. Several early studies using bacteria and mammalian cells, however, have indicated that replicating cells exposed to UV light initially synthesize smaller DNA strands than undamaged cells (reviewed in reference 32). At a later stage, these molecules are converted into DNA of high molecular weight, possibly via late, postreplicative, filling of the lesion-containing gap by socalled postreplication repair. The presence of single-stranded gaps in both sister chromatids behind a replication fork was recently visualized by electron microscopy on DNA of UVexposed budding yeast Saccharomyces cerevisiae (34).Damage avoidance and translesion synthesis are two pathways that allow cells to replicate damaged templates (3, 12). Damage avoidance (also called template switching-dependent synthesis) uses the undamaged sister chromatid as a template. This can be achieved by replication fork regression or by strand invasion with the sister chromatid and results in error-free bypass of DNA lesions (7, 65). Translesion synthesis, on the other hand, is characterized by insertion of a nucleotide opposite the lesion by specialized DNA polymerases of the Y family (48). The reduced stringency of the active site and a lack of proofreading activity of translesion synthesis polymerases imply that translesion synthesis is an inherently mutagenic process.The Y family poly...

Restarting stalled replication forks is vital to avoid fatal replication errors. Previously, it was demonstrated that hydroxyurea-stalled replication forks rescue replication either by an active restart mechanism or by new origin firing. To our surprise, using the DNA fibre assay, we only detect a slightly reduced fork speed on a UV-damaged template during the first hour after UV exposure, and no evidence for persistent replication fork arrest. Interestingly, no evidence for persistent UV-induced fork stalling was observed even in translesion synthesis defective, Polηmut cells. In contrast, using an assay to measure DNA molecule elongation at the fork, we observe that continuous DNA elongation is severely blocked by UV irradiation, particularly in UV-damaged Polηmut cells. In conclusion, our data suggest that UV-blocked replication forks restart effectively through re-priming past the lesion, leaving only a small gap opposite the lesion. This allows continuation of replication on damaged DNA. If left unfilled, the gaps may collapse into DNA double-strand breaks that are repaired by a recombination pathway, similar to the fate of replication forks collapsed after hydroxyurea treatment.

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