Changes in DNA superhelicity during DNA replication are mediated primarily by the activities of DNA helicases and topoisomerases. If these activities are defective, the progression of the replication fork can be hindered or blocked, which can lead to double-strand breaks, elevated recombination in regions of repeated DNA, and genome instability. Hereditary diseases like Werner's and Bloom's Syndromes are caused by defects in DNA helicases, and these diseases are associated with genome instability and carcinogenesis in humans. Here we report a Saccharomyces cerevisiae gene, MGS1 (Maintenance of Genome Stability 1), which encodes a protein belonging to the AAA ؉ class of ATPases, and whose central region is similar to Escherichia coli RuvB, a Holliday junction branch migration motor protein. The Mgs1 orthologues are highly conserved in prokaryotes and eukaryotes. The Mgs1 protein possesses DNA-dependent ATPase and single-strand DNA annealing activities. An mgs1 deletion mutant has an elevated rate of mitotic recombination, which causes genome instability. The mgs1 mutation is synergistic with a mutation in top3 (encoding topoisomerase III), and the double mutant exhibits severe growth defects and markedly increased genome instability. In contrast to the mgs1 mutation, a mutation in the sgs1 gene encoding a DNA helicase homologous to the Werner and Bloom helicases suppresses both the growth defect and the increased genome instability of the top3 mutant. Therefore, evolutionarily conserved Mgs1 may play a role together with RecQ family helicases and DNA topoisomerases in maintaining proper DNA topology, which is essential for genome stability.

The RecQ protein family is a highly conserved group of DNA helicases that play roles in maintaining genomic stability. In this study, we present biochemical and genetic evidence that Escherichia coli RecQ processes stalled replication forks and participates in SOS signaling. Cells that carry dnaE486, a mutation in the DNA polymerase III ␣-catalytic subunit, induce an RecA-dependent SOS response and become highly filamented at the semirestrictive temperature (38°C). An recQ mutation suppresses the induction of SOS response and the filamentation in the dnaE486 mutant at 38°C, causing appearance of a high proportion of anucleate cells. In vitro, RecQ binds and unwinds forked DNA substrates with a gap on the leading strand more efficiently than those with a gap on the lagging strand or Holliday junction DNA. RecQ unwinds the template duplex ahead of the fork, and then the lagging strand is unwound. Consequently, this process generates a single-stranded DNA (ssDNA) gap on the lagging strand adjacent to a replication fork. These results suggest that RecQ functions to generate an initiating signal that can recruit RecA for SOS induction and recombination at stalled replication forks, which are required for the cell cycle checkpoint and resumption of DNA replication.[Keywords: RecQ DNA helicase; DNA replication; SOS induction; DNA homologous recombination] Supplemental material is available at http://www.genesdev.org.

Saccharomyces cerevisiae Mgs1 protein, which possesses DNA-dependent ATPase and single strand DNA annealing activities, plays a role in maintaining genomic stability. We found that mgs1 is synthetic lethal with rad6 and exhibits a synergistic growth defect with rad18 and rad5, which are members of the RAD6 epistasis group important for tolerance of DNA damage during DNA replication. The mgs1 mutant is not sensitive to DNA-damaging agents, but the mgs1 rad5 double mutant has increased sensitivity to hydroxyurea and a greatly increased spontaneous mutation rate. Growth defects of mgs1 rad18 double mutants are suppressed by a mutation in SRS2, encoding a DNA helicase, or by overexpression of Rad52. Moreover, mgs1 mutation suppresses the temperature sensitivity of mutants in POL3, encoding DNA polymerase d. mgs1 also suppresses the growth defect of a pol3 mutant caused by expression of Escherichia coli RuvC, a bacterial Holliday junction resolvase. These ®ndings suggest that Mgs1 is essential for preventing genome instability caused by replication fork arrest in cells de®cient in the RAD6 pathway and may modulate replication fork movement catalyzed by yeast polymerase d.

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Proliferating cell nuclear antigen (PCNA), a sliding clamp required for processive DNA synthesis, provides attachment sites for various other proteins that function in DNA replication, DNA repair, cell cycle progression and chromatin assembly. It has been shown that differential posttranslational modifications of PCNA by ubiquitin or SUMO play a pivotal role in controlling the choice of pathway for rescuing stalled replication forks. Here, we explored the roles of Mgs1 and PCNA in replication fork rescue. We provide evidence that Mgs1 physically associates with PCNA and that Mgs1 helps suppress the RAD6 DNA damage tolerance pathway in the absence of exogenous DNA damage. We also show that PCNA sumoylation inhibits the growth of mgs1 rad18 double mutants, in which PCNA sumoylation and the Srs2 DNA helicase coordinately prevent RAD52-dependent homologous recombination. The proposed roles for Mgs1, Srs2, and modified PCNA during replication arrest highlight the importance of modulating the RAD6 and RAD52 pathways to avoid genome instability.Progression of the replication fork is often impeded by DNA lesions caused by exogenous or endogenous DNA-damaging agents. Replication forks also stall when they encounter tightly bound proteins or aberrant DNA structures (7). Stalled replication forks activate the DNA damage tolerance pathway, which promotes the reinitiation of DNA synthesis with or without removing the replication-blocking lesion.Genetic studies in Saccharomyces cerevisiae indicate that Rad6 and Rad18 play central roles in DNA damage tolerance pathway. This pathway is mediated by the protein products of RAD6, RAD18, RAD5, MMS2, and UBC13, as well as several other gene products (4, 9, 22). Rad6 is a ubiquitin E2-conjugating enzyme that forms a stable complex with Rad18, an E3 ligase that binds DNA (2, 3). The Ubc13-Mms2 heterodimer is also an E2-conjugating enzyme (14). Rad5 is a DNA-dependent ATPase that functions as an E3 ligase and associates with the Ubc13-Mms2 complex, recruiting this complex to chromatin in response to DNA damage (27). Previous study demonstrated that PCNA is a substrate for RAD6-dependent ubiquitination (13). When DNA synthesis on one or both strands is arrested by DNA damage, PCNA is mono-ubiquitinated on lysine 164 (Lys164) by the Rad6-Rad18 complex. Mono-ubiquitinated PCNA may target stalled replication forks to initiate error-prone DNA repair via translesion DNA synthesis, a process that requires low-fidelity polymerases, Pol or Pol, to synthesize across the damage (10,15,25,29). Alternatively, the Ubc13-Mms2 complex and Rad5 modulate the polyubiquitination of Lys164 through lysine 63-linked ubiquitin chains. When modified in this manner, PCNA promotes error-free DNA repair (13,25).PCNA is also sumoylated at Lys164 (and secondarily at Lys127) via a mechanism dependent on a distinct E2-conjugating enzyme, Ubc9, and an E3 ligase, Siz1 (13). Although ubiquitination and sumoylation both occur on Lys164, there is evidence suggesting that the two modifications do not appear to have antagon...