Defects in DNA Lesion Bypass Lead to Spontaneous Chromosomal Rearrangements and Increased Cell Death

Abstract

Rev3 polymerase and Mph1 DNA helicase participate in error-prone and error-free pathways, respectively, for the bypassing of template lesions during DNA replication. Here we have investigated the role of these pathways and their genetic interaction with recombination factors, other nonreplicative DNA helicases, and DNA damage checkpoint components in the maintenance of genome stability, viability, and sensitivity to the DNA-damaging agent methyl methanesulfonate (MMS). We find that cells lacking Rev3 and Mph1 exhibit a synergistic, Srs2-dependent increase in the rate of accumulating spontaneous, gross chromosomal rearrangements, suggesting that the suppression of point mutations by deletion of REV3 may lead to chromosomal rearrangements. While mph1⌬ is epistatic to homologous recombination (HR) genes, both Rad51 and Rad52, but not Rad59, are required for normal growth of the rev3⌬ mutant and are essential for survival of rev3⌬ cells during exposure to MMS, indicating that Mph1 acts in a Rad51-dependent, Rad59-independent subpathway of HR-mediated lesion bypass. Deletion of MPH1 helicase leads to synergistic DNA damage sensitivity increases in cells with chl1⌬ or rrm3⌬ helicase mutations, whereas mph1⌬ is hypostatic to sgs1⌬. Previously reported slow growth of mph1⌬ srs2⌬ cells is accompanied by G 2 /M arrest and fully suppressed by disruption of the Mec3-dependent DNA damage checkpoint. We propose a model for replication fork rescue mediated by translesion DNA synthesis and homologous recombination that integrates the role of Mph1 in unwinding D loops and its genetic interaction with Rev3 and Srs2-regulated pathways in the suppression of spontaneous genome rearrangements and in mutation avoidance.Nonreplicative DNA helicases play an important role in the maintenance of genome stability from bacteria to humans, most likely by affecting the formation and/or resolution of recombination intermediates and by facilitating replication fork progression through chromosomal regions with a propensity to adopt unusual DNA structures or those bound by proteins. In Saccharomyces cerevisiae, this group of DNA helicases includes the 3Ј-to-5Ј helicases Sgs1 and Srs2 and the 5Ј-to-3Ј DNA helicase Rrm3. In the absence of any two of these three helicases, unresolved recombination intermediates accumulate and lead to extremely slow growth that is fully suppressed by deletion of genes encoding early homologous recombination (HR) factors (4,6,17,20,37,46). In the absence of Sgs1, cells exhibit increased rates of mitotic recombination, frequent chromosome missegregation, accumulation of extrachromosomal ribosomal DNA (rDNA) circles, and increased rates of gross chromosomal rearrangements (GCRs) involving nonhomologous chromosomes (5,24,25,38,40,43,49,50). Based on the increased crossover frequency during HO endonucleaseinduced double-strand breaks (DSBs) in cells lacking Sgs1, it has also been proposed that Sgs1 may function in decatenation of Holliday junctions (HJs) to yield noncrossovers (12,22). Like Sgs1, Srs2 acts to favor noncro...

A Novel Rrm3 Function in Restricting DNA Replication via an Orc5-Binding Domain Is Genetically Separable from Rrm3 Function as an ATPase/Helicase in Facilitating Fork Progression

“…Whereas deletions of MGM101 , HDA1 or SET3 had no effect on sensitivity of wildtype or rrm3Δ cells to HU or MMS (S2 Fig), deletion of MPH1 caused a synergistic increase in HU and MMS sensitivity of the rrm3Δ mutant (S3 Fig), consistent with our previous finding [39]. In the absence of Mph1, rrm3Δ cells progressed very slowly through an undisturbed cell cycle and accumulated in G2/M when they were released from a 2-hour incubation in 100 mM HU (S3B and S3C Fig).…”

The NuA4 Complex Promotes Translesion Synthesis (TLS)-Mediated DNA Damage Tolerance

“…The migration patterns of cells lacking REV3 look very similar to wild type (Figure 2, C and D) and consistent with rev3D mutants having a similar rate of chromosomal rearrangements compared to wild type (Schmidt et al 2010). However, there was a measurable defect in the recovery of ubc13D cells (Figure 2, C-F), which is consistent with error-free being the dominant pathway for lesion bypass during S phase (Huang et al 2013).…”

Rad5-dependent DNA Repair Functions of the Saccharomyces cerevisiae FANCM Protein Homolog Mph1

“…In particular, the DNA helicase Srs2 shows a synergistic 4 interaction with Mph1 when double mutants are challenged with the DNA alkylation agent methyl methanesulfonate (MMS) (42)(43)(44)(45)(46). We hypothesized that redundancy with Srs2 might mask the ICL repair functions of Mph1 and Chl1.…”