Srs2 Plays a Critical Role in Reversible G₂ Arrest upon Chronic and Low Doses of UV Irradiation via Two Distinct Homologous Recombination-Dependent Mechanisms in Postreplication Repair-Deficient Cells

Abstract

. These results indicate that Srs2 plays an important role in checkpoint-mediated reversible G 2 arrest in PRR-deficient cells via two separate HR-dependent mechanisms. The first (required to suppress HR during PRR) is regulated by PCNA sumoylation, whereas the second (required for HR-dependent recovery following CLUV exposure) is regulated by CDK1-dependent phosphorylation.DNA damage occurs frequently in all organisms as a consequence of both endogenous metabolic processes and exogenous DNA-damaging agents. In nature, the steady-state level of DNA damage is usually very low. However, chronic lowlevel DNA damage can lead to age-related genome instability as a consequence of the accumulation of DNA damage (12,27). Increasing evidence implicates DNA damage-related replication stress in genome instability (7,21). Replication stress occurs when an active fork encounters DNA lesions or proteins tightly bound to DNA. These obstacles pose a threat to the integrity of the replication fork and are thus a potential source of genome instability, which can contribute to tumorigenesis and aging in humans (4, 11). Confronted with this risk, cells have developed fundamental DNA damage response mechanisms in order to faithfully complete DNA replication (8).In budding yeast Saccharomyces cerevisiae, the Rad6-dependent postreplication repair (PRR) pathway is subdivided into three subpathways, which allow replication to resume by bypassing the lesion without repairing the damage (3,22,33). Translesion synthesis (TLS) pathways dependent on the DNA polymerases eta and zeta promote error-free or mutagenic bypass depending on the DNA lesion and are activated upon monoubiquitination of proliferating cell nuclear antigen (PCNA) at Lys164 (K164) (5, 16, 37). The Rad5 (E3) and Ubc13 (E2)/Mms2 (E2 variant)-dependent pathway promotes error-free bypass by template switching and is activated by polyubiquitination of PCNA via a Lys63-linked ubiquitin chain (16,38,41). It remains mechanistically unclear how polyubiquitinated PCNA promotes template switching at the molecular level. In addition to its ubiquitin E3 activity, Rad5 also has a helicase domain and was recently shown to unwind and reanneal fork structures in vitro (6). This led to the proposal that Rad5 helicase activity is required at replication forks to promote fork regression and subsequent template switching. It is possible that PCNA polyubiquitination acts to facilitate Rad5-dependent template switching by inhibiting monoubiquitination-dependent TLS activity and/or by recruiting alternative proteins to the fork.In addition to modification by ubiquitin, PCNA can also be sumoylated on Lys164 by the SUMO E3 ligase Siz1 (16). A second sumoylation site, Lys127, is also targeted by an alternative SUMO E3 ligase, Siz2, albeit with lower efficiency (16,30). PCNA SUMO modification results in recruitment of the Srs2 helicase and subsequent inhibition of Rad51-dependent recombination events (29, 32). The modification can therefore allow the replicative bypass of lesions by promoting the RAD...

The Preference for Error-Free or Error-Prone Postreplication Repair in Saccharomyces cerevisiae Exposed to Low-Dose Methyl Methanesulfonate Is Cell Cycle Dependent

“…In their low-dose UV studies, Hishida et al discovered that the UV sensitivity of rad18 cells can be suppressed by deleting the HR inhibitor SRS2 (40). We extend this to show that the inability to repair ssDNA gaps by HR in rad18 cells is due to an SRS2-dependent block; deletion of SRS2 rescues viability in rad18 cells only when HR is active (via GAL-RAD57) (Fig.…”

“…One candidate for this repression is the Srs2 helicase, which has been shown to suppress HR during DNA replication (39). Deletion of SRS2 has been shown to rescue a rad18⌬ mutant after low-dose UV treatment (40). We asked whether a similar effect may occur in low-dose MMS, and especially, whether delayed induction of HR at various time points during the low-dose MMS treatment may be sufficient to rescue rad18⌬ cells in the absence of SRS2.…”

“…Our motivation for providing a formal definition for what constitutes a "low dose" of a DNA-damaging agent stemmed from difficulties we experienced in comparing our data with other studies of low-level exposure that span a variety of dose rates, genotoxic agents, and cells/tissues. For example, in previous studies evaluating the role of PRR in response to low-dose UV treatment, Hishida et al based their choice of UV irradiation dose (0.1 J/m 2 /min) on the natural phenomenon (sunlight exposure) that they were attempting to mimic (4,40); a clear analog for this dose using a chemical carcinogen such as MMS was not immediately apparent. By providing a physiological and agent-agnostic definition of "low-dose" DNA damage based upon the sensitivity of wild-type and mutant cells, we were able to reconcile our MMS results with these previous studies; both 0.1 J/m 2 /min UV exposure and 0.001% MMS treatment fail to induce any discernible cellular sensitivity in a wild-type cell population; however, mutant studies reveal an exquisite and profound dependence on PRR for survival in response to low-dose damage ( Fig.…”

DNA Damage Tolerance Pathway Choice Through Uls1 Modulation of Srs2 SUMOylation in Saccharomyces cerevisiae

“…There is increasing evidence that during unperturbed replication and at very low levels of DNA damage, Rad18-dependent DDT is the pathway of choice, with the error-free Rad5-(and Rad51-) dependent template switch operating during S phase, and error-prone synthesis active mainly on ssDNA gaps in G 2 (Hishida et al 2010;Huang et al 2013;Lehner and Jinks-Robertson 2014). We postulate that this error-free pathway is stimulated by Uls1.…”

Shared Genetic Pathways Contribute to the Tolerance of Endogenous and Low-Dose Exogenous DNA Damage in Yeast

“…The UV dose was attenuated using wire mesh placed between the light source and plates. The experimental UV doses used were below the level of detection of a UV meter (UVX-25, Ultraviolet Products), but the 33 (highest) dose generated results phenotypically similar to those in other studies (Hishida et al 2009(Hishida et al , 2010. Control plates were treated the same, but covered with aluminum foil to prevent UV exposure.…”