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

Abstract

DNA damage that escapes repair and blocks replicative DNA polymerases is tolerated by bypass mechanisms that fall into two general categories: error-free template switching and error-prone translesion synthesis. Prior studies of DNA damage responses in Saccharomyces cerevisiae have demonstrated that repair mechanisms are critical for survival when a single, high dose of DNA damage is delivered, while bypass/tolerance mechanisms are more important for survival when the damage level is low and continuous (acute and chronic damage, respectively). In the current study, epistatic interactions between DNA-damage tolerance genes were examined and compared when haploid yeast cells were exposed to either chronic ultraviolet light or chronic methyl methanesulfonate. Results demonstrate that genes assigned to error-free and error-prone bypass pathways similarly promote survival in the presence of each type of chronic damage. In addition to using defined sources of chronic damage, rates of spontaneous mutations generated by the Pol z translesion synthesis DNA polymerase (complex insertions in a frameshift-reversion assay) were used to infer epistatic interactions between the same genes. Similar epistatic interactions were observed in analyses of spontaneous mutation rates, suggesting that chronic DNA-damage responses accurately reflect those used to tolerate spontaneous lesions. These results have important implications when considering what constitutes a safe and acceptable level of exogenous DNA damage. D AMAGE to cellular DNA that results from normal metabolic processes is classified as spontaneous, while that resulting from exogenous physical or chemical agents is considered induced. Spontaneous damage occurs at low, continuous levels and hence is chronic in nature. Though induced damage is typically delivered in a single, acute dose, it can also be chronic, with examples including daily exposure to solar radiation or cigarette smoke. Spontaneous and induced damage are dealt with by two general, evolutionarily conserved mechanisms: one that permanently removes the damage and one that temporarily bypasses the damage, allowing it to be tolerated (for a general overview, see Ciccia and Elledge 2010). Most damage removal occurs via conserved excision repair pathways, but a small number of damages can be directly reversed enzymatically (e.g., thymine-thymine dimer reversal by photolyase). Damage removal by excision repair generates a single-strand gap that is filled using the complementary, undamaged strand as a template and hence is a high-fidelity process. Such repair only operates, however, when a relevant lesion is present in double-strand DNA. Damage that escapes repair and is encountered during replication can block the progress of DNA synthesis, leading to the formation of a single-strand gap opposite the lesion, replication fork collapse, cell-cycle arrest, and/or death. DNA-damage bypass/tolerance pathways thus become critically important during S and G2 phases, as these allow completion of replication and ...

Spatiotemporal regulation of PCNA ubiquitination in damage tolerance pathways

“…However, these observations do not indicate that DDT pathways primarily operate in G2 phase in normal cells. Instead, PCNA ubiquitination-mediated HDR appears to operate predominantly in S phase even behind replication forks (Karras and Jentsch 2010;Huang et al 2013;Lehner and Jinks-Robertson 2014). Other studies have demonstrated that RAD5-deficient cells challenged with relatively high doses of DNA-damaging agents progress slowly through S phase or fail to complete S phase with an accumulation of replication intermediates.…”

“…In S. cerevisiae, many studies have shown that a deficiency in PCNA ubiquitination-mediated DDT does not impede bulk replication of genomes containing damaged DNA but instead induces a G2 phase cell cycle arrest via checkpoint activation, suggesting that PCNA ubiquitination-mediated DDT is involved in the repair of gaps generated during replication of lesion-containing genomic DNA. This phenotype of DDT-deficient cells appears when cells are challenged with relatively low doses of DNA-damaging agents (Hishida et al 2009;Daigaku et al 2010;Karras and Jentsch 2010;Huang et al 2013;Lehner and Jinks-Robertson 2014). Under such conditions, genetically manipulated yeast strains in which PCNA is ubiquitinated or TLS pols are expressed only in G2, but not in S phase, tolerate damaging agents as well as the wildtype strain, demonstrating that the replication fork structure is not required for the operation of TLS or HDR, and instead that the gaps left behind the replication fork are the substrates for TLS and HDR (Daigaku et al 2010;Karras and Jentsch 2010).…”

“…The molecular mechanisms controlling pathway choice remain highly elusive. In S. cerevisiae, defects in PCNA ubiquitination-mediated HDR are generally rescued by the remaining TLS activity and vice versa, suggesting that TLS and PCNA ubiquitination-mediated HDR are interchangeable (Broomfield et al 1998;Torres-Ramos et al 2002;Karras and Jentsch 2010;Huang et al 2013;Lehner and Jinks-Robertson 2014;Ortiz-Baz an et al 2014). However, some studies demonstrated that one pathway is dominant and that defects due to its absence cannot be rescued by the remaining pathway, suggesting that TLS and PCNA ubiquitination-mediated HDR are not interchangeable in those cases.…”

Regulation of HLTF-mediated PCNA polyubiquitination by RFC and PCNA monoubiquitination levels determines choice of damage tolerance pathway

“…The possibility that TS precedes TLS was proposed based on experiments in which cells exposed to acute methyl methanesulfonate (MMS) treatment (0.033%, 30 min) were released into S phase ( 23 ). However, another study with CLUV showed a synergistic effect in TLS- and TS-deficient mutants, indicating that TLS and TS are interchangeable for survival ( 24 ). Under exposure to low-dose MMS (0.001%), cells have a preference for TS, which operates earlier, whereas TLS is executed later.…”

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.…”