The Mechanism of Nucleotide Excision Repair-Mediated UV-Induced Mutagenesis in Nonproliferating Cells

Abstract

Following the irradiation of nondividing yeast cells with ultraviolet (UV) light, most induced mutations are inherited by both daughter cells, indicating that complementary changes are introduced into both strands of duplex DNA prior to replication. Early analyses demonstrated that such two-strand mutations depend on functional nucleotide excision repair (NER), but the molecular mechanism of this unique type of mutagenesis has not been further explored. In the experiments reported here, an ade2 adeX colonycolor system was used to examine the genetic control of UV-induced mutagenesis in nondividing cultures of Saccharomyces cerevisiae. We confirmed a strong suppression of two-strand mutagenesis in NER-deficient backgrounds and demonstrated that neither mismatch repair nor interstrand crosslink repair affects the production of these mutations. By contrast, proteins involved in the error-prone bypass of DNA damage (Rev3, Rev1, PCNA, Rad18, Pol32, and Rad5) and in the early steps of the DNA-damage checkpoint response (Rad17, Mec3, Ddc1, Mec1, and Rad9) were required for the production of two-strand mutations. There was no involvement, however, for the Pol h translesion synthesis DNA polymerase, the Mms2-Ubc13 postreplication repair complex, downstream DNA-damage checkpoint factors (Rad53, Chk1, and Dun1), or the Exo1 exonuclease. Our data support models in which UV-induced mutagenesis in nondividing cells occurs during the Pol z-dependent filling of lesion-containing, NER-generated gaps. The requirement for specific DNA-damage checkpoint proteins suggests roles in recruiting and/or activating factors required to fill such gaps. MUTAGENESIS associated with induced DNA damage is generally considered to occur during S phase, when polymerase-blocking lesions are bypassed in an error-prone manner by a translesion synthesis (TLS) DNA polymerase. In the yeast Saccharomyces cerevisiae, ultraviolet (UV)-induced mutagenesis is dependent on Pol z (Rev3-Rev7) and Rev1, with rev1, rev3, or rev7 mutants exhibiting a "reversionless" phenotype in response to UV irradiation (Lawrence 2002). Just as induced mutagenesis is considered to be a consequence of error-prone TLS, nucleotide excision repair (NER) is thought to counteract such mutagenesis by removing UV-induced lesions before they can be encountered during DNA replication.Although NER is generally considered to be an error-free, mutation-avoidance mechanism, data obtained .30 years ago demonstrated a pro-mutation role of NER in the UVinduced mutagenesis that occurs in nondividing yeast cells (Eckardt and Haynes 1977;James and Kilbey 1977;James et al. 1978;Eckardt et al. 1980). Either the induction of recessive lethal mutations in diploid strains (James and Kilbey 1977;James et al. 1978) or the induction of forward mutations in the de novo adenine biosynthetic pathway in haploid strains was monitored (Eckardt and Haynes 1977;Eckardt et al. 1980). Despite the differences in assay systems used, the authors reached the same conclusions. First, UVinduced mutations in nondi...

Eukaryotic DNA polymerase ζ

“…In addition, the DNA damage checkpoint is involved in efficient mutagenesis under certain conditions. First, the 9-1-1 checkpoint clamp is required for UV-mutagenesis in non-dividing cells [121]. Second, while the 9-1-1 clamp is dispensable for UV-mutagenesis in dividing cells, in nucleotide excision repair-defective mutants 9-1-1 becomes required for UV-mutagenesis [122].…”

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

“…Furthermore, the 75% cins reduction in each single mutant suggests that Rad18 and Rad5 may sometimes work together to promote Pol z-dependent bypass. We previously observed a similar, dual requirement with regard to UV-induced, NER-dependent mutagenesis that occurs in nongrowing cells (Kozmin and Jinks-Robertson 2013).…”

Coordination of DNA damage tolerance mechanisms with cell cycle progression in fission yeast

“…In addition, the primary UV lesions could be processed into structures that require mutagenic TLS by mechanisms that are independent of DNA replication. For instance, NER at closely-spaced lesions produces gapped structures with lesions in the single-stranded region that are similar to daughter-strand gaps and can elicit a checkpoint response 66,72 . Repair of these gaps by mutagenic TLS generates most of the UV-induced mutations in stationary phase cells, but accounts for only a small fraction of mutations in proliferating cells like those used in this study 16-19,73 …”