Telomere Dysfunction Drives Increased Mutation by Error-Prone Polymerases Rev1 and ζ in Saccharomyces cerevisiae

Abstract

Using a model system, we have shown that replicative senescence is accompanied by a 16-fold increase in base substitution and frameshift mutations near a chromosome end. The increase was dependent on errorprone polymerases required for the mutagenic response to DNA lesions that block the replication fork.S ACCHAROMYCES cerevisiae cells lacking telomerase, a ribonucleoprotein complex required for telomere replication, experience progressive telomere degradation that culminates in replicative senescence (Lendvay et al. 1996). Deletions that encompass the CAN1 locus located $32 kb from the telomere on the left arm of chromosome V accumulated during senescence (Hackett et al. 2001;Hackett and Greider 2003) and have been attributed to replication fork stalling (Motegi et al. 2006). Since replication fork stalling also generates mutations (Quah et al. 1980), we investigated the effect of telomere dysfunction on the generation of mutations at the CAN1 locus.Mutation rate analysis: We examined the behavior of mutants defective for EST2, which encodes the catalytic subunit of telomerase (Counter et al. 1997) (Table 1). We determined the mutation rate at the CAN1 locus using an assay that selects against deletions in serial cultures of wild-type and est2 mutant cells. We observed no significant change from the wild-type rate of CAN1 mutation in est2 cultures before senescence (P ¼ 0.07), a 16-fold increase during senescence (P , 0.0001), and the restoration of wild-type levels upon recovery (P ¼ 0.09) ( Figure 1A). No senescence-dependent changes in the mutation rate at the CYH2 locus, located 310 kb from its telomere on the left arm of chromosome VII, were observed in est2 mutant cultures before (P ¼ 0.68), during (P ¼ 1.0), or after replicative senescence (P ¼ 1.0) (Figure 2), suggesting that the mutagenic effect is restricted to telomere-proximal sequences. The can1 mutation spectrum observed for senescent est2 mutant cells was similar to that of wild type ( Table 2), suggesting that the mechanism of senescence-dependent mutagenesis in est2 cells may be similar to the mechanism of spontaneous mutagenesis in wild-type cells. Since 50-70% of spontaneous mutagenesis has been attributed to the action of error-prone polymerases (Quah et al. 1980), we investigated whether they were involved in the mechanism underlying senescence-dependent mutagenesis. We examined the effects of mutations in the RAD30, REV7, and REV1 genes, which are required for error-prone polymerase function in yeast (Haracska et al. 2000;Johnson et al. 2000;Goodman 2002;Prakash et al. 2005). Mutation rates in est2 rad30 cultures before (P ¼ 0.32), during (P ¼ 0.34), and after senescence (P ¼ 0.15) were not significantly different from those in est2 cultures (Figure 1, A and B), suggesting that Pol h does not contribute to senescencedependent mutagenesis. In contrast, the rev1 and rev7 mutations completely suppressed senescence-dependent mutagenesis, as the CAN1 mutation rates were not significantly different from those of rev1 and rev7 mutants bef...

AID and Reactive Oxygen Species Can Induce DNA Breaks within Human Chromosomal Translocation Fragile Zones

“…This demonstrates the high sensitivity and low background of the ZBE assay and is consistent with previously reported spontaneous recombination rates from various constructs on different chromosomes (Chan and Kolodner, 2011; Meyer and Bailis, 2007; Yadav et al, 2014). Addition of the GAL1 promoter increases the ZBE rate at least 3.6-fold to 1.8 ×10 −10 (Figure 1C), confirming previous reports that transcription stimulates recombination (Kim and Jinks-Robertson, 2011; Thomas and Rothstein, 1989).…”

Mutagenic and Recombinagenic Responses to Defective DNA Polymerase δ Are Facilitated by the Rev1 Protein in pol3-t Mutants of Saccharomyces cerevisiae

“…The connection between dysfunctional DNA replication and genome instability has been firmly established in yeast by the elevated rates of many types of mutagenic and clastogenic events observed in a variety of DNA replication mutants (Aguilera and Klein 1988;Gordenin et al 1992;Ruskin and Fink 1993;Reagan et al 1995;Ohya et al 2002;Meyer and Bailis 2007). In particular, mutations that disrupt the polymerase function of Pol d confer increased rates of mutation and recombination (Gordenin et al 1992;Tran et al 1995Tran et al , 1996Tran et al , 1997Kokoska et al 1998Kokoska et al , 2000Lobachev et al 1998Lobachev et al , 2000Galli et al 2003), consistent with incom- 18.8 (9.3-24.1) 4.2 9.6 (6.7-11.4) 5.7 pol3-t rev7D…”

“…It is also important for seeing the mutations associated with double-strand break (DSB) repair events at the mating-type locus in yeast (Holbeck and Strathern 1997;Rattray et al 2002) and the immunoglobulin genes in mammals (Diaz et al 2001;Zan et al 2001). Perhaps the most relevant biochemical property of Pol z is its extraordinary ability to extend from mispaired bases Acharya et al 2006), which may make it ideal for catalyzing an extension following base insertion opposite a lesion by Pol h. The capacity to drive DNA synthesis from mismatch-containing substrates is likely to be what enables Pol z to function during translesion synthesis (Baynton et al 1998), homologous recombination (Rattray and Strathern 2002;Rattray et al 2003;Sonoda et al 2003;Wu et al 2003), and gross chromosomal rearrangement (Meyer and Bailis 2007). Importantly, a null allele of the REV3 gene was also previously shown to suppress the mutagenic effect of mutations in the POL3 gene, including pol3-t (Pavlov et al 2001;Northam et al 2006), suggesting that Pol z may be engaged following spontaneous replicative polymerase failure.…”

Mutation as a Stress Response and the Regulation of Evolvability

“…Additionally, telomere shortening in est2 mutants is associated with increased point mutagenesis in telomere-proximal regions, mediated by the error-prone polymerases Rev1 and Rev3/Rev7 (Meyer and Bailis, 2007), the first of which is a Y-family DNA polymerase homologous with DinB, and all of which are specialized, error-prone translesion polymerases. These authors did not measure mutation of the same gene close to and distant from the telomere, so the differences in mutagenesis upon telomere shortening might be due to either the mutation-target sequences or to telomere proximity per se, in either case, activated by stress.…”