Academic journal article Genetics

Participation of DNA Polymerase [Zeta] in Replication of Undamaged DNA in Saccharomyces Cerevisiae

Academic journal article Genetics

Participation of DNA Polymerase [Zeta] in Replication of Undamaged DNA in Saccharomyces Cerevisiae

Article excerpt

ABSTRACT

Translesion synthesis DNA polymerases contribute to DNA damage tolerance by mediating replication of damaged templates. Due to the low fidelity of these enzymes, lesion bypass is often mutagenic. We have previously shown that, in Saccharomyces cerevisiae, the contribution of the error-prone DNA polymerase ζ (Polζ) to replication and mutagenesis is greatly enhanced if the normal replisome is defective due to mutations in replication genes. Here we present evidence that this defective-replisome-induced mutagenesis (DRIM) results from the participation of Polζ in the copying of undamaged DNA rather than from mutagenic lesion bypass. First, DRIM is not elevated in strains that have a high level of endogenous DNA lesions due to defects in nucleotide excision repair or base excision repair pathways. Second, DRIM remains unchanged when the level of endogenous oxidative DNA damage is decreased by using anaerobic growth conditions. Third, analysis of the spectrum of mutations occurring during DRIM reveals the characteristic error signature seen during replication of undamaged DNA by Polζ in vitro. These results extend earlier findings in Escherichia coli indicating that Y-family DNA polymerases can contribute to the copying of undamaged DNA. We also show that exposure of wild-type yeast cells to the replication inhibitor hydroxyurea causes a Polζ-dependent increase in mutagenesis. This suggests that DRIM represents a response to replication impediment per se rather than to specific defects in the replisome components.

TRANSLESION synthesis (TLS) DNA polymerases have the ability to bypass lesions in template DNA that block DNA synthesis by replicative enzymes (Prakash et al. 2005). TLS polymerases are present in all domains of life and include eukaryotic polymerase ζ (Polζ), Polη, Polι, Polκ, and Rev1. Because of the lower selectivity of their active sites, TLS polymerases often introduce errors when bypassing lesions or copying undamaged DNA in vitro (Shcherbakova and Fijalkowska 2006). In yeast and human cells, DNA synthesis by Polζ is responsible for nearly all mutagenesis induced by exogenous genotoxicants (Waters et al. 2009). Polζ has been isolated from Saccharomyces cerevisiae as a complex of two subunits encoded by the REV3 and REV7 genes. This complex can synthesize, with a low efficiency, past several types ofDNAlesions(Nelsonet al. 1996;Guo et al. 2001). At the same time, Polζ is very efficient in extending primers containing a mismatched terminal nucleotide (Johnson et al. 2000; Guo et al. 2001; Haracska et al. 2001; Simhadri et al. 2002). In accordance with these properties, the main function of Polζ in TLS is proposed to be the extension from nucleotides incorporated opposite DNA lesions by other DNApolymerases(LawrenceandMaher2001;Bresson and Fuchs 2002; Prakash and Prakash 2002). The mismatch extension and TLS activity of Polζ is stimulated by the Rev1 protein (Acharya et al. 2006). Rev1 is a deoxycytidyl transferase involved in multiple protein- protein interactions with other DNA polymerases, and its essential function in TLS is thought to be structural rather thancatalytic(Waters et al. 2009).TheTLS activity of Polζ is also stimulated by proliferating cell nuclear antigen (PCNA), the DNA polymerase processivity factor (Garg et al. 2005; Northam et al. 2006).

In addition to the mutagenesis induced by environmental agents, Polζ is required for the vast majority of mutations provoked by endogenous DNA damage. For example, Polζ is responsible for the increase in mutation rate caused by defects in nucleotide excision repair (NER) (Roche et al. 1994; Harfe and Jinks-Robertson 2000), base excision repair (BER) (Xiao et al. 2001), post-replicative DNA repair (Roche et al. 1995; Broomfield et al. 1998; Xiao et al. 1999), homologous recombination (Roche et al. 1995; Harfe and Jinks- Robertson 2000), the overproduction of 3-methyladenine DNA glycosylase (Glassner et al. 1998), and the expression of altered uracil-DNA glycosylases that remove undamaged cytosines and thymines in a BERdefective strain (Auerbach et al. …

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