The critical mutagenic translesion DNA polymerase Rev1 is highly expressed during G ₂ /M phase rather than S phase

Abstract

The Rev1 protein lies at the root of mutagenesis in eukaryotes. Together with DNA polymerase (Rev3͞7), Rev1 function is required for the active introduction of the majority of mutations into the genomes of eukaryotes from yeast to humans. Rev1 and polymerase are error-prone translesion DNA polymerases, but Rev1's DNA polymerase catalytic activity is not essential for mutagenesis. Rather, Rev1 is thought to contribute to mutagenesis principally by engaging in crucial protein-protein interactions that regulate the access of translesion DNA polymerases to the primer terminus. This inference is based on the requirement of the N-terminal BRCT (BRCA1 C-terminal) domain of Saccharomyces cerevisiae Rev1 for mutagenesis and the interaction of the Cterminal region of mammalian Rev1 with several other translesion DNA polymerases. Here, we report that S. cerevisiae Rev1 is subject to pronounced cell cycle control in which the levels of Rev1 protein are Ϸ50-fold higher in G2 and throughout mitosis than during G1 and much of S phase. Differential survival of a rev1⌬ strain after UV irradiation at various points in the cell cycle indicates that this unanticipated regulation is physiologically relevant. This unexpected finding has important implications for the regulation of mutagenesis and challenges current models of error-prone lesion bypass as a process involving polymerase switching that operates mainly during S phase to rescue stalled replication forks.cell cycle ͉ mutagenesis ͉ translesion synthesis ͉ DNA damage

The mismatch repair system promotes DNA polymerase ζ-dependent translesion synthesis in yeast

“…The demonstration in yeast that reinitiation of DNA synthesis can occur during leading-as well as lagging-strand synthesis (37) suggests that TLS may be primarily a gap-filling process to deal with discontinuities that accumulate on both strands behind the fork. The finding that yeast Rev1 is most abundant in G 2 /M would be consistent with a gap-filling mechanism that functions largely outside of S phase (38). Although the results reported here do not directly address the polymerase-switch versus gap-filling models of TLS, we suggest they are more consistent with the latter model.…”

The DNA Polymerase Activity ofSaccharomyces cerevisiaeRev1 is Biologically Significant

“…A W1588-4C (MATa leu2-3,112 ade2-1 can1-100 his3-11,15 ura3-1 trp1-1 RAD5) (Zhao et al 1998) derivative (also bar1DTLEU2 and rev1DTkanMX4) (Waters and Walker 2006;D'Souza et al 2008) is the isogenic parent of all strains involving the integrated REV1 and mutated rev1 alleles. These integrations at the REV1 locus were tagged with a C-terminal--TEV-ProA-His 7 epitope tag (marked with HIS3) using pYM10 (Knop et al 1999), similar to that previously described (Waters and Walker 2006;D'Souza et al 2008). Vector DNA was removed by selecting for 5-fluoroorotic acid (5-FOA) resistant colonies (ura auxotrophs).…”

Proficient and Accurate Bypass of Persistent DNA Lesions by DinB DNA Polymerases

“…71 Although it is clear that regression of stalled replication forks also plays an important role in TLS, 72 these observations have led to the speculation that TLS polymerases may act post-replicatively at such gaps. 73 Such a model is supported by the recent observation that in S. cerevisiae the Y-family member Rev1 is most highly expressed not in S-phase but rather in G 2 /M, after the majority of DNA replication has taken place. 73 This model is also particularly attractive given the weak activity of Y-family DNA polymerases, as it allows replication and TLS to be carried out in parallel rather than in series.…”

“…73 Such a model is supported by the recent observation that in S. cerevisiae the Y-family member Rev1 is most highly expressed not in S-phase but rather in G 2 /M, after the majority of DNA replication has taken place. 73 This model is also particularly attractive given the weak activity of Y-family DNA polymerases, as it allows replication and TLS to be carried out in parallel rather than in series. 74 Further research will give considerable insight into these and other problems.…”