Novel role for the C terminus of Saccharomyces cerevisiae Rev1 in mediating protein-protein interactions.

In all eukaryotes examined to date, Rev1 and the REV3/REV7-encoded polymerase zeta (Polζ) are absolutely required for the mutagenic bypass of DNA lesions, a process called translesion DNA synthesis (TLS). Even on undamaged DNA, most TLS occurs at a low fidelity, relative to replicative DNA synthesis, and represents a major source of DNA damage-induced base substitutions and frameshift mutations (7, 36). In the yeast Saccharomyces cerevisiae, the majority of mutagenic DNA lesion bypass is carried out by Polζ, a member of the B family of DNA polymerases, and the Y-family polymerase Rev1 (21). Both Polζ and Rev1 are part of the “error-prone” branch of the RAD6 epistasis pathway (20). Inactivating mutations in the rev genes render cells sensitive to DNA-damaging agents and confer a “reversionless” phenotype after DNA damage (23, 25). Together, Polζ and Rev1 functions are required for the generation of 95% to 98% of UV radiation (UV)-induced base pair substitutions (22, 24). Though the yeast Polζ can, in some cases, insert nucleotides across damaged bases, it possesses a unique facility to extend DNA synthesis from terminally mismatched primers and, in general, from distorted DNA structures (15, 45). The Polζ enzyme consists of a catalytic subunit, Rev3, and an accessory subunit, Rev7, that enhances the polymerase activity of Rev3 by about 200-fold (33). Polζ replicates past abasic sites in the DNA template, an activity that is greatly stimulated by Rev1 in vitro (32). The Rev1 polymerase is endowed with the unique ability to insert predominantly dCMP both across an undamaged template G and abasic sites as well as a variety of other DNA lesions (11, 21, 32). In this capacity, Rev1 uses a novel mechanism of DNA synthesis whereby the incoming dCTP pairs with an arginine rather than the template base, and the template G is evicted from the DNA helix (30). Intriguingly, the catalytic activity of Rev1 appears not to be essential for the bypass of DNA damage, as mutations inactivating this activity leave Rev1 still competent to participate in translesion synthesis, (12; L. Waters, S. D'Souza and G. C. Walker, unpublished observations), albeit with an altered mutation spectrum (35). In contrast to the polymerase domain, the N-terminal BRCT motif of yeast Rev1 is required for mutagenesis. A G193R point mutation in the Rev1 BRCT motif (the rev1-1 allele) abrogates the ability of Rev1 to function in induced mutagenesis and confers a moderate sensitivity to DNA damaging agents (19, 25). BRCT domains (BRCA1 C terminus) are ubiquitous motifs that facilitate physical interactions among proteins involved in the cellular response to DNA damage (4, 44). Interestingly, the Rev1-1 protein retains its catalytic dCMP transferase activity in vitro (31). Thus, the TLS function of yeast Rev1 is thought to require protein-protein interactions, rather than its polymerase activity, and has been proposed to nucleate a protein complex at the site of a stalled replication fork, possibly to facilitate a switch between the replicative DNA polymerase and other TLS polymerases (12). However, the recent observation that the yeast Rev1 protein levels fluctuate dramatically in the yeast cell cycle and peak during the G2/M phase suggests that Rev1 functions mainly in the G2/M phase rather than during the S phase of the cell cycle when replication occurs (46). In addition to its BRCT and polymerase domains, Rev1 possesses a C-terminal region which, in higher eukaryotes, has been shown to interact with a variety of translesion DNA polymerases, including Polκ, Polλ, Polη, and Polι and the Polζ accessory subunit, Rev7 (9, 29, 34, 40, 41). Unlike human and mouse Rev1, relatively little is known about the C terminus of the Saccharomyces cerevisiae Rev1 protein. Due to the poor sequence conservation of the C termini between yeast Rev1 and its vertebrate homologs, it has been suggested that the C terminus of yeast Rev1 plays no part in the process of mutagenic bypass or protein-protein interactions (16, 29, 41). In this study, we assess the contribution of the C terminus of yeast Rev1 in DNA damage tolerance by overproducing a C-terminal fragment in cells exposed to DNA damage. We find that overproduction of the extreme C terminus of the yeast Rev1 protein confers a strong dominant-negative phenotype on the survival and reversion frequencies of otherwise wild-type cells exposed to DNA damage. We show that the dominant-negative phenotype of the Rev1 C terminus requires both REV1 and REV7, providing the first in vivo evidence for a critical function of the C terminus of Saccharomyces cerevisiae Rev1 in DNA damage tolerance. We use immunoprecipitation experiments to demonstrate that two novel regions of the Rev1 protein, the extreme C terminus and the BRCT region, independently interact with the Rev7 protein, the accessory subunit of Polζ. Consistent with an earlier observation (1) showing a direct interaction between the Rev1 polymerase-associated domain (PAD) and Rev7, we report that the Rev1 PAD also immunoprecipitates Rev7. Our results thus significantly expand the study of the interaction between Rev1 and Rev7 and have important implications for the process of translesion DNA synthesis.