Requirement of RAD5 and MMS2 for Postreplication Repair of UV-Damaged DNA in Saccharomyces cerevisiae

Abstract

UV lesions in the template strand block the DNA replication machinery. Genetic studies of the yeast Saccharomyces cerevisiae have indicated the requirement of the Rad6-Rad18 complex, which contains ubiquitinconjugating and DNA-binding activities, in the error-free and mutagenic modes of damage bypass. Here, we examine the contributions of the REV3, RAD30, RAD5, and MMS2 genes, all of which belong to the RAD6 epistasis group, to the postreplication repair of UV-damaged DNA. Discontinuities, which are formed in DNA strands synthesized from UV-damaged templates, are not repaired in the rad5⌬ and mms2⌬ mutants, thus indicating the requirement of the Rad5 protein and the Mms2-Ubc13 ubiquitin-conjugating enzyme complex in this repair process. Some discontinuities accumulate in the absence of RAD30-encoded DNA polymerase (Pol) but not in the absence of REV3-encoded DNA Pol. We concluded that replication through UV lesions in yeast is mediated by at least three separate Rad6-Rad18-dependent pathways, which include mutagenic translesion synthesis by Pol, error-free translesion synthesis by Pol, and postreplication repair of discontinuities by a Rad5-dependent pathway. We suggest that newly synthesized DNA possessing discontinuities is restored to full size by a "copy choice" type of DNA synthesis which requires Rad5, a DNA-dependent ATPase, and also PCNA and Pol␦. The possible roles of the Rad6-Rad18 and the Mms2-Ubc13 enzyme complexes in Rad5-dependent damage bypass are discussed.Replication of damaged DNA templates can occur by translesion synthesis. This process is usually mutagenic but can be error free as well (see below). In Escherichia coli, the umuCencoded DNA polymerase V (PolV) promotes mutagenic translesion synthesis (29,35). Error-prone translesion synthesis, however, accounts for only a minor portion of the damage bypass in E. coli, whereas much of the damage bypass occurs by means of error-free mechanisms. Error-free bypass mechanisms come into play when the replication machinery terminates synthesis at the site of a DNA lesion and replication restarts downstream of the lesion. This results in the formation of a gap in the newly synthesized strand across from the DNA lesion (30). In E. coli, this gap is filled in by RecA-dependent recombination mechanisms by which the DNA strand from the undamaged sister duplex is transferred to the gapped strand in the damaged duplex (31). In mammalian cells, a copy choice type of DNA synthesis has been invoked as the major mechanism for the filling in of gaps formed in the newly synthesized strand opposite DNA lesions (11). In this mechanism, the newly synthesized daughter strand of the undamaged complementary sequence is used as the template to bypass the lesion. Once the lesion is bypassed, DNA polymerase switches back to copying the damaged template strand.Genetic studies of the yeast Saccharomyces cerevisiae have indicated the requirement of the RAD6 and RAD18 genes in error-free, as well as mutagenic, damage bypass processes (22,28). rad6 and rad18 mutants exhibit a ...

Genetic evidence for a role of Saccharomyces cerevisiae Mph1 in recombinational DNA repair under replicative stress

“…In response to stalled replication forks, PCNA is monoubiquitinated on Lys164 by the Rad6-Rad18 complex, thus activating TLS (Hoege et al, 2002;Stelter and Ulrich, 2003;Watanabe et al, 2004). Monoubiquitinated PCNA can be polyubiquitinated by the Rad5-Ubc13-Mms2 complex (Hofmann and Pickart, 1999) to allow error-free mechanisms (Torres-Ramos et al, 2002). Alternatively, K164 of PCNA can be SUMOylated to regulate recombination at the fork (Pfander et al, 2005;Moldovan et al, 2006) via a direct interaction with Srs2 (Pfander et al, 2005), a 3 -5 DNA helicase (Rong and Klein, 1993), thought to exert a negative control on HR (Krejci et al, 2003;Veaute et al, 2003).…”

The Chfr mitotic checkpoint protein functions with Ubc13-Mms2 to form Lys63-linked polyubiquitin chains

“…A subset of these ubiquitin ligases catalyses the formation of Lys63-linked polyubiquitin chains and functions with a specific ubiquitin-conjugating enzyme consisting of a Ubc13-Mms2 heterodimer. The substrates of these ubiquitin ligases are usually not targeted for degradation, but are involved in signaling cellular stress (Hofmann and Pickart, 1999;Brusky et al, 2000;Deng et al, 2000;Ulrich and Jentsch, 2000;Xiao et al, 2000;Pickart, 2001;Wang et al, 2001;Torres-Ramos et al, 2002). As Chfr plays a role in the response of cells to mitotic stress, we examined its specificity for ubiquitin-conjugating enzymes and the type of polyubiquitin chains that it catalyses.…”

Human SHPRH is a ubiquitin ligase for Mms2–Ubc13-dependent polyubiquitylation of proliferating cell nuclear antigen

“…The Mms2-Ubc13-Rad5-dependent pathway promotes the repair of discontinuities that form in the newly synthesized strand opposite from DNA lesions (10). Although the mechanism by which the Rad5 pathway operates is not known, it likely utilizes a template switching mechanism, wherein the newly synthesized daughter strand of the undamaged complementary sequence is used as the template for bypassing the lesion (10,11).…”

“…Pol promotes lesion bypass primarily via its role as an extender, wherein, after the insertion of a nucleotide opposite the DNA lesion by another polymerase, Pol performs the extension of the nascent primer terminus (3,9). The Mms2-Ubc13-Rad5-dependent pathway promotes the repair of discontinuities that form in the newly synthesized strand opposite from DNA lesions (10). Although the mechanism by which the Rad5 pathway operates is not known, it likely utilizes a template switching mechanism, wherein the newly synthesized daughter strand of the undamaged complementary sequence is used as the template for bypassing the lesion (10,11).…”