Functional and Physical Interaction of Yeast Mgs1 with PCNA: Impact on RAD6-Dependent DNA Damage Tolerance

Abstract

Proliferating cell nuclear antigen (PCNA), a sliding clamp required for processive DNA synthesis, provides attachment sites for various other proteins that function in DNA replication, DNA repair, cell cycle progression and chromatin assembly. It has been shown that differential posttranslational modifications of PCNA by ubiquitin or SUMO play a pivotal role in controlling the choice of pathway for rescuing stalled replication forks. Here, we explored the roles of Mgs1 and PCNA in replication fork rescue. We provide evidence that Mgs1 physically associates with PCNA and that Mgs1 helps suppress the RAD6 DNA damage tolerance pathway in the absence of exogenous DNA damage. We also show that PCNA sumoylation inhibits the growth of mgs1 rad18 double mutants, in which PCNA sumoylation and the Srs2 DNA helicase coordinately prevent RAD52-dependent homologous recombination. The proposed roles for Mgs1, Srs2, and modified PCNA during replication arrest highlight the importance of modulating the RAD6 and RAD52 pathways to avoid genome instability.Progression of the replication fork is often impeded by DNA lesions caused by exogenous or endogenous DNA-damaging agents. Replication forks also stall when they encounter tightly bound proteins or aberrant DNA structures (7). Stalled replication forks activate the DNA damage tolerance pathway, which promotes the reinitiation of DNA synthesis with or without removing the replication-blocking lesion.Genetic studies in Saccharomyces cerevisiae indicate that Rad6 and Rad18 play central roles in DNA damage tolerance pathway. This pathway is mediated by the protein products of RAD6, RAD18, RAD5, MMS2, and UBC13, as well as several other gene products (4, 9, 22). Rad6 is a ubiquitin E2-conjugating enzyme that forms a stable complex with Rad18, an E3 ligase that binds DNA (2, 3). The Ubc13-Mms2 heterodimer is also an E2-conjugating enzyme (14). Rad5 is a DNA-dependent ATPase that functions as an E3 ligase and associates with the Ubc13-Mms2 complex, recruiting this complex to chromatin in response to DNA damage (27). Previous study demonstrated that PCNA is a substrate for RAD6-dependent ubiquitination (13). When DNA synthesis on one or both strands is arrested by DNA damage, PCNA is mono-ubiquitinated on lysine 164 (Lys164) by the Rad6-Rad18 complex. Mono-ubiquitinated PCNA may target stalled replication forks to initiate error-prone DNA repair via translesion DNA synthesis, a process that requires low-fidelity polymerases, Pol or Pol, to synthesize across the damage (10,15,25,29). Alternatively, the Ubc13-Mms2 complex and Rad5 modulate the polyubiquitination of Lys164 through lysine 63-linked ubiquitin chains. When modified in this manner, PCNA promotes error-free DNA repair (13,25).PCNA is also sumoylated at Lys164 (and secondarily at Lys127) via a mechanism dependent on a distinct E2-conjugating enzyme, Ubc9, and an E3 ligase, Siz1 (13). Although ubiquitination and sumoylation both occur on Lys164, there is evidence suggesting that the two modifications do not appear to have antagon...

Human Wrnip1 Is Localized in Replication Factories in a Ubiquitin-binding Zinc Finger-dependent Manner

“…1C), further indicating that the recombinant protein can recapitulate the behavior of endogenous Wrnip1. In the same fractionation experiment, Wrnip1 and Wrnip1-EGFP were also found to co-fractionate with PCNA and polymerase , both of which have been previously shown to be in complex with Wrnip1 (9,30).…”

“…In S. cerevisiae, mgs1 was found to be synthetically lethal with the rad6 and rad18 genes involved in DNA damage tolerance, indicating that Mgs1/Wrnip1 might deal with stalled replication forks (8). A further study demonstrated that yeast Mgs1 can physically interact with the DNA loading clamp PCNA 4 and suggested that Mgs1 may participate in a DNA damage tolerance pathway alternative to the Rad6-Rad18 one (9). The inferred role of Mgs1/Wrnip1 in replication has been strengthened by the evidence that Mgs1 in vitro stimulates the activity of Fen1, an endonuclease indispensable for the removal of Okazaki fragments during lagging-strand replication (10).…”

Eukaryotic DNA damage tolerance and translesion synthesis through covalent modifications of PCNA

“…The authors suggested an alternative model in which PCNA monoubiquitination may disrupt its interactions with a protein(s) that inhibits binding to the TLS polymerases. To date no such candidate protein has been identified, although we notice a recent report [130] that Mgs1, a protein with homology to E. coli RuvB and eukaryotic clamp loader protein RFC, as well as DNA-dependent ATPase activity and DNA-annealing activities [131,132], associates with PCNA and appears to repress the RAD6 pathway in the absence of exogenous damage. Other concerns with the above paradigm include the stability of monoubiquitinated PCNA, particularly in mammalian cells, that extends past the expected time required to bypass the damage [122], which would allow persistent TLS with unnecessarily increased mutation rates.…”

Werner Helicase-interacting Protein 1 Binds Polyubiquitin via Its Zinc Finger Domain

“…Given the ability of the UBZ domain to bind multiple types of polyubiquitin chains, as shown in this study, the specificity is more likely conferred by the substrate protein rather than a unique polyubiquitin chain assembly. PCNA is an interesting candidate for the ubiquitinated scaffold on which UBZ domain-containing proteins assemble, since Pol, Pol, RAD18, and WRNIP1/ MGS1 all bind PCNA (11,23,54,55).…”

“…Yeasts that overexpress Mgs1p are generally healthy but exhibit marked sensitivity to the DNA-damaging agents hydroxyurea, methyl methanesulfonate, and UV radiation (22,23). This increase in DNA damage sensitivity upon Mgs1p overexpression is dependent on the presence of RAD18, indicating that an overabundance of Mgs1p cripples the postreplication repair pathway (23).…”