title: Rad18/Rad5/Mms2-mediated polyubiquitination of PCNA is implicated in replication completion during replication stress.
abstract: Ubiquitination of proteins was previously shown to modulate various processes of DNA metabolism. PCNA, a processivity factor with essential functions in replication and repair, is modified with ubiquitin at K164. In addition, PCNA is sumoylated at K127 and K164. We found that the rad18delta mutation suppresses the temperature sensitivity of the polymerase delta mutants hys2-1 and cdc2-1 as well as the synthetic lethality of cdc2-1 pol32delta mutants, suggesting a role for Rad18 in modulating DNA replication. As Rad18 mediates ubiquitination of PCNA, we examined whether PCNA modifications affected its function in replication. Multicopy PCNA alleviated the replication defects of rfc5-1 strains, but not those of poldelta mutants. In contrast, multicopy PCNA-K164R had reduced ability to suppress the replication defects of rfc5-1, but alleviated those of poldelta mutants. The roles of sumoylated and ubiquitinated PCNA in rfc5-1 and hys2-1 mutants were addressed by using mutant backgrounds that selectively affected sumoylation (siz1delta), ubiquitination (rad18delta), polyubiquitination (rad5delta, mms2delta), or the ability of cells to perform translesion synthesis (polzetadelta, poletadelta). Our results are consistent with the idea that the Rad18/Rad5/Mms2 polyubiquitination pathway is important for replication completion, perhaps by promoting a template switch type of DNA synthesis.
authors.affiliation: Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, 980-8578, Japan. branzei@postman.riken.go.jp
authors.forename: Dana
authors.initials: D
authors.lastname: Branzei
authors.forename: Masayuki
authors.initials: M
authors.lastname: Seki
authors.forename: Takemi
authors.initials: T
authors.lastname: Enomoto
mesh_terms: D000251:Adenosine Triphosphatases / Q000502:physiology*; D002455:Cell Division / Q000502:physiology; D004265:DNA Helicases; D004261:DNA Replication / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D016521:Electrophoresis, Gel, Pulsed-Field; D016297:Mutagenesis, Site-Directed; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism* / Q000502:physiology*; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases
chemical_list: D004268:DNA-Binding Proteins; C112520:MMS2 protein, S cerevisiae; D018809:Proliferating Cell Nuclear Antigen; C063607:RAD18 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases; D000251:Adenosine Triphosphatases; C076225:RAD5 protein, S cerevisiae; D004265:DNA Helicases