title: Essential domains of Schizosaccharomyces pombe Rad8 required for DNA damage response.
abstract: Schizosaccharomyces pombe Rad8 is a conserved protein homologous to S. cerevisiae Rad5 and human HLTF that is required for error-free postreplication repair by contributing to polyubiquitylation of PCNA. It has three conserved domains: an E3 ubiquitin ligase motif, a SNF2-family helicase domain, and a family-specific HIRAN domain. Data from humans and budding yeast suggest that helicase activity contributes to replication fork regression and template switching for fork restart. We constructed specific mutations in the three conserved domains and found that both the E3 ligase and HIRAN domains are required for proper response to DNA damage caused by a variety of agents. In contrast, mutations in the helicase domain show no phenotypes in a wild-type background. To determine whether Rad8 functionally overlaps with other helicases, we compared the phenotypes of single and double mutants with a panel of 23 nonessential helicase mutants, which we categorized into five phenotypic groups. Synthetic phenotypes with rad8∆ were observed for mutants affecting recombination, and a rad8 helicase mutation affected the HU response of a subset of recombination mutants. Our data suggest that the S. pombe Rad8 ubiquitin ligase activity is important for response to a variety of damaging agents, while the helicase domain plays only a minor role in modulating recombination-based fork restart during specific forms of replication stress.
authors.affiliation: Program in Molecular and Computational Biology, University of Southern California, Los Angeles, California 90089-2910.
authors.forename: Lin
authors.initials: L
authors.lastname: Ding
authors.affiliation: Program in Molecular and Computational Biology, University of Southern California, Los Angeles, California 90089-2910 forsburg@usc.edu.
authors.forename: Susan L
authors.identifier: http://orcid.org/0000-0002-4895-8598
authors.initials: SL
authors.lastname: Forsburg
mesh_terms: D004249:DNA Damage; D004265:DNA Helicases / Q000235:genetics*; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D009153:Mutagens / Q000494:pharmacology; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D012568:Schizosaccharomyces / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics*
chemical_list: D009153:Mutagens; D029702:Schizosaccharomyces pombe Proteins; D008741:Methyl Methanesulfonate; D044767:Ubiquitin-Protein Ligases; D004265:DNA Helicases
keywords: HIRAN domain; SNF2 postreplication repair; helicases; ubiquitin E3 ligase