title: Regulation of Rfa2 phosphorylation in response to genotoxic stress in Candida albicans.
abstract: Successful pathogens must be able to swiftly respond to and repair DNA damages inflicted by the host defence. The replication protein A (RPA) complex plays multiple roles in DNA damage response and is regulated by phosphorylation. However, the regulators of RPA phosphorylation remain unclear. Here, we investigated Rfa2 phosphorylation in the pathogenic fungus Candida albicans. Rfa2, a RFA subunit, is phosphorylated when DNA replication is inhibited by hydroxyurea and dephosphorylated during the recovery. By screening a phosphatase mutant library, we found that Pph3 associates with different regulatory subunits to differentially control Rfa2 dephosphorylation in stressed and unstressed cells. Site-directed mutagenesis revealed T11, S18, S29, and S30 being critical for Rfa2 phosphorylation in response to genotoxic insult. We obtained evidence that the genome integrity checkpoint kinase Mec1 and the cyclin-dependent kinase Clb2-Cdc28 mediate Rfa2 phosphorylation. Although cells expressing either a phosphomimetic or a non-phosphorylatable version of Rfa2 had defects, the latter exhibited greater sensitivity to genotoxic challenge, failure to repair DNA damages and to deactivate Rad53-mediated checkpoint pathways in a dosage-dependent manner. These mutants were also less virulent in mice. Our results provide important new insights into the regulatory mechanism and biological significance of Rfa2 phosphorylation in C. albicans.
authors.affiliation: Key Laboratory of Cell Proliferation and Regulation Biology, College of Life Sciences, Beijing Normal University, Beijing, China; Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore.
authors.forename: Jiaxin
authors.initials: J
authors.lastname: Gao
authors.forename: Haitao
authors.initials: H
authors.lastname: Wang
authors.forename: Ada Hang-Heng
authors.initials: AH
authors.lastname: Wong
authors.forename: Guisheng
authors.initials: G
authors.lastname: Zeng
authors.forename: Zhenxing
authors.initials: Z
authors.lastname: Huang
authors.forename: Yanming
authors.initials: Y
authors.lastname: Wang
authors.forename: Jianli
authors.initials: J
authors.lastname: Sang
authors.forename: Yue
authors.initials: Y
authors.lastname: Wang
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D002176:Candida albicans / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity; D002177:Candidiasis / Q000382:microbiology*; D004249:DNA Damage; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D010766:Phosphorylation; D051716:Replication Protein A / Q000235:genetics / Q000378:metabolism; D014774:Virulence
chemical_list: D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D051716:Replication Protein A