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Regulation of Rfa2 phosphorylation in response to genotoxic stress in C andida albicans
Author(s) -
Gao Jiaxin,
Wang Haitao,
Wong Ada HangHeng,
Zeng Guisheng,
Huang Zhenxing,
Wang Yanming,
Sang Jianli,
Wang Yue
Publication year - 2014
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/mmi.12749
Subject(s) - biology , phosphorylation , dephosphorylation , candida albicans , dna damage , dna replication , dna repair , cyclin dependent kinase 1 , kinase , microbiology and biotechnology , g2 m dna damage checkpoint , mutagenesis , protein subunit , hyperphosphorylation , corpus albicans , phosphatase , mutant , dna , cell cycle , biochemistry , genetics , cell cycle checkpoint , gene
Summary 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 C andida 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 .