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Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis
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Regulation of Rfa2 phosphorylation in response to genotoxic stress in Candida albicans

Jiaxin Gao1, Haitao Wang, Ada Hang-Heng Wong

  • 1Key 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.

Molecular Microbiology
|August 12, 2014
PubMed

Insights

Pathogenic fungi like Candida albicans repair DNA damage via Rfa2 phosphorylation, regulated by Pph3 and Mec1. Non-phosphorylatable Rfa2 impairs DNA repair and reduces virulence.

Area of Science:

  • Microbiology
  • Molecular Biology
  • Genetics

Background:

  • Pathogenic fungi must efficiently repair DNA damage to survive host defenses.
  • The Replication Protein A (RPA) complex is crucial for DNA damage response and its function is modulated by phosphorylation.
  • The specific regulators of RPA phosphorylation in fungi are not well understood.

Purpose of the Study:

  • To investigate the regulation and biological significance of Rfa2 phosphorylation in the pathogenic fungus Candida albicans.
  • To identify kinases and phosphatases involved in controlling Rfa2 phosphorylation.
  • To determine the role of Rfa2 phosphorylation in DNA repair and fungal virulence.

Main Methods:

  • Screening of a phosphatase mutant library in Candida albicans.
  • Site-directed mutagenesis to identify critical phosphorylation sites on Rfa2.
  • Analysis of Rfa2 phosphorylation in response to genotoxic stress (hydroxyurea).
  • Assessment of DNA repair efficiency, checkpoint activation, and virulence in Rfa2 mutants.

Main Results:

  • Rfa2 is phosphorylated during replication stress and dephosphorylated upon recovery.
  • The phosphatase Pph3, with regulatory subunits, controls Rfa2 dephosphorylation.
  • T11, S18, S29, and S30 are critical sites for Rfa2 phosphorylation.
  • Mec1 and Clb2-Cdc28 kinase pathways mediate Rfa2 phosphorylation.
  • Non-phosphorylatable Rfa2 mutants show increased sensitivity to genotoxic agents, impaired DNA repair, and reduced virulence in a mouse model.

Conclusions:

  • Rfa2 phosphorylation is a key regulatory mechanism in Candida albicans' response to DNA damage.
  • Pph3, Mec1, and Clb2-Cdc28 play significant roles in controlling Rfa2 phosphorylation status.
  • Rfa2 phosphorylation is essential for maintaining genome integrity and virulence in this important fungal pathogen.

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