Rfa2 is specifically dephosphorylated by Pph3 in Candida albicans

Rfa2 is a ssDNA (single-stranded DNA)-binding protein that plays an important role in DNA replication, recombination and repair. Rfa2 is regulated by phosphorylation, which alters its protein–protein interaction and protein–DNA interaction. In the present study, we found that the Pph3–Psy2 phosphatase complex is responsible for Rfa2 dephosphorylation both during normal G(1)-phase and under DNA replication stress in Candida albicans. Phosphorylated Rfa2 extracted from pph3Δ or psy2Δ G(1) cells exhibited diminished binding affinity to dsDNA (double-stranded DNA) but not to ssDNA. We also discovered that Cdc28 (cell division cycle 28) and Mec1 are responsible for Rfa2 phosphorylation in G(1)-phase and under DNA replication stress respectively. Moreover, MS revealed that the domain of Rfa2 that was phosphorylated in G(1)-phase differed from that phosphorylated under the stress conditions. The results of the present study imply that differential phosphorylation plays a crucial role in RPA (replication protein A) regulation.