Characterization of Pph3-mediated dephosphorylation of Rad53 during methyl methanesulfonate-induced DNA damage repair in Candida albicans

Abstract

Genotoxic stress causes DNA damage or stalled DNA replication and filamentous growth in the pathogenic fungus The DNA checkpoint kinase Rad53 critically regulates by phosphorylation effectors that execute the stress response. Rad53 itself is activated by phosphorylation and inactivated by dephosphorylation. Previous studies have suggested that the phosphatase Pph3 dephosphorylates Rad53. Here, we used mass spectrometry and mutagenesis to identify Pph3 dephosphorylation sites on Rad53 in We found that serine residues 351, 461 and 477, which were dephosphorylated in wild-type cells during the recovery from DNA damage caused by methyl methanesulfonate (MMS), remained phosphorylated in cells. Phosphomimetic mutation of the three residues () impaired Rad53 dephosphorylation, exit from cell cycle arrest, dephosphorylation of two Rad53 effectors Dun1 and Dbf4, and the filament-to-yeast growth transition during the recovery from MMS-induced DNA damage. The phenotypes observed in the mutant also occurred in the mutant. Together, our findings reveal a molecular mechanism by which Pph3 controls DNA damage response in .

Ser/Thr protein phosphatases in fungi: structure, regulation and function

“…As in S. cerevisiae , Pph3/Psy2 are required for the response to DNA damage caused by methyl methanesulfonate but not by HU [201]. More recent work has revealed Rad53 Ser residues 351, 461 and 477 as likely targets for Pph3-mediated dephosphorylation [202]. Pph3 is also responsible for dephosphorylation of Rfa2, a subunit of the replication protein A complex that is phosphorylated by Mec1 and the cyclin-dependent kinase Clb2-Cdc28 in response to the genotoxic insult [203].…”

Dissecting Nucleosome Function with a Comprehensive Histone H2A and H2B Mutant Library

“…It has been reported the dephosphorylation of Rad53 by Pph3 is required for checkpoint inactivation after MMS but not HU exposure ( Wang et al 2012 ), while Glc7 is needed for the disappearance of hyperphosphorylated Rad53 to recover from HU but not MMS exposure ( Bazzi et al 2010 ). In addition, some other evidence supports the idea that different phosphorylation states of Rad53 may exist under different toxic stresses and that these patterns are regulated by the activities of various kinases and phosphatases ( Smolka et al 2005 ; Keogh et al 2006 ; Yao et al 2017 ). Further studies to unravel how exactly these enzymes are engaged in response to different regents will be required.…”

Protein-Protein Interactions in Candida albicans

“…et al, 2015). An expert with this technology is Prof. Wang who has used Co-IP a lot in his lab and showed several interactions (Zheng et al, 2004, 2007; Li et al, 2007, 2008, 2012; Sinha et al, 2007; Bai et al, 2011; Zeng et al, 2012; Wang et al, 2013, 2016; Gao et al, 2014; Huang et al, 2014a, b; Guan et al, 2015; Au Yong et al, 2016; Liu et al, 2016; Yao et al, 2016, 2017; Yang et al, 2018). Several tags have been used, such as a Flag-tag (Umeyama et al, 2002; Singh et al, 2011), Myc-tag (Cheetham et al, 2007, 2011; Sinha et al, 2007; Kos et al, 2016), GFP or derived tags (Bishop et al, 2010; Greig et al, 2015), HA-tag (Ni et al, 2004; Askew et al, 2011; Sellam et al, 2019), TAP-tag (see below) or the protein A tag with a TEV protease site (Blackwell et al, 2003).…”