Rfa2 is specifically dephosphorylated by Pph3 in <i>Candida albicans</i>

Wang, Haitao; Gao, Jiaxin; Wong, Ada Hang‐Heng; Hu, Kang-Di; Li, Wanjie; Wang, Yue; Sang, Jianli

doi:10.1042/bj20120952

Cited by 11 publications

(16 citation statements)

References 49 publications

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“…PP4 was also implicated in dephosphorylation of RPA2 in C. albicans and mammals (Lee et al, 2010; Wang et al, 2013), as well as mammalian 53BP1, KAP1, and CHD4 (Lee et al, 2012, 2014). Other mechanisms that downregulate the checkpoint act by degrading Mrc1 or human CLASPIN (Fong et al, 2013; Mailand et al, 2006; Peschiaroli et al, 2006), or by sequestering Rad9 by Rtt107-Slx4 in yeast (Ohouo et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

Yeast PP4 Interacts with ATR Homolog Ddc2-Mec1 and Regulates Checkpoint Signaling

et al. 2015

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SUMMARY Mec1-Ddc2 (ATR-ATRIP) controls the DNA damage checkpoint and shows differential cell-cycle regulation in yeast. To find regulators of Mec1-Ddc2, we exploited a mec1 mutant that retains catalytic activity in G2 and recruitment to stalled replication forks, but which is compromised for the intra-S phase checkpoint. Two screens, one for spontaneous survivors and an E-MAP screen for synthetic growth effects, identified loss of PP4 phosphatase, pph3Δ and psy2Δ, as the strongest suppressors of mec1-100 lethality on HU. Restored Rad53 phosphorylation accounts for part, but not all, of the pph3Δ-mediated survival. Phosphoproteomic analysis confirmed that 94% of the mec1-100-compromised targets on HU are PP4 regulated, including a phosphoacceptor site within Mec1 itself, mutation of which confers damage sensitivity. Physical interaction between Pph3 and Mec1, mediated by cofactors Psy2 and Ddc2, is shown biochemically and through FRET in subnuclear repair foci. This establishes a physical and functional Mec1-PP4 unit for regulating the checkpoint response.

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Section: Introductionmentioning

confidence: 99%

Yeast PP4 Interacts with ATR Homolog Ddc2-Mec1 and Regulates Checkpoint Signaling

et al. 2015

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“…RPA2, which is a homolog of ScRfa2 in animal cells, undergoes phosphorylation upon DNA damage stress [26]. Previous studies in C. albicans demonstrated that Rad53 and Rfa2 are phosphorylated by Mec1, a homolog of ATM, in response to DNA damage [18,21,29]. Thus, the phosphorylation of Rad53 and Rfa2 is crucial for the DNA damage response.…”

Section: Discussionmentioning

confidence: 93%

“…5 and 6), suggesting that the role of Ppt1 in C. albicans is not conserved. Previously, we found that the protein phosphatases Glc7, Pph3 and Ptc2 are directly involved in cell cycle progression and the DNA damage response in C. albicans, implying that yeast cells mainly rely on these protein phosphatases in response to genotoxins [18,23,29]. Studies in S. cerevisiae have shown that Ppt1 specifically dephosphorylates Hsp90 via its TPR domain [16], and Hsp90 contributes to the DNA damage response to radiation in animal cells [31].…”

Section: Discussionmentioning

confidence: 98%

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Role of Ppt1 in multiple stress responses in Candida albicans

Gao

et al. 2014

Chin. Sci. Bull.

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To study the function of CaPpt1, we deleted PPT1 gene from the Candida albicans genome by sequentially replacing the entire coding region with the selectable markers ARG4 and HIS1. The results showed that the deletion of Ppt1 did not affect the hyphal formation of C. albicans under serum induction and caused enhanced sensitivity to DNA damage, Calcofluor white and saltinduced stress. We also found that Ppt1 was not required for the phenotypic response of cells treated with the genotoxins, methylmethane sulfonate and hydroxyurea. Flow cytometric analyses indicated that ppt1D cells and wild-type cells showed similar G 2 /M arrest profiles when exposed to DNA damage stress. Ppt1 was not required for the activation of the DNA damage response pathway, as indicated by normal phosphorylation of Rad53 and Rfa2 in ppt1D cells under DNA damage stress. We suggest that Ppt1 plays important roles in response to various stress conditions in C. albicans.

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“…Dephosphorylation of C. albicans Rfa2 requires the Pph3–Psy2 phosphatase complex (Wang et al 2013; Gao et al 2014), and the PP2AC and PP4C phosphatases are necessary to dephosphorylate human RPA2 during the DNA-damage response (Feng et al 2009; Lee et al 2010). Mass spectrometry analysis of proteins isolated from unstressed C. albicans pph3 Δ mutant cells revealed five potential targets of phosphorylation in Rfa2, none of which were located within the first 40 amino acids.…”

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confidence: 99%

The DNA Damage Response and Checkpoint Adaptation in Saccharomyces cerevisiae: Distinct Roles for the Replication Protein A2 (Rfa2) N-Terminus

et al. 2015

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In response to DNA damage, two general but fundamental processes occur in the cell: (1) a DNA lesion is recognized and repaired, and (2) concomitantly, the cell halts the cell cycle to provide a window of opportunity for repair to occur. An essential factor for a proper DNA-damage response is the heterotrimeric protein complex Replication Protein A (RPA). Of particular interest is hyperphosphorylation of the 32-kDa subunit, called RPA2, on its serine/threonine-rich amino (N) terminus following DNA damage in human cells. The unstructured N-terminus is often referred to as the phosphorylation domain and is conserved among eukaryotic RPA2 subunits, including Rfa2 in Saccharomyces cerevisiae. An aspartic acid/alanine-scanning and genetic interaction approach was utilized to delineate the importance of this domain in budding yeast. It was determined that the Rfa2 N-terminus is important for a proper DNA-damage response in yeast, although its phosphorylation is not required. Subregions of the Rfa2 N-terminus important for the DNA-damage response were also identified. Finally, an Rfa2 N-terminal hyperphosphorylation-mimetic mutant behaves similarly to another Rfa1 mutant (rfa1-t11) with respect to genetic interactions, DNA-damage sensitivity, and checkpoint adaptation. Our data indicate that post-translational modification of the Rfa2 N-terminus is not required for cells to deal with "repairable" DNA damage; however, post-translational modification of this domain might influence whether cells proceed into M-phase in the continued presence of unrepaired DNA lesions as a "last-resort" mechanism for cell survival.KEYWORDS Replication Protein A; DNA damage; checkpoint adaptation; phosphorylation; genetic interaction C ELLS encounter environmental stress on a continual basis and have evolved mechanisms to monitor the integrity of the genome and prevent temporary DNA lesions from becoming permanent DNA mutations. A central factor in genome monitoring is the protein complex Replication Protein A (RPA). The canonical RPA complex is composed of three subunits named RPA1, RPA2, and RPA3, also often referred to by their apparent molecular weights as RPA70, RPA32, and RPA14, respectively (Wold 1997;Iftode et al. 1999;Fanning et al. 2006;Zou et al. 2006;Oakley and Patrick 2010). Originally identified as a protein complex essential for in vitro SV40 DNA replication (Wold and Kelly 1988;Wold et al. 1989;Weinberg et al. 1990), this complex is also essential for DNA repair/ recombination (Longhese et al. 1994;Firmenich et al. 1995;Sung 1997;Umezu et al. 1998) and has roles in cell-cycle regulation (Longhese et al. 1996;Lee et al. 1998;Anantha et al. 2008;Anantha and Borowiec 2009). This is consistent with the major biochemical function of RPA, which is highaffinity binding to single-strand DNA (ssDNA), an intermediate of replication, repair/recombination, and substrate for checkpoint activation (Smith et al. 2010;Flynn and Zou 2010;Mimitou and Symington 2011;Ashton et al. 2013). In addition to acting as a "sensor" of DNA damag...

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Rfa2 is specifically dephosphorylated by Pph3 in Candida albicans

Cited by 11 publications

References 49 publications

Yeast PP4 Interacts with ATR Homolog Ddc2-Mec1 and Regulates Checkpoint Signaling

Yeast PP4 Interacts with ATR Homolog Ddc2-Mec1 and Regulates Checkpoint Signaling

Role of Ppt1 in multiple stress responses in Candida albicans

The DNA Damage Response and Checkpoint Adaptation in Saccharomyces cerevisiae: Distinct Roles for the Replication Protein A2 (Rfa2) N-Terminus

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