Protein phosphatase Pph3 and its regulatory subunit Psy2 regulate Rad53 dephosphorylation and cell morphogenesis during recovery from DNA damage in Candida albicans

The ability of the pathogenic fungus Candida albicans to switch cellular morphologies is important for infection and virulence. Recent studies have revealed that C. albicans yeast cells can switch to filamentous growth under genotoxic stress in a manner dependent on the DNA replication/damage checkpoint. Here, we have investigated the functions of Pph3 (orf19.4378) and Psy2 (orf19.3685), whose orthologues in Saccharomyces cerevisiae mediate the dephosphorylation of the DNA damage checkpoint kinase Rad53 and the histone variant H2AX during recovery from DNA damage. Deleting PPH3 or PSY2 causes hypersensitivity to DNA-damaging agents, including cisplatin, methylmethane sulfonate (MMS), and UV light. In addition, pph3Δ and psy2Δ cells exhibit strong filamentous growth under genotoxic stress. Flow cytometry analysis shows that the mutant cells have lost the ability to adapt to genotoxic stress and remain arrested even after the stress is withdrawn. Furthermore, we show that Pph3 and Psy2 are required for the dephosphorylation of Rad53, but not H2AX, during DNA damage recovery. Taken together, these results show that C. albicans Pph3 and Psy2 have important roles in mediating genotoxin-induced filamentous growth and regulating Rad53 dephosphorylation.     

Characterization of the role of a trimeric protein phosphatase complex in recovery from cisplatin-induced versus noncrosslinking DNA damage

Cisplatin (cis-diamminedichloroplatinum) and related chemotherapeutic DNA-crosslinking agents are widely used to treat human cancers. Saccharomyces cerevisiae with separate deletions of the genes encoding the trimeric protein serine/threonine phosphatase (Pph)3p-platinum sensitivity (Psy)4p-Psy2p complex, are more sensitive than the isogenic wild-type (WT) strain to cisplatin. We show here that cisplatin causes an enhanced intra-S-phase cell cycle delay in these three deletion mutants. The C-terminal tail of histone 2AX (H2AX) is hyperphosphorylated in the same mutants, and Pph3p is found to interact with phosphorylated H2AX (gammaH2AX). After cisplatin treatment is terminated, pph3Delta, psy4Delta and psy2Delta mutants are delayed as compared with the WT strain in the dephosphorylation of Rad53p. In contrast, only pph3Delta and psy2Delta cells are more sensitive than WT cells to methylmethanesulfonate, a noncrosslinking DNA-alkylating agent that is known to cause a Rad53p-dependent intra-S-phase cell cycle delay. Dephosphorylation of Rad53p and the recovery of chromosome replication are delayed in the same mutants, but not in psy4Delta cells. By comparison with their mammalian orthologues, the regulatory subunit Psy4p is likely to inhibit Pph3p catalytic activity. The presence of a weak but active Pph3p-Psy2p complex may allow psy4Delta cells to escape from the Rad53p-mediated cell cycle arrest. Overall, our data suggest that the trimeric Pph3p-Psy4p-Psy2p complex may dephosphorylate both gammaH2AX and Rad53p, the differences lying in the more stable interaction of the Pph3 phosphatase with gammaH2AX as opposed to a transient interaction with Rad53p.     

Phosphatase Complex Pph3/Psy2 Is Involved in Regulation of Efficient Non-Homologous End-Joining Pathway in the Yeast Saccharomyces cerevisiae

One of the main mechanisms for double stranded DNA break (DSB) repair is through the non-homologous end-joining (NHEJ) pathway. Using plasmid and chromosomal repair assays, we showed that deletion mutant strains for interacting proteins Pph3p and Psy2p had reduced efficiencies in NHEJ. We further observed that this activity of Pph3p and Psy2p appeared linked to cell cycle Rad53p and Chk1p checkpoint proteins. Pph3/Psy2 is a phosphatase complex, which regulates recovery from the Rad53p DNA damage checkpoint. Overexpression of Chk1p checkpoint protein in a parallel pathway to Rad53p compensated for the deletion of PPH3 or PSY2 in a chromosomal repair assay. Double mutant strains Δpph3/Δchk1 and Δpsy2/Δchk1 showed additional reductions in the efficiency of plasmid repair, compared to both single deletions which is in agreement with the activity of Pph3p and Psy2p in a parallel pathway to Chk1p. Genetic interaction analyses also supported a role for Pph3p and Psy2p in DNA damage repair, the NHEJ pathway, as well as cell cycle progression. Collectively, we report that the activity of Pph3p and Psy2p further connects NHEJ repair to cell cycle progression.     

Critical role of DNA checkpoints in mediating genotoxic-stress-induced filamentous growth in Candida albicans

The polymorphic fungus Candida albicans switches from yeast to filamentous growth in response to a range of genotoxic insults, including inhibition of DNA synthesis by hydroxyurea (HU) or aphidicolin (AC), depletion of the ribonucleotide-reductase subunit Rnr2p, and DNA damage induced by methylmethane sulfonate (MMS) or UV light (UV). Deleting RAD53, which encodes a downstream effector kinase for both the DNA-replication and DNA-damage checkpoint pathways, completely abolished the filamentous growth caused by all the genotoxins tested. Deleting RAD9, which encodes a signal transducer of the DNA-damage checkpoint, specifically blocked the filamentous growth induced by MMS or UV but not that induced by HU or AC. Deleting MRC1, the counterpart of RAD9 in the DNA-replication checkpoint, impaired DNA synthesis and caused cell elongation even in the absence of external genotoxic insults. Together, the results indicate that the DNA-replication/damage checkpoints are critically required for the induction of filamentous growth by genotoxic stress. In addition, either of two mutations in the FHA1 domain of Rad53p, G65A, and N104A, nearly completely blocked the filamentous-growth response but had no significant deleterious effect on cell-cycle arrest. These results suggest that the FHA domain, known for its ability to bind phosphopeptides, has an important role in mediating genotoxic-stress-induced filamentous growth and that such growth is a specific, Rad53p-regulated cellular response in C. albicans.     

Phosphatases,DNA Damage Checkpoints and Checkpoint Deactivation

Cells have evolved intricate and specialized responses to DNA damage, central to which are the DNA damage checkpoints that arrest cell cycle progression and facilitate the repair process. Activation of these damage checkpoints relies heavily on the activity of Ser/Thr kinases, such as Chk1 and Chk2 (Saccharomyces cerevisiae Rad53), which are themselves activated by phosphorylation. Only more recently have we begun to understand how cells disengage the checkpoints to reenter the cell cycle. Here, we review progress toward understanding the functions of phosphatases in checkpoint deactivation in S. cerevisiae, focusing on the non-redundant roles of the type 2A phosphatase Pph3 and the PP2C phosphatases Ptc2 and Ptc3 in the deactivation of Rad53. We discuss how these phosphatases may specifically recognize different phosphorylated forms of Rad53 and how each may independently regulate different facets of the checkpoint response. In conjunction with the independent dephosphorylation of other checkpoint proteins, such regulation may allow a more tailored response to DNA damage that is coordinated with the repair process, ultimately resulting in the resumption of growth.     

Distinct phosphatases mediate the deactivation of the DNA damage checkpoint kinase Rad53

The DNA damage checkpoint regulates DNA replication and arrests cell cycle progression in response to genotoxic stress. In Saccharomyces cerevisiae, the protein kinase Rad53 plays a central role in preventing genomic instability and maintaining viability in the presence of replication stress and DNA damage. Activation of Rad53 depends on phosphorylation by the upstream kinase Mec1, followed by autophosphorylation on multiple residues. Also critical for cell viability, the molecular mechanism of Rad53 deactivation remains incompletely understood. Rad53 dephosphorylation after repair of a persistent double strand break in G(2)/M has been shown to depend on the presence of the PP2C-type phosphatases Ptc2 and Ptc3. More recently, the PP2A-like protein phosphatase Pph3 has been shown to be required to dephosphorylate Rad53 after DNA methylation damage in S phase. However, we show here that Ptc2/3 are dispensable for Rad53 deactivation after replication stress or DNA methylation damage. Pph3 is also dispensable for the deactivation of Rad53 after replication stress. In addition, Rad53 kinase activity is still deactivated in pph3 null cells after DNA methylation damage, despite persistent Rad53 hyperphosphorylation. Finally, a strain in which the three phosphatases are deleted shows a severe defect in Rad53 kinase deactivation after DNA methylation damage but not after replication stress. In all, our results suggest that distinct phosphatases operate to return Rad53 to its basal state after different genotoxic stresses and that a yet unidentified phosphatase may be responsible for the deactivation of Rad53 after replication stress.     

The Protein Factor-arrest 11 (Far11) Is Essential for the Toxicity of Human Caspase-10 in Yeast and Participates in the Regulation of Autophagy and the DNA Damage Signaling

The heterologous expression of human caspase-10 in Saccharomyces cerevisiae induces a lethal phenotype, which includes some hallmarks of apoptosis and autophagy, alterations in the intra-S checkpoint, and cell death. To determine the cellular processes and pathways that are responsible of the caspase-10-induced cell death we have designed a loss-of-function screening system to identify genes that are essential for the lethal phenotype. We observed that the ER-Golgi-localized family of proteins Far, MAPK signaling, the autophagy machinery, and several kinases and phosphatases are essential for caspase-10 toxicity. We also found that the expression of caspase-10 elicits a simultaneous activation of the MAP kinases Fus3, Kss1, and Slt2. Furthermore, the protein Far11, which is a target of MAP kinases, is essential for the dephosphorylation of Atg13 and, consequently, for the induction of autophagy. In addition, Far11 participates in the regulation of the DNA damage response through the dephosphorylation of Rad53. Finally, we have also demonstrated that Far11 is able to physically interact with the phosphatases Pph21, Pph22, and Pph3. Overall, our results indicate that the expression of human caspase-10 in S. cerevisiae activates an intracellular death signal that depends on the Far protein complex and that Far11 may function as a regulator subunit of phosphatases in different processes, thus representing a mechanistic link between them.     

The essentiality landscape of cell cycle related genes in human pluripotent and cancer cells

Mechanisms controlling DNA resection at sites of damage and affecting genome stability have been the subject of deep investigation, though their complexity is not yet fully understood. Specifically, the regulatory role of post-translational modifications in the localization, stability and function of DNA repair proteins is an important aspect of such complexity. Here, we took advantage of the superior resolution of phosphorylated proteins provided by Phos-Tag technology to study pathways controlling the reversible phosphorylation of yeast Exo1, an exonuclease involved in a number of DNA repair pathways. We report that Rad53, a checkpoint kinase downstream of Mec1, is responsible for Exo1 phosphorylation in response to DNA replication stress and we demonstrate a role for the type-2A protein phosphatase Pph3 in the dephosphorylation of both Rad53 and Exo1 during checkpoint recovery. Fluorescence microscopy studies showed that Rad53-dependent phosphorylation is not required for the recruitment or the release of Exo1 from the nucleus, whereas 14-3-3 proteins are necessary for Exo1 nuclear translocation. By shedding light on the mechanism of Exo1 control, these data underscore the importance of post-translational modifications and protein interactions in the regulation of DNA end resection.     

A Rad53 Independent Function of Rad9 Becomes Crucial for Genome Maintenance in the Absence of the RecQ Helicase Sgs1

The conserved family of RecQ DNA helicases consists of caretaker tumour suppressors, that defend genome integrity by acting on several pathways of DNA repair that maintain genome stability. In budding yeast, Sgs1 is the sole RecQ helicase and it has been implicated in checkpoint responses, replisome stability and dissolution of double Holliday junctions during homologous recombination. In this study we investigate a possible genetic interaction between SGS1 and RAD9 in the cellular response to methyl methane sulphonate (MMS) induced damage and compare this with the genetic interaction between SGS1 and RAD24. The Rad9 protein, an adaptor for effector kinase activation, plays well-characterized roles in the DNA damage checkpoint response, whereas Rad24 is characterized as a sensor protein also in the DNA damage checkpoint response. Here we unveil novel insights into the cellular response to MMS-induced damage. Specifically, we show a strong synergistic functionality between SGS1 and RAD9 for recovery from MMS induced damage and for suppression of gross chromosomal rearrangements, which is not the case for SGS1 and RAD24. Intriguingly, it is a Rad53 independent function of Rad9, which becomes crucial for genome maintenance in the absence of Sgs1. Despite this, our dissection of the MMS checkpoint response reveals parallel, but unequal pathways for Rad53 activation and highlights significant differences between MMS- and hydroxyurea (HU)-induced checkpoint responses with relation to the requirement of the Sgs1 interacting partner Topoisomerase III (Top3). Thus, whereas earlier studies have documented a Top3-independent role of Sgs1 for an HU-induced checkpoint response, we show here that upon MMS treatment, Sgs1 and Top3 together define a minor but parallel pathway to that of Rad9.     

Limiting amounts of budding yeast Rad53 S-phase checkpoint activity results in increased resistance to DNA alkylation damage

The Saccharomyces cerevisiae protein kinase Rad53 plays a key role in maintaining genomic integrity after DNA damage and is an essential component of the ‘intra-S-phase checkpoint’. In budding yeast, alkylating chemicals, such as methyl methanesulfonate (MMS), or depletion of nucleotides by hydroxyurea (HU) stall DNA replication forks and thus activate Rad53 during S-phase. This stabilizes stalled DNA replication forks and prevents the activation of later origins of DNA replication. Here, we report that a reduction in the level of Rad53 kinase causes cells to behave very differently in response to DNA alkylation or to nucleotide depletion. While cells lacking Rad53 are unable to activate the checkpoint response to HU or MMS, so that they rapidly lose viability, a reduction in Rad53 enhances cell survival only after DNA alkylation. This reduction in the level of Rad53 allows S-phase cells to maintain the stability of DNA replication forks upon MMS treatment, but does not prevent the collapse of forks in HU. Our results may have important implications for cancer therapies, as they suggest that partial impairment of the S-phase checkpoint Rad53/Chk2 kinase provides cells with a growth advantage in the presence of drugs that damage DNA.     

白色念珠菌CaBEM1基因的克隆及其在酿酒酵母丝状生长中的作用

白色念珠菌在不同的生长条件下能发生显著的形态变化 ,这种变化由多种调控因子与信号转导途径所调控。酿酒酵母的G1期细胞周期蛋白Cln1和Cln2参与其形态发生 ,cln1/cln1、cln2 /cln2双缺失株不能形成菌丝。把白色念珠菌基因组文库导入cln1/cln1、cln2 /cln2缺失株 ,筛选能校正菌丝形成缺陷的基因 ,分离得到白色念珠菌中的CaBEM 1基因。从核苷酸序列推导 ,CaBEM1编码一种 6 32个氨基酸的蛋白质 ,氨基酸序列分析表明在其N端有 2个SH3结构域 ,中部有 1个PX结构域 ,C端有 1个PB1结构域 ;CaBem1的氨基酸序列与酿酒酵母的Bem1同源性达 38% ,与裂殖酵母的Scd2同源性达 32 %。在酿酒酵母的缺失株中异源表达CaBEM1,能够部分校正它们在氮源缺乏条件下的菌丝形成缺陷。这种菌丝形成的校正作用绕过MAPK途径和cAMP/PKA途径 ,表明CaBem1在菌丝形成中的作用可能位于这两条信号转导途径的下游     

Acute ethanol stress induces sumoylation of conserved chromatin structural proteins in Saccharomyces cerevisiae

Stress is ubiquitous to life and can irreparably damage essential biomolecules and organelles in cells. To survive, organisms must sense and adapt to stressful conditions. One highly conserved adaptive stress response is through the posttranslational modification of proteins by the small ubiquitin-like modifier (SUMO). Here, we examine the effects of acute ethanol stress on protein sumoylation in the budding yeast Saccharomyces cerevisiae. We found that cells exhibit a transient sumoylation response after acute exposure to ≤7.5% vol/vol ethanol. By contrast, the sumoylation response becomes chronic at 10% ethanol exposure. Mass spectrometry analyses identified 18 proteins that are sumoylated after acute ethanol exposure, with 15 known to associate with chromatin. Upon further analysis, we found that the chromatin structural proteins Smc5 and Smc6 undergo ethanol-induced sumoylation that depends on the activity of the E3 SUMO ligase Mms21. Using cell-cycle arrest assays, we observed that Smc5 and Smc6 ethanol-induced sumoylation occurs during G1 and G2/M phases but not S phase. Acute ethanol exposure also resulted in the formation of Rad52 foci at levels comparable to Rad52 foci formation after exposure to the DNA alkylating agent methyl methanesulfonate (MMS). MMS exposure is known to induce the intra-S-phase DNA damage checkpoint via Rad53 phosphorylation, but ethanol exposure did not induce Rad53 phosphorylation. Ethanol abrogated the effect of MMS on Rad53 phosphorylation when added simultaneously. From these studies, we propose that acute ethanol exposure induces a change in chromatin leading to sumoylation of specific chromatin structural proteins.     

The ATM-related Tel1 protein of Saccharomyces cerevisiae controls a checkpoint response following phleomycin treatment

MEC1 and TEL1 encode ATR- and ATM-related proteins in the budding yeast Saccharomyces cerevisiae, respectively. Phleomycin is an agent that catalyzes double-strand breaks in DNA. We show here that both Mec1 and Tel1 regulate the checkpoint response following phleomycin treatment. MEC1 is required for Rad53 phosphorylation and cell-cycle progression delay following phleomycin treatment in G1, S or G2/M phases. The tel1Δ mutation confers a defect in the checkpoint responses to phleomycin treatment in S phase. In addition, the tel1Δ mutation enhances the mec1 defect in activation of the phleomycin-induced checkpoint pathway in S phase. In contrast, the tel1Δ mutation confers only a minor defect in the checkpoint responses in G1 phase and no apparent defect in G2/M phase. Methyl methanesulfonate (MMS) treatment also activates checkpoints, inducing Rad53 phosphorylation in S phase. MMS-induced Rad53 phosphorylation is not detected in mec1Δ mutants during S phase, but occurs in tel1Δ mutants similar to wild-type cells. Finally, Xrs2 is phosphorylated after phleomycin treatment in a TEL1-dependent manner during S phase, whereas no significant Xrs2 phosphorylation is detected after MMS treatment. Together, our results support a model in which Tel1 contributes to checkpoint control in response to phleomycin-induced DNA damage in S phase.     

Identification of an exoribonuclease homolog, CaKEM1/CaXRN1, in Candida albicans and its characterization in filamentous growth

KEM1/XRN1 and RAT1 are two known exoribonuclease genes in Saccharomyces cereivsiae and encode a cytoplasmic and nuclear exoribonuclease, respectively. CaKEM1/CaXRN1 and CaRAT1, the Candida albicans homologs of 5'-->3' exoribonuclease genes, were identified by protein sequence comparisons and by functional complementation of the S. cerevisiae kem1/xrn1 null mutation. The deduced amino acid sequences of CaKEM1 and CaRAT1 show 51% and 55% identities to those of the S. cerevisiae KEM1 and RAT1, respectively. The exonuclease motifs were found to be highly conserved in CaKem1p and CaRat1p. We disrupted two chromosomal copies of CaKEM1 in a diploid C. albicans strain and demonstrate that C. albicans kem1/kem1 mutants are defective in filamentous growth on filamentous-inducing media. These results imply that CaKEM1 is involved in filamentous growth of C. albicans.     

Regulation of Rfa2 phosphorylation in response to genotoxic stress in Candida albicans

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 Candida 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.