Phosphatase 2A negatively regulates mitotic exit in Saccharomyces cerevisiae

In budding yeast Saccharomyces cerevisiae, Cdc5 kinase is a component of mitotic exit network (MEN), which inactivates cyclin-dependent kinase (CDK) after chromosome segregation. cdc5-1 mutants arrest at telophase at the nonpermissive temperature due to the failure of CDK inactivation. To identify more negative regulators of MEN, we carried out a genetic screen for genes that are toxic to cdc5-1 mutants when overexpressed. Genes that encode the B-regulatory subunit (Cdc55) and the three catalytic subunits (Pph21, Pph22, and Pph3) of phosphatase 2A (PP2A) were isolated. In addition to cdc5-1, overexpression of CDC55, PPH21, or PPH22 is also toxic to other temperature-sensitive mutants that display defects in mitotic exit. Consistently, deletion of CDC55 partially suppresses the temperature sensitivity of these mutants. Moreover, in the presence of spindle damage, PP2A mutants display nuclear localized Cdc14, the key player in MEN pathway, indicative of MEN activation. All the evidence suggests the negative role of PP2A in mitotic exit. Finally, our genetic and biochemical data suggest that PP2A regulates the phosphorylation of Tem1, which acts at the very top of MEN pathway.     

The spindle assembly checkpoint regulates the phosphorylation state of a subset of DNA checkpoint proteins in Saccharomyces cerevisiae

The DNA and the spindle assembly checkpoints play key roles in maintaining genomic integrity by coordinating cell responses to DNA lesions and spindle dysfunctions, respectively. These two surveillance pathways seem to operate mostly independently of one another, and little is known about their potential physiological connections. Here, we show that in Saccharomyces cerevisiae, the activation of the spindle assembly checkpoint triggers phosphorylation changes in two components of the DNA checkpoint, Rad53 and Rad9. These modifications are independent of the other DNA checkpoint proteins and are abolished in spindle checkpoint-defective mutants, hinting at specific functions for Rad53 and Rad9 in the spindle damage response. Moreover, we found that after UV irradiation, Rad9 phosphorylation is altered and Rad53 inactivation is accelerated when the spindle checkpoint is activated, which suggests the implication of the spindle checkpoint in the regulation of the DNA damage response.     

The Saccharomyces cerevisiae Sae2 protein promotes resection and bridging of double strand break ends

When eukaryotic chromosomes undergo double strand breaks (DSBs), several evolutionarily conserved proteins, among which the MRX complex, are recruited to the break site, leading to checkpoint activation and DNA repair. The function of the Saccharomyces cerevisiae Sae2 protein, which is known to work together with the MRX complex in meiotic DSB processing and in specific mitotic DSB repair events, is only beginning to be elucidated. Here we provide new insights into the role of Sae2 in mitotic DSB repair. We show that repair by single strand annealing of a single DSB, which is generated by the HO endonuclease between direct repeats, is defective both in the absence of Sae2 and in the presence of the hypomorphic rad50s allele altering the Rad50 subunit of MRX. Moreover, SAE2 overexpression partially suppresses the rad50s single strand annealing repair defects, suggesting that the latter might arise from defective MRX-Sae2 interactions. Finally, SAE2 deletion slows down resection of an HO-induced DSB and impairs DSB end bridging. Thus, Sae2 participates in DSB single strand annealing repair by ensuring both resection and intrachromosomal association of the broken ends.     

Mediator of DNA damage checkpoint protein 1 regulates BRCA1 localization and phosphorylation in DNA damage checkpoint control

BRCA1 is a tumor suppressor involved in DNA repair and damage-induced checkpoint controls. In response to DNA damage, BRCA1 relocalizes to nuclear foci at the sites of DNA lesions. However, little is known about the regulation of BRCA1 relocalization following DNA damage. Here we show that mediator of DNA damage checkpoint protein 1 (MDC1), previously named NFBD1 or Kiaa0170, is a proximate mediator of DNA damage responses that regulates BRCA1 function. MDC1 regulates ataxia-telangiectasia-mutated (ATM)-dependent phosphorylation events at the site of DNA damage. Importantly down-regulation of MDC1 abolishes the relocalization and hyperphosphorylation of BRCA1 following DNA damage, which coincides with defective G(2)/M checkpoint control in response to DNA damage. Taken together these data suggest that MDC1 regulates BRCA1 function in DNA damage checkpoint control.     

Rad9 phosphorylation sites couple Rad53 to the Saccharomyces cerevisiae DNA damage checkpoint

Rad9 is required for the MEC1/TEL1-dependent activation of Saccharomyces cerevisiae DNA damage checkpoint pathways mediated by Rad53 and Chk1. DNA damage induces Rad9 phosphorylation, and Rad53 specifically associates with phosphorylated Rad9. We report here that multiple Mec1/Tel1 consensus [S/T]Q sites within Rad9 are phosphorylated in response to DNA damage. These Rad9 phosphorylation sites are selectively required for activation of the Rad53 branch of the checkpoint pathway. Consistent with the in vivo function in recruiting Rad53, Rad9 phosphopeptides are bound by Rad53 forkhead-associated (FHA) domains in vitro. These data suggest that functionally independent domains within Rad9 regulate Rad53 and Chk1, and support the model that FHA domain-mediated recognition of Rad9 phosphopeptides couples Rad53 to the DNA damage checkpoint pathway.     

Spontaneous chromosome loss in Saccharomyces cerevisiae is suppressed by DNA damage checkpoint functions.

Genomic instability is one of the hallmarks of cancer cells and is often the causative factor in revealing recessive gene mutations that progress cells along the pathway to unregulated growth. Genomic instability can take many forms, including aneuploidy and changes in chromosome structure. Chromosome loss, loss and reduplication, and deletions are the majority events that result in loss of heterozygosity (LOH). Defective DNA replication, repair, and recombination can significantly increase the frequency of spontaneous genomic instability. Recently, DNA damage checkpoint functions that operate during the S-phase checkpoint have been shown to suppress spontaneous chromosome rearrangements in haploid yeast strains. To further study the role of DNA damage checkpoint functions in genomic stability, we have determined chromosome loss in DNA damage checkpoint-deficient yeast strains. We have found that the DNA damage checkpoints are essential for preserving the normal chromosome number and act synergistically with homologous recombination functions to ensure that chromosomes are segregated correctly to daughter cells. Failure of either of these processes increases LOH events. However, loss of the G2/M checkpoint does not result in an increase in chromosome loss, suggesting that it is the various S-phase DNA damage checkpoints that suppress chromosome loss. The mec1 checkpoint function mutant, defective in the yeast ATR homolog, results in increased recombination through a process that is distinct from that operative in wild-type cells.     

The checkpoint Saccharomyces cerevisiae Rad9 protein contains a tandem tudor domain that recognizes DNA

DNA damage checkpoints are signal transduction pathways that are activated after genotoxic insults to protect genomic integrity. At the site of DNA damage, ‘mediator’ proteins are in charge of recruiting ‘signal transducers’ to molecules ‘sensing’ the damage. Budding yeast Rad9, fission yeast Crb2 and metazoan 53BP1 are presented as mediators involved in the activation of checkpoint kinases. Here we show that, despite low sequence conservation, Rad9 exhibits a tandem tudor domain structurally close to those found in human/mouse 53BP1 and fission yeast Crb2. Moreover, this region is important for the resistance of Saccharomyces cerevisiae to different genotoxic stresses. It does not mediate direct binding to a histone H3 peptide dimethylated on K79, nor to a histone H4 peptide dimethylated on lysine 20, as was demonstrated for 53BP1. However, the tandem tudor region of Rad9 directly interacts with single-stranded DNA and double-stranded DNAs of various lengths and sequences through a positively charged region absent from 53BP1 and Crb2 but present in several yeast Rad9 homologs. Our results argue that the tandem tudor domains of Rad9, Crb2 and 53BP1 mediate chromatin binding next to double-strand breaks. However, their modes of chromatin recognition are different, suggesting that the corresponding interactions are differently regulated.     

Characterization of DNA damage-stimulated self-interaction of Saccharomyces cerevisiae checkpoint protein Rad17p

Saccharomyces cerevisiae Rad17p is necessary for cell cycle checkpoint arrests in response to DNA damage. Its known interactions with the checkpoint proteins Mec3p and Ddc1p in a PCNA-like complex indicate a sensor role in damage recognition. In a novel application of the yeast two-hybrid system and by immunoprecipitation, we show here that Rad17p is capable of increased self-interaction following DNA damage introduced by 4-nitroquinoline-N-oxide, camptothecin or partial inactivation of DNA ligase I. Despite overlap of regions required for Rad17p interactions with Rad17p or Mec3p, single amino acid substitutions revealed that Rad17p x Rad17p complex formation is independent of Mec3p. E128K (rad17-1) was found to inhibit Rad17p interaction with Mec3p but not with Rad17p. On the other hand, Phe-121 is essential for Rad17p self-interaction, and its function in checkpoint arrest but not for Mec3p interaction. These differential effects indicate that Rad17p-Rad17p interaction plays a role that is independent of the Rad17p x Mec3p x Ddc1p complex, although our results are also compatible with Rad17p-mediated supercomplex formation of the Rad17p x Mec3p x Ddc1p heterotrimer in response to DNA damage.     

The conserved Mec1/Rad53 nuclear checkpoint pathway regulates mitochondrial DNA copy number in Saccharomyces cerevisiae

How mitochondrial DNA (mtDNA) copy number is determined and modulated according to cellular demands is largely unknown. Our previous investigations of the related DNA helicases Pif1p and Rrm3p uncovered a role for these factors and the conserved Mec1/Rad53 nuclear checkpoint pathway in mtDNA mutagenesis and stability in Saccharomyces cerevisiae. Here, we demonstrate another novel function of this pathway in the regulation of mtDNA copy number. Deletion of RRM3 or SML1, or overexpression of RNR1, which recapitulates Mec1/Rad53 pathway activation, resulted in an approximately twofold increase in mtDNA content relative to the corresponding wild-type yeast strains. In addition, deletion of RRM3 or SML1 fully rescued the approximately 50% depletion of mtDNA observed in a pif1 null strain. Furthermore, deletion of SML1 was shown to be epistatic to both a rad53 and an rrm3 null mutation, placing these three genes in the same genetic pathway of mtDNA copy number regulation. Finally, increased mtDNA copy number via the Mec1/Rad53 pathway could occur independently of Abf2p, an mtDNA-binding protein that, like its metazoan homologues, is implicated in mtDNA copy number control. Together, these results indicate that signaling through the Mec1/Rad53 pathway increases mtDNA copy number by altering deoxyribonucleoside triphosphate pools through the activity of ribonucleotide reductase. This comprises the first linkage of a conserved signaling pathway to the regulation of mitochondrial genome copy number and suggests that homologous pathways in humans may likewise regulate mtDNA content under physiological conditions.     

HERC2-USP20 axis regulates DNA damage checkpoint through Claspin

The DNA damage response triggers cell-cycle checkpoints, DNA repair and apoptosis using multiple post-translational modifications as molecular switches. However, how ubiquitination regulates ATR signaling in response to replication stress and single-strand break is still unclear. Here, we identified the deubiquitination enzyme (DUB) USP20 as a pivotal regulator of ATR-related DDR pathway. Through screening a panel of DUBs, we identified USP20 as critical for replication stress response. USP20 is phosphorylated by ATR, resulting in disassociation of the E3 ubiquitin ligase HERC2 from USP20 and USP20 stabilization. USP20 in turn deubiquitinates and stabilizes Claspin and enhances the activation of ATR-Chk1 signaling. These findings reveal USP20 to be a novel regulator of ATR-dependent DNA damage signaling.     

UBC9 autosumoylation negatively regulates sumoylation of septins in Saccharomyces cerevisiae

Sumoylation regulates a wide range of cellular processes. However, little is known about the regulation of the SUMO machinery. In this study, we demonstrate that two lysine residues (Lys-153 and Lys-157) in the C-terminal region of the yeast E2-conjugating enzyme Ubc9 are the major and minor autosumoylation sites, respectively. Surprisingly, mutation of Lys-157 (ubc9(K157R)) significantly stimulates the level of Ubc9 autosumoylation at Lys-153. The functional role of Ubc9 autosumoylation is exemplified in our findings that cell cycle-dependent sumoylation of cytoskeletal septin proteins is inversely correlated with the Ubc9 autosumoylation level and that mutation of the Ubc9 autosumoylation sites results in aberrant cell morphology. Our study elucidates a regulatory mechanism that utilizes automodification of the E2 enzyme of the sumoylation machinery to control substrate sumoylation.     

MAD2 interacts with DNA repair proteins and negatively regulates DNA damage repair

MAD2 (mitotic arrest deficient 2) is a key regulator of mitosis. Recently, it had been suggested that MAD2-induced mitotic arrest mediates DNA damage response and that upregulation of MAD2 confers sensitivity to DNA-damaging anticancer drug-induced apoptosis. In this study, we report a potential novel role of MAD2 in mediating DNA nucleotide excision repair through physical interactions with two DNA repair proteins, XPD (xeroderma pigmentosum complementation group D) and ERCC1. First, overexpression of MAD2 resulted in decreased nuclear accumulation of XPD, a crucial step in the initiation of DNA repair. Second, immunoprecipitation experiments showed that MAD2 was able to bind to XPD, which led to competitive suppression of binding activity between XPD and XPA, resulting in the prevention of physical interactions between DNA repair proteins. Third, unlike its role in mitosis, the N-terminus domain seemed to be more important in the binding activity between MAD2 and XPD. Fourth, phosphorylation of H2AX, a process that is important for recruitment of DNA repair factors to DNA double-strand breaks, was suppressed in MAD2-overexpressing cells in response to DNA damage. These results suggest a negative role of MAD2 in DNA damage response, which may be accounted for its previously reported role in promoting sensitivity to DNA-damaging agents in cancer cells. However, the interaction between MAD2 and ERCC1 did not show any effect on the binding activity between ERCC1 and XPA in the presence or absence of DNA damage. Our results suggest a novel function of MAD2 by interfering with DNA repair proteins.     

The spindle checkpoint protein Mad2 regulates APC/C activity during prometaphase and metaphase of meiosis I in Saccharomyces cerevisiae

In many eukaryotes, disruption of the spindle checkpoint protein Mad2 results in an increase in meiosis I nondisjunction, suggesting that Mad2 has a conserved role in ensuring faithful chromosome segregation in meiosis. To characterize the meiotic function of Mad2, we analyzed individual budding yeast cells undergoing meiosis. We find that Mad2 sets the duration of meiosis I by regulating the activity of APC(Cdc20). In the absence of Mad2, most cells undergo both meiotic divisions, but securin, a substrate of the APC/C, is degraded prematurely, and prometaphase I/metaphase I is accelerated. Some mad2Δ cells have a misregulation of meiotic cell cycle events and undergo a single aberrant division in which sister chromatids separate. In these cells, both APC(Cdc20) and APC(Ama1) are prematurely active, and meiosis I and meiosis II events occur in a single meiotic division. We show that Mad2 indirectly regulates APC(Ama1) activity by decreasing APC(Cdc20) activity. We propose that Mad2 is an important meiotic cell cycle regulator that ensures the timely degradation of APC/C substrates and the proper orchestration of the meiotic divisions.     

Saccharomyces cerevisiae Sae2- and Tel1-dependent single-strand DNA formation at DNA break promotes microhomology-mediated end joining

Microhomology-mediated end joining (MMEJ) joins DNA ends via short stretches [5-20 nucleotides (nt)] of direct repeat sequences, yielding deletions of intervening sequences. Non-homologous end joining (NHEJ) and single-strand annealing (SSA) are other error prone processes that anneal single-stranded DNA (ssDNA) via a few bases (<5 nt) or extensive direct repeat homologies (>20 nt). Although the genetic components involved in MMEJ are largely unknown, those in NHEJ and SSA are characterized in some detail. Here, we surveyed the role of NHEJ or SSA factors in joining of double-strand breaks (DSBs) with no complementary DNA ends that rely primarily on MMEJ repair. We found that MMEJ requires the nuclease activity of Mre11/Rad50/Xrs2, 3' flap removal by Rad1/Rad10, Nej1, and DNA synthesis by multiple polymerases including Pol4, Rad30, Rev3, and Pol32. The mismatch repair proteins, Rad52 group genes, and Rad27 are dispensable for MMEJ. Sae2 and Tel1 promote MMEJ but inhibit NHEJ, likely by regulating Mre11-dependent ssDNA accumulation at DNA break. Our data support the role of Sae2 and Tel1 in MMEJ and genome integrity.     

RAD53 regulates DBF4 independently of checkpoint function in Saccharomyces cerevisiae

The Cdc7p and Dbf4p proteins form an active kinase complex in Saccharomyces cerevisiae that is essential for the initiation of DNA replication. A genetic screen for mutations that are lethal in combination with cdc7-1 led to the isolation of seven lsd (lethal with seven defect) complementation groups. The lsd7 complementation group contained two temperature-sensitive dbf4 alleles. The lsd1 complementation group contained a new allele of RAD53, which was designated rad53-31. RAD53 encodes an essential protein kinase that is required for the activation of DNA damage and DNA replication checkpoint pathways, and that is implicated as a positive regulator of S phase. Unlike other RAD53 alleles, we demonstrate that the rad53-31 allele retains an intact checkpoint function. Thus, the checkpoint function and the DNA replication function of RAD53 can be functionally separated. The activation of DNA replication through RAD53 most likely occurs through DBF4. Two-hybrid analysis indicates that the Rad53p protein binds to Dbf4p. Furthermore, the steady-state level of DBF4 message and Dbf4p protein is reduced in several rad53 mutant strains, indicating that RAD53 positively regulates DBF4. These results suggest that two different functions of the cell cycle, initiation of DNA replication and the checkpoint function, can be coordinately regulated through the common intermediate RAD53.     

Tor1 regulates protein solubility in Saccharomyces cerevisiae

Accumulation of insoluble protein in cells is associated with aging and aging-related diseases; however, the roles of insoluble protein in these processes are uncertain. The nature and impact of changes to protein solubility during normal aging are less well understood. Using quantitative mass spectrometry, we identify 480 proteins that become insoluble during postmitotic aging in Saccharomyces cerevisiae and show that this ensemble of insoluble proteins is similar to those that accumulate in aging nematodes. SDS-insoluble protein is present exclusively in a nonquiescent subpopulation of postmitotic cells, indicating an asymmetrical distribution of this protein. In addition, we show that nitrogen starvation of young cells is sufficient to cause accumulation of a similar group of insoluble proteins. Although many of the insoluble proteins identified are known to be autophagic substrates, induction of macroautophagy is not required for insoluble protein formation. However, genetic or chemical inhibition of the Tor1 kinase is sufficient to promote accumulation of insoluble protein. We conclude that target of rapamycin complex 1 regulates accumulation of insoluble proteins via mechanisms acting upstream of macroautophagy. Our data indicate that the accumulation of proteins in an SDS-insoluble state in postmitotic cells represents a novel autophagic cargo preparation process that is regulated by the Tor1 kinase.     

Dampening DNA damage checkpoint signalling via coordinated BRCT domain interactions

In response to DNA damage, checkpoint signalling protects genome integrity at the cost of repressing cell cycle progression and DNA replication. Mechanisms for checkpoint down‐regulation are therefore necessary for proper cellular proliferation. We recently uncovered a phosphatase‐independent mechanism for dampening checkpoint signalling, where the checkpoint adaptor Rad9 is counteracted by the repair scaffolds Slx4‐Rtt107. Here, we establish the molecular requirements for this new mode of checkpoint regulation. We engineered a minimal multi‐BRCT‐domain (MBD) module that recapitulates the action of Slx4‐Rtt107 in checkpoint down‐regulation. MBD mimics the damage‐induced Dpb11‐Slx4‐Rtt107 complex by synergistically interacting with lesion‐specific phospho‐sites in Ddc1 and H2A. We propose that efficient recruitment of Dpb11‐Slx4‐Rtt107 or MBD via a cooperative ‘two‐site‐docking’ mechanism displaces Rad9. MBD also interacts with the Mus81 nuclease following checkpoint dampening, suggesting a spatio‐temporal coordination of checkpoint signalling and DNA repair via a combinatorial mode of BRCT‐domains interactions.