Role of Mammalian Rad9 in Genomic Stability and Ionizing Radiation Response

Eukaryotic cells have evolved DNA damage response mechanisms utilizing proficient DNA repair and cell cycle checkpoints in order to maintain genomic stability. The Schizosaccharomyces pombe Rad9 gene was initially identified as encoding a cell cycle checkpoint protein. When the mammalian homologue of S. pombe Rad9 was inactivated, however, chromosomal instability was observed even in the absence of DNA damaging agents. Both an increase in chromosome end-to-end associations and telomere loss were observed in cells with inactivated mammalian Rad9. This telomere instability correlated with enhanced S- and G2-phase specific cell killing, delayed kinetics of γ-H2AX foci appearance and disappearance, and reduced chromosomal repair after ionizing radiation (IR) exposure, suggesting that Rad9 plays a role in cell cycle phase specific DNA damage repair. Inactivation of mammalian Rad9 also resulted in decreased homologous recombinational (HR) repair, which occurs predominantly in the S- and G2-phase of the cell cycle. These newly defined functions of mammalian Rad9 are discussed in relation to telomere stability and HR repair as a mechanism for promoting cell survival after IR exposure.     

The role of Schizosaccharomyces pombe Rad32, the Mre11 homologue, and other DNA damage response proteins in non-homologous end joining and telomere length maintenance.

The Schizosaccharomyces pombe homologue of Mre11, Rad32, is required for repair of UV- and ionising radiation-induced DNA damage and meiotic recombination. In this study we have investigated the role of Rad32 and other DNA damage response proteins in non-homologous end joining (NHEJ) and telomere length maintenance in S.pombe. We show that NHEJ in S.pombe occurs by an error-prone mechanism, in contrast to the accurate repair observed in Saccharomyces cerevisiae. Deletion of the rad32 gene results in a modest reduction in NHEJ activity and the remaining repair events that occur are accurate. Mutations in two of the phosphoesterase motifs in Rad32 have no effect on the efficiency or accuracy of end joining, suggesting that the role of Rad32 protein may be to recruit another nuclease(s) for processing during the end joining reaction. We also analysed NHEJ in other DNA damage response mutants and showed that the checkpoint mutant rad3-d and two recombination mutants defective in rhp51 and rhp54 (homologues of S.cerevisiae RAD51 and RAD54, respectively) are not affected. However disruption of rad22, rqh1 and rhp9 / crb2 (homologues of the S.cerevisiae RAD52, SGS1 and RAD9 genes) resulted in increased NHEJ activity. Telomere lengths in the rad32, rhp9 and rqh1 null alleles were reduced to varying extents intermediate between the lengths observed in wild-type and rad3 null cells.     

cDNA Cloning and Gene Mapping of Human Homologs forSchizosaccharomyces pombe rad17, rad1,andhus1and Cloning of Homologs from Mouse,Caenorhabditis elegans,andDrosophila melanogaster

Mutations in DNA repair/cell cycle checkpoint genes can lead to the development of cancer. The cloning of human homologs of yeast DNA repair/cell cycle checkpoint genes should yield candidates for human tumor suppressor genes as well as identifying potential targets for cancer therapy. TheSchizosaccharomyces pombegenesrad17, rad1,andhus1have been identified as playing roles in DNA repair and cell cycle checkpoint control pathways. We have cloned the cDNA for the human homolog ofS. pombe rad17,RAD17, which localizes to chromosomal location 5q13 by fluorescencein situhybridization and radiation hybrid mapping; the cDNA for the human homolog ofS. pombe rad1,RAD1, which maps to 5p14–p13.2; and the cDNA for the human homolog ofS. pombe hus1,HUS1, which maps to 7p13–p12. The human gene loci have previously been identified as regions containing tumor suppressor genes. In addition, we report the cloning of the cDNAs for genes related toS. pombe rad17, rad9, rad1,andhus1from mouse,Caenorhabditis elegans,andDrosophila melanogaster.These includeRad17andRad9fromD. melanogaster,hpr-17 and hpr-1 fromC. elegans,and RAD1 and HUS1 from mouse. The identification of homologs of theS. pomberad checkpoint genes from mammals, arthropods, and nematodes indicates that this cell cycle checkpoint pathway is conserved throughout eukaryotes.     

Rad3 and Sty1 function in Schizosaccharomyces pombe: an integrated response to DNA damage and environmental stress?

In Schizosaccharomyces pombe , the Ataxia Telangiectasia-mutated (Atm)/Atm and Rad 3 Related (Atr) homologue Rad3 is an essential regulator of the response to DNA damage and stalled replication forks. Rad3 activates the downstream kinases Chk1 and Cds1. These kinases in turn inhibit cell cycle progression by mediating Cdc2 phosphorylation. Studies in both yeast and mammalian cells suggest additional roles for Rad3 in regulating cellular responses to environmental stress. In S. pombe , cellular responses to various environmental stresses are regulated primarily through the stress-activated MAP kinase p38 homologue Sty1. An important function of Sty1 is to drive cells rapidly through mitosis by facilitating the accumulation of Cdc25. Interestingly, Sty1 is activated simultaneously with Rad3 following exposure to UV radiation or ionizing radiation (IR). Similarly, exposure to environmental stresses induces the expression of rad3 ⁺, cds1 ⁺ and other checkpoint regulator genes. It is currently unclear how the pathways regulated by Sty1 and Rad3 and their opposing effects on mitosis are integrated. Recent studies suggest that Sty1 and Rad3 function together to regulate the expression of several stress response genes following exposure to IR. In this review, we discuss current knowledge on the interaction of Rad3/Atm and Sty1/p38 in regulating cellular responses to environmental stress and DNA damage.     

Molecular characterization of the Schizosaccharomyces pombe nbs1+ gene involved in DNA repair and telomere maintenance

The human MRN complex is a multisubunit nuclease that is composed of Mre11, Rad50, and Nbs1 and is involved in homologous recombination and DNA damage checkpoints. Mutations of the MRN genes cause genetic disorders such as Nijmegen breakage syndrome. Here we identified a Schizosaccharomyces pombe nbs1(+) homologue by screening for mutants with mutations that caused methyl methanesulfonate (MMS) sensitivity and were synthetically lethal with the rad2Delta mutation. Nbs1 physically interacts with the C-terminal half of Rad32, the Schizosaccharomyces pombe Mre11 homologue, in a yeast two-hybrid assay. nbs1 mutants showed sensitivities to gamma-rays, UV, MMS, and hydroxyurea and displayed telomere shortening similar to the characteristics of rad32 and rad50 mutants. nbs1, rad32, and rad50 mutant cells were elongated and exhibited abnormal nuclear morphology. These findings indicate that S. pombe Nbs1 forms a complex with Rad32-Rad50 and is required for homologous recombination repair, telomere length regulation, and the maintenance of chromatin structure. Amino acid sequence features and some characteristics of the DNA repair function suggest that the S. pombe Rad32-Rad50-Nbs1 complex has functional similarity to the corresponding MRN complexes of higher eukaryotes. Therefore, S. pombe Nbs1 will provide an additional model system for studying the molecular function of the MRN complex associated with genetic diseases.     

Distinct Cdk1 requirements during single-strand annealing, noncrossover, and crossover recombination

Repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) in haploid cells is generally restricted to S/G2 cell cycle phases, when DNA has been replicated and a sister chromatid is available as a repair template. This cell cycle specificity depends on cyclin-dependent protein kinases (Cdk1 in Saccharomyces cerevisiae), which initiate HR by promoting 5'-3' nucleolytic degradation of the DSB ends. Whether Cdk1 regulates other HR steps is unknown. Here we show that yku70Δ cells, which accumulate single-stranded DNA (ssDNA) at the DSB ends independently of Cdk1 activity, are able to repair a DSB by single-strand annealing (SSA) in the G1 cell cycle phase, when Cdk1 activity is low. This ability to perform SSA depends on DSB resection, because both resection and SSA are enhanced by the lack of Rad9 in yku70Δ G1 cells. Furthermore, we found that interchromosomal noncrossover recombinants are generated in yku70Δ and yku70Δ rad9Δ G1 cells, indicating that DSB resection bypasses Cdk1 requirement also for carrying out these recombination events. By contrast, yku70Δ and yku70Δ rad9Δ cells are specifically defective in interchromosomal crossover recombination when Cdk1 activity is low. Thus, Cdk1 promotes DSB repair by single-strand annealing and noncrossover recombination by acting mostly at the resection level, whereas additional events require Cdk1-dependent regulation in order to generate crossover outcomes.     

Cell cycle progression in the presence of irreparable DNA damage is controlled by a Mec1- and Rad53-dependent checkpoint in budding yeast.

We studied the response of nucleotide excision repair (NER)-defective rad14Delta cells to UV irradiation in G(1) followed by release into the cell cycle. Only a subset of checkpoint proteins appears to mediate cell cycle arrest and regulate the timely activation of replication origins in the presence of unrepaired UV-induced lesions. In fact, Mec1 and Rad53, but not Rad9 and the Rad24 group of checkpoint proteins, are required to delay cell cycle progression in rad14Delta cells after UV damage in G(1). Consistently, Mec1-dependent Rad53 phosphorylation after UV irradiation takes place in rad14Delta cells also in the absence of Rad9, Rad17, Rad24, Mec3 and Ddc1, and correlates with entry into S phase. Two-dimensional gel analysis indicates that late replication origins are not fired in rad14Delta cells UV-irradiated in G(1) and released into the cell cycle, which instead initiate DNA replication from early origins and accumulate replication and recombination intermediates. Progression through S phase of UV-treated NER-deficient mec1 and rad53 mutants correlates with late origin firing, suggesting that unregulated DNA replication in the presence of irreparable UV-induced lesions might result from a failure to prevent initiation at late origins.     

Role of exonucleolytic degradation in group I intron homing in phage T4

The Schizosaccharomyces pombe checkpoint gene named rad3(+) encodes an ATM-homologous protein kinase that shares a highly conserved motif with proteins involved in DNA metabolism. Previous studies have shown that Rad3 fulfills its function via the regulation of the Chk1 and Cds1 protein kinases. Here we describe a novel role for Rad3 in the control of telomere integrity. Mutations in the rad3(+) gene alleviated telomeric silencing and produced shortened lengths in the telomere repeat tracts. Genetic analysis revealed that the other checkpoint rad mutations rad1, rad17, and rad26 belong to the same phenotypic class with rad3 with regard to control of the telomere length. Of these mutations, rad3 and rad26 have a drastic effect on telomere shortening. tel1(+), another ATM homologue in S. pombe, carries out its telomere maintenance function in parallel with the checkpoint rad genes. Furthermore, either a single or double disruption of cds1(+) and chk1(+) caused no obvious changes in the telomeric DNA structure. Our results demonstrate a novel role of the S. pombe ATM homologues that is independent of chk1(+) and cds1(+).     

DNA damage-dependent and -independent phosphorylation of the hRad9 checkpoint protein

Cell cycle checkpoints are regulatory mechanisms that maintain genomic integrity by preventing cell cycle progression when genetic anomalies are present. The hRad9 protein is the human homologue of Schizosaccharomyces pombe Rad9, a checkpoint protein required for preventing the onset of mitosis if DNA damage is present or if DNA replication is incomplete. Genetic and biochemical analyses indicate that hRad9 is a component of the checkpoint response in humans and has possible roles in regulating the cell cycle, apoptosis, and DNA repair. Previous studies indicate that hRad9 is modified by phosphorylation, both in the absence of exogenous stress and in response to various genotoxins. In this study, we report the mapping of several sites of constitutive phosphorylation of hRad9 to (S/T)PX(R/P) sequences near the C terminus of the protein. We also demonstrate that a serine to alanine mutation at residue 272 abrogates an ionizing radiation (IR)-induced phosphorylation of hRad9 and further show that phosphorylation at (S/T)P sites is not a prerequisite for IR-induced phosphorylation of serine 272. Finally, we report that hRad9 undergoes cell cycle-regulated hyper-phosphorylation in G(2)/M that is enhanced by IR but distinct from that on serine 272. Unlike the IR-induced phosphorylation at serine 272, this event is dependent on serine 277 and threonine 292, two C-terminal (S/T)P sites in hRad9.     

ATM-dependent phosphorylation of human Rad9 is required for ionizing radiation-induced checkpoint activation

ATM (ataxia-telangiectasia-mutated) is a Ser/Thr kinase involved in cell cycle checkpoints and DNA repair. Human Rad9 (hRad9) is the homologue of Schizosaccharomyces pombe Rad9 protein that plays a critical role in cell cycle checkpoint control. To examine the potential signaling pathway linking ATM and hRad9, we investigated the modification of hRad9 in response to DNA damage. Here we show that hRad9 protein is constitutively phosphorylated in undamaged cells and undergoes hyperphosphorylation upon treatment with ionizing radiation (IR), ultraviolet light (UV), and hydroxyurea (HU). Interestingly, hyperphosphorylation of hRad9 induced by IR is dependent on ATM. Ser(272) of hRad9 is phosphorylated directly by ATM in vitro. Furthermore, hRad9 is phosphorylated on Ser(272) in response to IR in vivo, and this modification is delayed in ATM-deficient cells. Expression of hRad9 S272A mutant protein in human lung fibroblast VA13 cells disturbs IR-induced G(1)/S checkpoint activation and increased cellular sensitivity to IR. Together, our results suggest that the ATM-mediated phosphorylation of hRad9 is required for IR-induced checkpoint activation.     

Sensor and effector kinases in DNA damage checkpoint regulate capacity for homologous recombination repair of fission yeast in G2 phase

Although the G2/M DNA damage checkpoint is currently viewed as a set of coordinated cellular responses affecting both cell cycle progression and non-cell cycle targets, the relative contributions of the two target categories to DNA repair and cell survival after exposure to ionizing radiation have not been clearly addressed. We investigated how rad3 (ATR ortholog) or chk1/cds1 (CHK1/CHK2 orthologs) null mutations change the kinetics of double-strand break (DSB) repair in Schizosaccharomyces pombe cells under conditions of forced G2 arrest. After 200-Gy γ-ray irradiation, DSBs were repaired in rad3Δ cdc25-22 or chk1Δ cds1Δ cdc25-22 cells, almost as efficiently as in cdc25-22 cells at the restrictive temperature. In contrast, little repair was observed in the checkpoint-deficient cells up to 4h after higher-dose (500Gy) irradiation, whereas repair was still efficient in the control cdc25-22 cells. Immediate loss of viability appeared not be responsible for the repair defect after the higher dose, since both checkpoint-proficient and deficient cells with cdc25-22 allele synchronously resumed cycling with a similar time course when released to the permissive temperature 4h after irradiation. Recruitment of repair proteins Rad11 (Rpa1 ortholog), Rad22 (Rad52 ortholog), and Rhp54 (Rad54 ortholog) to the damage sites was not significantly impaired in the checkpoint-deficient cells, whereas their release was profoundly delayed. Our results suggest that sensor and effector kinases in the damage checkpoint machinery affect the efficiency of repair downstream of, or in parallel with the core repair reaction.     

A novel mutant allele of Schizosaccharomyces pombe rad26 defective in monitoring S-phase progression to prevent premature mitosis.

A semipermissive growth condition was defined for a Schizosaccharomyces pombe strain carrying a thermosensitive allele of DNA polymerase delta (pol delta ts03). Under this condition, DNA polymerase delta is semidisabled and causes a delay in S-phase progression. Using a genetic strategy, we have isolated a panel of mutants that enter premature mitosis when DNA replication is incomplete but which are not defective for arrest in G2/M following DNA damage. We characterized the aya14 mutant, which enters premature mitosis when S phase is arrested by genetic or chemical means. However, this mutant is sensitive to neither UV nor gamma irradiation. Two genomic clones, rad26+ and cds1+, were found to suppress the hydroxyurea sensitivity of the aya14 mutant. Genetic analysis indicates that aya14 is a novel allele of the cell cycle checkpoint gene rad26+, which we have named rad26.a14. cds1+ is a suppressor which suppresses the S-phase feedback control defect of rad26.a14 when S phase is inhibited by either hydroxyurea or cdc22, but it does not suppress the defect when S phase is arrested by a mutant DNA polymerase. Analyses of rad26.a14 in a variety of cdc mutant backgrounds indicate that strains containing rad26.a14 bypass S-phase arrest but not G1 or late S/G2 arrest. A model of how Rad26 monitors S-phase progression to maintain the dependency of cell cycle events and coordinates with other rad/hus checkpoint gene products in responding to radiation damage is proposed.     

Roles of the checkpoint sensor clamp Rad9-Rad1-Hus1 (911)-complex and the clamp loaders Rad17-RFC and Ctf18-RFC in Schizosaccharomyces pombe telomere maintenance

While telomeres must provide mechanisms to prevent DNA repair and DNA damage checkpoint factors from fusing chromosome ends and causing permanent cell cycle arrest, these factors associate with functional telomeres and play critical roles in the maintenance of telomeres. Previous studies have established that Tel1 (ATM) and Rad3 (ATR) kinases play redundant but essential roles for telomere maintenance in fission yeast. In addition, the Rad9-Rad1-Hus1 (911) and Rad17-RFC complexes work downstream of Rad3 (ATR) in fission yeast telomere maintenance. Here, we investigated how 911, Rad17-RFC and another RFC-like complex Ctf18-RFC contribute to telomere maintenance in fission yeast cells lacking Tel1 and carrying a novel hypomorphic allele of rad3 (DBD-rad3), generated by the fusion between the DNA binding domain (DBD) of the fission yeast telomere capping protein Pot1 and Rad3. Our investigations have uncovered a surprising redundancy for Rad9 and Hus1 in allowing Rad1 to contribute to telomere maintenance in DBD-rad3 tel1 cells. In addition, we found that Rad17-RFC and Ctf18-RFC carry out redundant telomere maintenance functions in DBD-rad3 tel1 cells. Since checkpoint sensor proteins are highly conserved, genetic redundancies uncovered here may be relevant to telomere maintenance and detection of DNA damage in other eukaryotes.     

Telomere binding of checkpoint sensor and DNA repair proteins contributes to maintenance of functional fission yeast telomeres

Telomeres, the ends of linear chromosomes, are DNA double-strand ends that do not trigger a cell cycle arrest and yet require checkpoint and DNA repair proteins for maintenance. Genetic and biochemical studies in the fission yeast Schizosaccharomyces pombe were undertaken to understand how checkpoint and DNA repair proteins contribute to telomere maintenance. On the basis of telomere lengths of mutant combinations of various checkpoint-related proteins (Rad1, Rad3, Rad9, Rad17, Rad26, Hus1, Crb2, Chk1, Cds1), Tel1, a telomere-binding protein (Taz1), and DNA repair proteins (Ku70, Rad32), we conclude that Rad3/Rad26 and Tel1/Rad32 represent two pathways required to maintain telomeres and prevent chromosome circularization. Rad1/Rad9/Hus1/Rad17 and Ku70 are two additional epistasis groups, which act in the Rad3/Rad26 pathway. However, Rad3/Rad26 must have additional target(s), as cells lacking Tel1/Rad32, Rad1/Rad9/Hus1/Rad17, and Ku70 groups did not circularize chromosomes. Cells lacking Rad3/Rad26 and Tel1/Rad32 senesced faster than a telomerase trt1Delta mutant, suggesting that these pathways may contribute to telomere protection. Deletion of taz1 did not suppress chromosome circularization in cells lacking Rad3/Rad26 and Tel1/Rad32, also suggesting that two pathways protect telomeres. Chromatin immunoprecipitation analyses found that Rad3, Rad1, Rad9, Hus1, Rad17, Rad32, and Ku70 associate with telomeres. Thus, checkpoint sensor and DNA repair proteins contribute to telomere maintenance and protection through their association with telomeres.     

Analysis of Rad3 and Chk1 protein kinases defines different checkpoint responses.

Eukaryotic cells respond to DNA damage and S phase replication blocks by arresting cell-cycle progression through the DNA structure checkpoint pathways. In Schizosaccharomyces pombe, the Chk1 kinase is essential for mitotic arrest and is phosphorylated after DNA damage. During S phase, the Cds1 kinase is activated in response to DNA damage and DNA replication blocks. The response of both Chk1 and Cds1 requires the six 'checkpoint Rad' proteins (Rad1, Rad3, Rad9, Rad17, Rad26 and Hus1). We demonstrate that DNA damage-dependent phosphorylation of Chk1 is also cell-cycle specific, occurring primarily in late S phase and G2, but not during M/G1 or early S phase. We have also isolated and characterized a temperature-sensitive allele of rad3. Rad3 functions differently depending on which checkpoint pathway is activated. Following DNA damage, rad3 is required to initiate but not maintain the Chk1 response. When DNA replication is inhibited, rad3 is required for both initiation and maintenance of the Cds1 response. We have identified a strong genetic interaction between rad3 and cds1, and biochemical evidence shows a physical interaction is possible between Rad3 and Cds1, and between Rad3 and Chk1 in vitro. Together, our results highlight the cell-cycle specificity of the DNA structure-dependent checkpoint response and identify distinct roles for Rad3 in the different checkpoint responses. Keywords: ATM/ATR/cell-cycle checkpoints/Chk1/Rad3     

A role for homologous recombination proteins in cell cycle regulation

Eukaryotic cells respond to DNA breaks, especially double-stranded breaks (DSBs), by activating the DNA damage response (DDR), which encompasses DNA repair and cell cycle checkpoint signaling. The DNA damage signal is transmitted to the checkpoint machinery by a network of specialized DNA damage-recognizing and signal-transducing molecules. However, recent evidence suggests that DNA repair proteins themselves may also directly contribute to the checkpoint control. Here, we investigated the role of homologous recombination (HR) proteins in normal cell cycle regulation in the absence of exogenous DNA damage. For this purpose, we used Chinese Hamster Ovary (CHO) cells expressing the Fluorescent ubiquitination-based cell cycle indicators (Fucci). Systematic siRNA-mediated knockdown of HR genes in these cells demonstrated that the lack of several of these factors alters cell cycle distribution, albeit differentially. The knock-down of MDC1, Rad51 and Brca1 caused the cells to arrest in the G2 phase, suggesting that they may be required for the G2/M transition. In contrast, inhibition of the other HR factors, including several Rad51 paralogs and Rad50, led to the arrest in the G1/G0 phase. Moreover, reduced expression of Rad51B, Rad51C, CtIP and Rad50 induced entry into a quiescent G0-like phase. In conclusion, the lack of many HR factors may lead to cell cycle checkpoint activation, even in the absence of exogenous DNA damage, indicating that these proteins may play an essential role both in DNA repair and checkpoint signaling.     

Identification and characterization of the rlp1+, the novel Rad51 paralog in the fission yeast Schizosaccharomyces pombe

A new DNA repair gene from fission yeast Schizosaccharomyces pombe rlp1+ (RecA-like protein) has been identified. Rlp1 shows homology to RecA-like proteins, and is the third S. pombe Rad51 paralog besides Rhp55 and Rhp57. The new gene encodes a 363 aa protein with predicted Mr of 41,700 and has NTP-binding motif. The rlp1Delta mutant is sensitive to methyl methanesulfonate (MMS), ionizing radiation (IR), and camptothecin (CPT), although to a lesser extent than the deletion mutants of rhp55+ and rhp51+ genes. In contrast to other recombinational repair mutants, the rlp1Delta mutant does not exhibit sensitivity to UV light and mitomycin C (MMC). Mitotic recombination is moderately reduced in rlp1 mutant. Epistatic analysis of MMS and IR-sensitivity of rlp1Delta mutant indicates that rlp1+ acts in the recombinational pathway of double-strand break (DSB) repair together with rhp51+, rhp55+, and rad22+ genes. Yeast two-hybrid analysis suggests that Rlp1 may interact with Rhp57 protein. We propose that Rlp1 have an accessory role in repair of a subset of DNA damage induced by MMS and IR, and is required for the full extent of DNA recombination and cell survival under condition of a replication fork collapse.     

Dissection of Rad9 BRCT domain function in the mitotic checkpoint response to telomere uncapping

In Saccharomyces cerevisiae, destabilizing telomeres, via inactivation of telomeric repeat binding factor Cdc13, induces a cell cycle checkpoint that arrests cells at the metaphase to anaphase transition—much like the response to an unrepaired DNA double strand break (DSB). Throughout the cell cycle, the multi-domain adaptor protein Rad9 is required for the activation of checkpoint effector kinase Rad53 in response to DSBs and is similarly necessary for checkpoint signaling in response to telomere uncapping. Rad53 activation in G1 and S phase depends on Rad9 association with modified chromatin adjacent to DSBs, which is mediated by Tudor domains binding histone H3 di-methylated at K79 and BRCT domains to histone H2A phosphorylated at S129. Nonetheless, Rad9 Tudor or BRCT mutants can initiate a checkpoint response to DNA damage in nocodazole-treated cells. Mutations affecting di-methylation of H3 K79, or its recognition by Rad9 enhance 5′ strand resection upon telomere uncapping, and potentially implicate Rad9 chromatin binding in the checkpoint response to telomere uncapping. Indeed, we report that Rad9 binds to sub-telomeric chromatin, upon telomere uncapping, up to 10 kb from the telomere. Rad9 binding occurred within 30 min after inactivating Cdc13, preceding Rad53 phosphorylation. In turn, Rad9 Tudor and BRCT domain mutations blocked chromatin binding and led to attenuated checkpoint signaling as evidenced by decreased Rad53 phosphorylation and impaired cell cycle arrest. Our work identifies a role for Rad9 chromatin association, during mitosis, in the DNA damage checkpoint response to telomere uncapping, suggesting that chromatin binding may be an initiating event for checkpoints throughout the cell cycle.     

Regulation of gross chromosomal rearrangements by ubiquitin and SUMO ligases in Saccharomyces cerevisiae

Gross chromosomal rearrangements (GCRs) are frequently observed in many cancers. Previously, we showed that inactivation of Rad5 or Rad18, ubiquitin ligases (E3) targeting for proliferating cell nuclear antigen (PCNA), increases the de novo telomere addition type of GCR (S. Smith, J. Y. Hwang, S. Banerjee, A. Majeed, A. Gupta, and K. Myung, Proc. Natl. Acad. Sci. USA 101:9039-9044, 2004). GCR suppression by Rad5 and Rad18 appears to be exerted by the RAD5-dependent error-free mode of bypass DNA repair. In contrast, Siz1 SUMO ligase and another ubiquitin ligase, Bre1, which target for PCNA and histone H2B, respectively, have GCR-supporting activities. Inactivation of homologous recombination (HR) proteins or the helicase Srs2 reduces GCR rates elevated by the rad5 or rad18 mutation. GCRs are therefore likely to be produced through the restrained recruitment of an HR pathway to stalled DNA replication forks. Since this HR pathway is compatible with Srs2, it is not a conventional form of recombinational pathway. Lastly, we demonstrate that selection of proper DNA repair pathways to stalled DNA replication forks is controlled by the Mec1-dependent checkpoint and is executed by cooperative functions of Siz1 and Srs2. We propose a mechanism for how defects in these proteins could lead to diverse outcomes (proper repair or GCR formation) through different regulation of DNA repair machinery.