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.     

Role of a Complex Containing Rad17, Mec3, and Ddc1 in the Yeast DNA Damage Checkpoint Pathway

Genetic analysis has suggested that RAD17, RAD24, MEC3, and DDC1 play similar roles in the DNA damage checkpoint control in budding yeast. These genes are required for DNA damage-induced Rad53 phosphorylation and considered to function upstream of RAD53 in the DNA damage checkpoint pathway. Here we identify Mec3 as a protein that associates with Rad17 in a two-hybrid screen and demonstrate that Rad17 and Mec3 interact physically in vivo. The amino terminus of Rad17 is required for its interaction with Mec3, and the protein encoded by the rad17-1 allele, containing a missense mutation at the amino terminus, is defective for its interaction with Mec3 in vivo. Ddc1 interacts physically and cosediments with both Rad17 and Mec3, indicating that these three proteins form a complex. On the other hand, Rad24 is not found to associate with Rad17, Mec3, and Ddc1. DDC1 overexpression can partially suppress the phenotypes of the rad24Δ mutation: sensitivity to DNA damage, defect in the DNA damage checkpoint and decrease in DNA damage-induced phosphorylation of Rad53. Taken together, our results suggest that Rad17, Mec3, and Ddc1 form a complex which functions downstream of Rad24 in the DNA damage checkpoint pathway.     

G2 / M checkpoint genes of Saccharomyces cerevisiae : further evidence for roles in DNA replication and / or repair

We have cloned, sequenced and disrupted the checkpoint genes RAD17, RAD24 and MEC3 of Saccharomyces cerevisiae. Mec3p shows no strong similarity to other proteins currently in the database. Rad17p is similar to Rec1 from Ustilago maydis, a 3′ to 5′ DNA exonuclease/checkpoint protein, and the checkpoint protein Rad1p from Schizosaccharomyces pombe (as we previously reported). Rad24p shows sequence similarity to replication factor C (RFC) subunits, and the S. pombe Rad17p checkpoint protein, suggesting it has a role in DNA replication and/or repair. This hypothesis is supported by our genetic experiments which show that overexpression of RAD24 strongly reduces the growth rate of yeast strains that are defective in the DNA replication/repair proteins Rfc1p (cdc44), DNA polα (cdc17) and DNA polδ (cdc2) but has much weaker effects on cdc6, cdc9, cdc15 and CDC ⁺ strains. The idea that RAD24 overexpression induces DNA damage, perhaps by interfering with replication/repair complexes, is further supported by our observation that RAD24 overexpression increases mitotic chromosome recombination in CDC ⁺ strains. Although RAD17, RAD24 and MEC3 are not required for cell cycle arrest when S phase is inhibited by hydroxyurea (HU), they do contribute to the viability of yeast cells grown in the presence of HU, possibly because they are required for the repair of HU-induced DNA damage. In addition, all three are required for the rapid death of cdc13 rad9 mutants. All our data are consistent with models in which RAD17, RAD24 and MEC3 are coordinately required for the activity of one or more DNA repair pathways that link DNA damage to cell cycle arrest.     

G2 / M checkpoint genes of Saccharomyces cerevisiae : further evidence for roles in DNA replication and / or repair

We have cloned, sequenced and disrupted the checkpoint genes RAD17, RAD24 and MEC3 of Saccharomyces cerevisiae. Mec3p shows no strong similarity to other proteins currently in the database. Rad17p is similar to Rec1 from Ustilago maydis, a 3′ to 5′ DNA exonuclease/checkpoint protein, and the checkpoint protein Rad1p from Schizosaccharomyces pombe (as we previously reported). Rad24p shows sequence similarity to replication factor C (RFC) subunits, and the S. pombe Rad17p checkpoint protein, suggesting it has a role in DNA replication and/or repair. This hypothesis is supported by our genetic experiments which show that overexpression of RAD24 strongly reduces the growth rate of yeast strains that are defective in the DNA replication/repair proteins Rfc1p (cdc44), DNA polα (cdc17) and DNA polδ (cdc2) but has much weaker effects on cdc6, cdc9, cdc15 and CDC ⁺ strains. The idea that RAD24 overexpression induces DNA damage, perhaps by interfering with replication/repair complexes, is further supported by our observation that RAD24 overexpression increases mitotic chromosome recombination in CDC ⁺ strains. Although RAD17, RAD24 and MEC3 are not required for cell cycle arrest when S phase is inhibited by hydroxyurea (HU), they do contribute to the viability of yeast cells grown in the presence of HU, possibly because they are required for the repair of HU-induced DNA damage. In addition, all three are required for the rapid death of cdc13 rad9 mutants. All our data are consistent with models in which RAD17, RAD24 and MEC3 are coordinately required for the activity of one or more DNA repair pathways that link DNA damage to cell cycle arrest. Received: 8 April 1997 / Accepted: 10 May 1997     

The human checkpoint protein hRad17 interacts with the PCNA-like proteins hRad1, hHus1, and hRad9

DNA damage activates cell cycle checkpoints that prevent progression through the cell cycle. In yeast, the DNA damage checkpoint response is regulated by a series of genes that have mammalian homologs, including rad1, rad9, hus1, and rad17. On the basis of sequence homology, yeast and human Rad1, Rad9, and Hus1 protein homologs are predicted to structurally resemble the sliding clamp PCNA. Likewise, Rad17 homologs have extensive homology with replication factor C (RFC) subunits (p36, p37, p38, p40, and p140), which form a clamp loader for PCNA. These observations predict that Rad1, Hus1, and Rad9 might interact with Rad17 as a clamp-clamp loader pair during the DNA damage response. In this report, we demonstrate that endogenous human Rad17 (hRad17) interacts with the PCNA-related checkpoint proteins hRad1, hRad9, and hHus1. Mutational analysis of hRad1 and hRad17 demonstrates that this interaction has properties similar to the interaction between RFC and PCNA, a well characterized clamp-clamp loader pair. Moreover, we show that DNA damage affects the association of hRad17 with the clamp-like checkpoint proteins. Collectively, these data provide the first experimental evidence that hRad17 interacts with the PCNA-like proteins hRad1, hHus1, and hRad9 in manner similar to the interaction between RFC and PCNA.     

Structure-based predictions of Rad1, Rad9, Hus1 and Rad17 participation in sliding clamp and clamp-loading complexes

The repair of damaged DNA is coupled to the completion of DNA replication by several cell cycle checkpoint proteins, including, for example, in fission yeast Rad1^Sp, Hus1^Sp, Rad9^Sp and Rad17^Sp. We have found that these four proteins are conserved with protein sequences throughout eukaryotic evolution. Using computational techniques, including fold recognition, comparative modeling and generalized sequence profiles, we have made high confidence structure predictions for the each of the Rad1, Hus1 and Rad9 protein families (Rad17^Sc, Mec3^Sc and Ddc1^Sc in budding yeast, respectively). Each of these families was found to share a common protein fold with that of PCNA, the sliding clamp protein that tethers DNA polymerase to its template. We used previously reported genetic and biochemical data for these proteins from yeast and human cells to predict a heterotrimeric PCNA-like ring structure for the functional Rad1/Rad9/Hus1 complex and to determine their exact order within it. In addition, for each individual protein family, contact regions with neighbors within the PCNA-like ring were identified. Based on a molecular model for Rad17^Sp, we concluded that members of this family, similar to the subunits of the RFC clamp-loading complex, are capable of coupling ATP binding with conformational changes required to load a sliding clamp onto DNA. This model substantiates previous findings regarding the behavior of Rad17 family proteins upon DNA damage and within the RFC complex of clamp-loading proteins.     

Genetic interaction of RAD53 protein kinase with histones is important for DNA replication

Studies in budding yeast suggest the protein kinase Rad53 plays novel roles in controlling initiation of DNA replication and in maintaining cellular histone levels, and these roles are independent of Rad53-mediated regulation of the checkpoint and of nucleotide levels. In order to elucidate the role of Rad53 in replication initiation, we isolated a novel allele of RAD53, rad53-rep, that separates the checkpoint function of RAD53 from the DNA replication function. rad53-rep mutants display a chromosome loss phenotype that is suppressed by increased origin dosage, providing further evidence that Rad53 plays a role in the initiation of DNA replication. Deletion of the major histone H3–H4 pair suppresses rad53-rep-cdc7-1 synthetic lethality, suggesting Rad53''s functions in degradation of excess cellular histone and in replication initiation are related. Rad53-rep is active as a protein kinase yet fails to interact with origins of replication and like the rad53Δ mutant, the rad53-rep mutant accumulates excess soluble histones, and it is sensitive to histone dosage. In contrast, a checkpoint defective allele of RAD53 with mutations in both FHA domains, binds origins and growth of this mutant is unaffected by histone dosage. Based on these observations, we hypothesize that the origin binding and the histone degradation activities of Rad53 are central to its function in DNA replication and are independent of its checkpoint functions. We propose a model in which Rad53 acts as a “nucleosome buffer”, interacting with origins of replication to prevent the binding of excess histones to origin DNA and to maintain proper chromatin configuration.Key words: DNA replication, Rad53, histones, checkpoint, origins of replication     

Cloning by functional complementation,and inactivation,of theSchizosaccharomyces pombe homologue of theSaccharomyces cerevisiae geneABC1

TheSaccharomyces cerevisiae geneABC1 is required for the correct functioning of thebc ₁ complex of the mitochondrial respiratory chain. By functional complementation of aS. cerevisiae abc1 ^? mutant, we have cloned aSchizosaccharomyces pombe cDNA, whose predicted product is 50% identical to the Abc1 protein. Significant homology is also observed with bacterial, nematode, and even human amino acid sequences of unknown function, suggesting that the Abc1 protein is conserved through evolution. The cloned cDNA corresponds to a singleS. pombe geneabc1Sp, located on chromosome II, expression of which is not regulated by the carbon source. Inactivation of theabc1Sp gene by homologous gene replacement causes a respiratory deficiency which is efficiently rescued by the expression of theS. cerevisiae ABC1 gene. The inactivated strain shows a drastic decrease in thebc ₁ complex activity, a decrease in cytochromeaa3 and a slow growth phenotype. To our knowledge, this is the first example of the inactivation of a respiratory gene inS. pombe. Our results highlight the fact thatS. pombe growth is highly dependent upon respiration, and thatS. pombe could represent a valuable model for studying nucleo-mitochondrial interactions in higher eukaryotes.     

Accumulation of sumoylated Rad52 in checkpoint mutants perturbed in DNA replication

Checkpoints are cellular surveillance and signaling pathways that regulate responses to DNA damage and perturbations of DNA replication. Here we show that high levels of sumoylated Rad52 are present in the mec1 sml1 and rad53 sml1 checkpoint mutants exposed to DNA-damaging agents such as methyl methanesulfonate (MMS) or the DNA replication inhibitor hydroxyurea (HU). The kinase-defective mutant rad53-K227A also showed high levels of Rad52 sumoylation. Elevated levels of Rad52 sumoylation occur in checkpoint mutants proceeding S phase being exposed DNA-damaging agent. Interestingly, chromatin immunoprecipitation (ChIP) on chip analyses revealed non-canonical chromosomal localization of Rad52 in the HU-treated rad53-K227A cells arrested in early S phase: Rad52 localization at dormant and early DNA replication origins. However, such unusual localization was not dependent on the sumoylation of Rad52. In addition, we also found that Rad52 could be highly sumoylated in the absence of Rad51. Double mutation of RAD51 and RAD53 exhibited the similar levels of Rad52 sumoylation to RAD53 single mutation. The significance and regulation mechanism of Rad52 sumoylation by checkpoint pathways will be discussed.     

Genetic interaction of RAD53 protein kinase with histones is important for DNA replication

Studies in budding yeast suggest the protein kinase Rad53 plays novel roles in controlling initiation of DNA replication and in maintaining cellular histone levels, and these roles are independent of Rad53-mediated regulation of the checkpoint and of nucleotide levels. In order to elucidate the role of Rad53 in replication initiation, we isolated a novel allele of RAD53, rad53-rep,that separates the checkpoint function of RAD53 from the DNA replication function. rad53-rep mutants display a chromosome loss phenotype that is suppressed by increased origin dosage, providing further evidence that Rad53 plays a role in the initiation of DNA replication. Deletion of the major histone H3-H4 pair suppresses rad53-rep-cdc7-1 synthetic lethality, suggesting Rad53's functions in degradation of excess cellular histone and in replication initiation are related. Rad53-rep is active as a protein kinase yet fails to interact with origins of replication and like the rad53D mutant, the rad53-rep mutant accumulates excess soluble histones, and it is sensitive to histone dosage. In contrast, a checkpoint defective allele of RAD53 with mutations in both FHA domains, binds origins, and growth of a rad53-FHA mutant is unaffected by histone dosage. Based on these observations, we hypothesize that the origin binding and the histone degradation activities of Rad53 are central to its function in DNA replication and are independent of its checkpoint functions. We propose a model in which Rad53 acts as a "nucleosome buffer," interacting with origins of replication to prevent the binding of excess histones to origin DNA and to maintain proper chromatin configuration.     

The mitotic DNA damage checkpoint proteins Rad17 and Rad24 are required for repair of double-strand breaks during meiosis in yeast

We show here that deletion of the DNA damage checkpoint genes RAD17 and RAD24 in Saccharomyces cerevisiae delays repair of meiotic double-strand breaks (DSBs) and results in an altered ratio of crossover-to-noncrossover products. These mutations also decrease the colocalization of immunostaining foci of the RecA homologs Rad51 and Dmc1 and cause a delay in the disappearance of Rad51 foci, but not of Dmc1. These observations imply that RAD17 and RAD24 promote efficient repair of meiotic DSBs by facilitating proper assembly of the meiotic recombination complex containing Rad51. Consistent with this proposal, extra copies of RAD51 and RAD54 substantially suppress not only the spore inviability of the rad24 mutant, but also the gamma-ray sensitivity of the mutant. Unexpectedly, the entry into meiosis I (metaphase I) is delayed in the checkpoint single mutants compared to wild type. The control of the cell cycle in response to meiotic DSBs is also discussed.     

Functional and Physical Interaction between Rad24 and Rfc5 in the Yeast Checkpoint Pathways

The RFC5 gene encodes a small subunit of replication factor C (RFC) complex in Saccharomyces cerevisiae and has been shown to be required for the checkpoints which respond to replication block and DNA damage. Here we describe the isolation of RAD24, known to play a role in the DNA damage checkpoint, as a dosage-dependent suppressor of rfc5-1. RAD24 overexpression suppresses the sensitivity of rfc5-1 cells to DNA-damaging agents and the defect in DNA damage-induced Rad53 phosphorylation. Rad24, like Rfc5, is required for the regulation of Rad53 phosphorylation in response to DNA damage. The Rad24 protein, which is structurally related to the RFC subunits, interacts physically with RFC subunits Rfc2 and Rfc5 and cosediments with Rfc5. Although the rad24Δ mutation alone does not cause a defect in the replication block checkpoint, it does enhance the defect in rfc5-1 mutants. Furthermore, overexpression of RAD24 suppresses the rfc5-1 defect in the replication block checkpoint. Taken together, our results demonstrate a physical and functional interaction between Rad24 and Rfc5 in the checkpoint pathways.     

Rad51 gain-of-function mutants that exhibit high affinity DNA binding cause DNA damage sensitivity in the absence of Srs2

We previously identified several rad51 gain-of-function alleles that partially suppress the requirement for RAD55 and RAD57 in DNA repair. To gain further insight into the mechanism of action of these alleles, we compared the activities of Rad51-V328A, Rad51-P339S and Rad51-I345T with wild-type Rad51, for DNA binding, filament stability, strand exchange and interaction with the antirecombinase helicase, Srs2. These alleles were chosen because they show the highest activity in suppression of ionizing radiation sensitivity of the rad57 mutant, and Val 328 and Ile 345 are conserved in the human Rad51 protein. All three mutant proteins exhibited higher affinity for single-stranded DNA (ssDNA) and showed more robust strand exchange activity with oligonucleotide substrates than wild-type Rad51, with the Rad51-I345T and Rad51-V328A proteins displaying higher activity than Rad51-P339S. However, the Srs2 antirecombinase was able to disrupt Rad51–ssDNA complexes formed with all the mutant proteins. In vivo, the rad51-I345T mutant strain exhibited high resistance to methyl methane sulfonate that was dependent on functional SRS2. These results suggest the Srs2 translocase is able to disrupt Rad51–ssDNA complexes at stalled replication forks, but in the absence of Srs2 the enhanced DNA binding of the Rad51-I345T protein is detrimental to cell survival.     

The Saccharomyces cerevisiae RAD9, RAD17 and RAD24 genes are required for suppression of mutagenic post-replicative repair during chronic DNA damage

In Saccharomyces cerevisiae, a DNA damage checkpoint in the S-phase is responsible for delaying DNA replication in response to genotoxic stress. This pathway is partially regulated by the checkpoint proteins Rad9, Rad17 and Rad24. Here, we describe a novel hypermutable phenotype for rad9Δ, rad17Δ and rad24Δ cells in response to a chronic 0.01% dose of the DNA alkylating agent MMS. We report that this hypermutability results from DNA damage introduction during the S-phase and is dependent on a functional translesion synthesis pathway. In addition, we performed a genetic screen for interactions with rad9Δ that confer sensitivity to 0.01% MMS. We report and quantify 25 genetic interactions with rad9Δ, many of which involve the post-replication repair machinery. From these data, we conclude that defects in S-phase checkpoint regulation lead to increased reliance on mutagenic translesion synthesis, and we describe a novel role for members of the S-phase DNA damage checkpoint in suppressing mutagenic post-replicative repair in response to sublethal MMS treatment.     

Differential expression and requirements for Schizosaccharomyces pombe RAD52 homologs in DNA repair and recombination

In fission yeast two RAD52 homologs have been identified, rad22A⁺ and rad22B⁺. Two-hybrid experiments and GST pull-down assays revealed physical interaction between Rad22A and Rad22B, which is dependent on the N-terminal regions. Interaction with Rhp51 is dependent on the C-terminal parts of either protein. Both Rad22A and Rad22B also interact with RPA. The expression of rad22B⁺ in mitotically dividing cells is very low in comparison with rad22A⁺ but is strongly enhanced after induction of meiosis, in contrast to rad22A⁺. Rad22B mutant cells are not hypersensitive to DNA-damaging agents (X-rays, UV and cisplatin) and display normal levels of recombination. In these respects the Schizosaccharomyces pombe rad22B mutant resembles the weak phenotype of vertebrate cells deficient for RAD52. Mutation of rad22A⁺ leads to severe sensitivity to DNA-damaging agents and to defects in recombination. In a rad22Arad22B double mutant a further increase in sensitivity to DNA-damaging agents and additional mitotic recombination defects were observed. The data presented here indicate that Rad22A and Rad22B have overlapping roles in repair and recombination, although specialized functions for each protein cannot be excluded.     

HDF1 and RAD17 Genes are Involved in DNA Double-strand Break Repair in Stationary Phase Saccharomyces cerevisiae

DNA repair, checkpoint pathways and protection mechanisms against different types of perturbations are critical factors for the prevention of genomic instability. The aim of the present work was to analyze the roles of RAD17 and HDF1 gene products during the late stationary phase, in haploid and diploid yeast cells upon gamma irradiation. The checkpoint protein, Rad17, is a component of a PCNA-like complex—the Rad17/Mec3/Ddc1 clamp—acting as a damage sensor; this protein is also involved in double-strand break (DBS) repair in cycling cells. The HDF1 gene product is a key component of the non-homologous end-joining pathway (NHEJ). Diploid and haploid rad17Δ/rad17Δ, and hdf1Δ Saccharomyces cerevisiae mutant strains and corresponding isogenic wild types were used in the present study. Yeast cells were grown in standard liquid nutrient medium, and maintained at 30°C for 21 days in the stationary phase, without added nutrients. Cell samples were irradiated with ⁶⁰Co γ rays at 5 Gy/s, 50 Gy ≤ Dabs ≤ 200 Gy. Thereafter, cells were incubated in PBS (liquid holding: LH, 0 ≤ t ≤ 24 h). DNA chromosomal analysis (by pulsed-field electrophoresis), and surviving fractions were determined as a function of absorbed doses, either immediately after irradiation or after LH. Our results demonstrated that the proteins Rad17, as well as Hdf1, play essential roles in DBS repair and survival after gamma irradiation in the late stationary phase and upon nutrient stress (LH after irradiation). In haploid cells, the main pathway is NHEJ. In the diploid state, the induction of LH recovery requires the function of Rad17. Results are compatible with the action of a network of DBS repair pathways expressed upon different ploidies, and different magnitudes of DNA damage.     

Genetic Analysis Workshop 13: Simulated longitudinal data on families for a system of oligogenic traits

Background

The Rad26/Rad3 complex in fission yeast detects genotoxic insults and initiates the cell cycle arrest and recovery activities of the DNA damage checkpoint. To investigate how the Rad26/Rad3 complex performs these functions, we constructed and characterized Rad26-GFP.

Results

Rad26-GFP localized to approximately six nuclear dots in cycling cells. Following treatment with a DNA damaging agent, Rad26-GFP localization changed. Damaged cells contained one or two bright Rad26-GFP spots, in addition to smaller, more numerous Rad26-GFP speckles. Genetic analyses demonstrated that these Rad26-GFP patterns (dots, spots and speckles) were unaffected by null mutations in other DNA damage checkpoint genes, including rad3 ⁺. Data obtained with our Rad26.T12-GFP fusion protein correlate spots with cell cycle arrest activities and speckles with DNA repair activities. In addition, physiological experiments demonstrated that rad26Δ and rad3Δ alleles confer sensitivity to a microtubule-depolymerizing drug.

Conclusion

We have discovered three distinct Rad26-GFP cellular structures. Formation of these structures did not require other checkpoint proteins. These data demonstrate that Rad26 can respond to genotoxic insult in the absence of Rad3 and the other checkpoint Rad proteins.     

Nitrosative stress induces a novel intra-S checkpoint pathway in Schizosaccharomyces pombe involving phosphorylation of Cdc2 by Wee1

Excess production of nitric oxide and reactive nitrogen intermediates causes nitrosative stress on cells. Schizosaccharomyces pombe was used as a model to study the cell cycle regulation under nitrosative stress response. We discovered a novel intra-S-phase checkpoint that is activated in S. pombe under nitrosative stress. The mechanism for this intra-S-phase checkpoint activation is distinctly different than previously reported for genotoxic stress in S. pombe by methyl methane sulfonate. Our flow cytometry data established the fact that Wee1 phosphorylates Cdc2 Tyr15 which leads to replication slowdown in the fission yeast under nitrosative stress. We checked the roles of Rad3, Rad17, Rad26, Swi1, Swi3, Cds1, and Chk1 under nitrosative stress but those were not involved in the activation of the DNA replication checkpoint. Rad24 was found to be involved in intra-S-phase checkpoint activation in S. pombe under nitrosative stress but that was independent of Cdc25.