A recombination repair gene of Schizosaccharomyces pombe, rhp57, is a functional homolog of the Saccharomyces cerevisiae RAD57 gene and is phylogenetically related to the human XRCC3 gene

To identify Schizosaccharomyces pombe genes involved in recombination repair, we identified seven mutants that were hypersensitive to both methyl methanesulfonate (MMS) and gamma-rays and that contained mutations that caused synthetic lethality when combined with a rad2 mutation. One of the mutants was used to clone the corresponding gene from a genomic library by complementation of the MMS-sensitive phenotype. The gene obtained encodes a protein of 354 amino acids whose sequence is 32% identical to that of the Rad57 protein of Saccharomyces cerevisiae. An rhp57 (RAD57 homolog of S. pombe) deletion strain was more sensitive to MMS, UV, and gamma-rays than the wild-type strain and showed a reduction in the frequency of mitotic homologous recombination. The MMS sensitivity was more severe at lower temperature and was suppressed by the presence of a multicopy plasmid bearing the rhp51 gene. An rhp51 rhp57 double mutant was as sensitive to UV and gamma-rays as an rhp51 single mutant, indicating that rhp51 function is epistatic to that of rhp57. These characteristics of the rhp57 mutants are very similar to those of S. cerevisiae rad57 mutants. Phylogenetic analysis suggests that Rhp57 and Rad57 are evolutionarily closest to human Xrcc3 of the RecA/Rad51 family of proteins.     

Role of the Schizosaccharomyces pombe F-Box DNA helicase in processing recombination intermediates

In an effort to identify novel genes involved in recombination repair, we isolated fission yeast Schizosaccharomyces pombe mutants sensitive to methyl methanesulfonate (MMS) and a synthetic lethal with rad2. A gene that complements such mutations was isolated from the S. pombe genomic library, and subsequent analysis identified it as the fbh1 gene encoding the F-box DNA helicase, which is conserved in mammals but not conserved in Saccharomyces cerevisiae. An fbh1 deletion mutant is moderately sensitive to UV, MMS, and gamma rays. The rhp51 (RAD51 ortholog) mutation is epistatic to fbh1. fbh1 is essential for viability in stationary-phase cells and in the absence of either Srs2 or Rqh1 DNA helicase. In each case, lethality is suppressed by deletion of the recombination gene rhp57. These results suggested that fbh1 acts downstream of rhp51 and rhp57. Following UV irradiation or entry into the stationary phase, nuclear chromosomal domains of the fbh1Delta mutant shrank, and accumulation of some recombination intermediates was suggested by pulsed-field gel electrophoresis. Focus formation of Fbh1 protein was induced by treatment that damages DNA. Thus, the F-box DNA helicase appears to process toxic recombination intermediates, the formation of which is dependent on the function of Rhp51.     

A new recombinational DNA repair gene from Schizosaccharomyces pombe with homology to Escherichia coli RecA.

A new DNA repair gene from Schizosaccharomyces pombe with homology to RecA was identified and characterized. Comparative analysis showed highest similarity to Saccharomyces cerevisiae Rad55p. rhp55(+) (rad homologue pombe 55) encodes a predicted 350-amino-acid protein with an M(r) of 38,000. The rhp55Delta mutant was highly sensitive to methyl methanesulfonate (MMS), ionizing radiation (IR), and, to a lesser degree, UV. These phenotypes were enhanced at low temperatures, similar to deletions in the S. cerevisiae RAD55 and RAD57 genes. Many rhp55Delta cells were elongated with aberrant nuclei and an increased DNA content. The rhp55 mutant showed minor deficiencies in meiotic intra- and intergenic recombination. Sporulation efficiency and spore viability were significantly reduced. Double-mutant analysis showed that rhp55(+) acts in one DNA repair pathway with rhp51(+) and rhp54(+), homologs of the budding yeast RAD51 and RAD54 genes, respectively. However, rhp55(+) is in a different epistasis group for repair of UV-, MMS-, or gamma-ray-induced DNA damage than is rad22(+), a putative RAD52 homolog of fission yeast. The structural and functional similarity suggests that rhp55(+) is a homolog of the S. cerevisiae RAD55 gene and we propose that the functional diversification of RecA-like genes in budding yeast is evolutionarily conserved.     

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.     

Saccharomyces cerevisiae exonuclease-1 plays a role in UV resistance that is distinct from nucleotide excision repair.

Two closely related genes, EXO1 and DIN 7, in the budding yeast Saccharomyces cerevisiae have been found to be sequence homologs of the exo1 gene from the fission yeast Schizosaccharomyces pombe . The proteins encoded by these genes belong to the Rad2/XPG and Rad27/FEN-1 families, which are structure-specific nucleases functioning in DNA repair. An XPG nuclease deficiency in humans is one cause of xeroderma pigmentosum and those afflicted display a hypersensitivity to UV light. Deletion of the RAD2 gene in S. cerevisiae also causes UV hypersensitivity, due to a defect in nucleotide excision repair (NER), but residual UV resistance remains. In this report, we describe evidence for the residual repair of UV damage to DNA that is dependent upon Exo1 nuclease. Expression of the EXO1 gene is UV inducible. Genetic analysis indicates that the EXO1 gene is involved in a NER-independent pathway for UV repair, as exo1 rad2 double mutants are more sensitive to UV than either the rad2 or exo1 single mutants. Since the roles of EXO1 in mismatch repair and recombination have been established, double mutants were constructed to examine the possible relationship between the role of EXO1 in UV resistance and its roles in other pathways for repair of UV damaged DNA. The exo1 msh2 , exo1 rad51 , rad2 rad51 and rad2 msh2 double mutants were all more sensitive to UV than their respective pairs of single mutants. This suggests that the observed UV sensitivity of the exo1 deletion mutant is unlikely to be due to its functional deficiencies in MMR, recombination or NER. Further, it suggests that the EXO1 , RAD51 and MSH2 genes control independent mechanisms for the maintenance of UV resistance.     

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.     

Characterization of RAD52 homologs in the fission yeast Schizosaccharomyces pombe

The RAD52 gene of Saccharomyces cerevisiae is essential for repair of DNA double-strand breaks (DSBs) by homologous recombination. Inactivation of this gene confers hypersensitivity to DSB-inducing agents and defects in most forms of recombination. The rad22+ gene in Schizosaccharomyces pombe (here referred to as rad22A+) has been characterized as a homolog of RAD52 in fission yeast. Here, we report the identification of a second RAD52 homolog in Schizosaccharomyces pombe, called rad22B+. The amino acid sequences of Rad22A and Rad22B show significant conservation (38% identity). Deletion mutants of respectively, rad22A and rad22B, show different phenotypes with respect to sensitivity to X-rays and the ability to perform homologous recombination as measured by the integration of plasmid DNA. Inactivation of rad22A+ leads to a severe sensitivity to X-rays and a strong decrease in recombination (13-fold), while the rad22B mutation does not result in a decrease in homologous recombination or a change in radiation sensitivity. In a rad22A-rad22B double mutant the radiation sensitivity is further enhanced in comparison with the rad22A single mutant. Overexpression of the rad22B+ gene results in partial suppression of the DNA repair defects of the rad22A mutant strain. Meiotic recombination and spore viability are only slightly affected in either single mutant, but outgrowth of viable spores is almost 31-fold reduced in the rad22A-rad22B double mutant. The results obtained imply a crucial role for rad22A+ in repair and recombination in vegetative cells just like RAD52 in S. cerevisiae. The rad22B+ gene presumably has an auxiliary role in the repair of DSBs. The drastic reduced spore viability in the double mutant suggests that meiosis in S. pombe is dependent on the presence of either rad22A+ or rad22B+.     

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.     

Structural and functional conservation of the human homolog of the Schizosaccharomyces pombe rad2 gene, which is required for chromosome segregation and recovery from DNA damage.

The rad2 mutant of Schizosaccharomyces pombe is sensitive to UV irradiation and deficient in the repair of UV damage. In addition, it has a very high degree of chromosome loss and/or nondisjunction. We have cloned the rad2 gene and have shown it to be a member of the Saccharomyces cerevisiae RAD2/S. pombe rad13/human XPG family. Using degenerate PCR, we have cloned the human homolog of the rad2 gene. Human cDNA has 55% amino acid sequence identity to the rad2 gene and is able to complement the UV sensitivity of the rad2 null mutant. We have thus isolated a novel human gene which is likely to be involved both in controlling the fidelity of chromosome segregation and in the repair of UV-induced DNA damage. Its involvement in two fundamental processes for maintaining chromosomal integrity suggests that it is likely to be an important component of cancer avoidance mechanisms.     

The Rad52 homologs Rad22 and Rti1 of Schizosaccharomyces pombe are not essential for meiotic interhomolog recombination, but are required for meiotic intrachromosomal recombination and mating-type-related DNA repair

Proteins of the RAD52 epistasis group play an essential role in repair of some types of DNA damage and genetic recombination. In Schizosaccharomyces pombe, Rad22 (a Rad52 ortholog) has been shown to be as necessary for repair and recombination events during vegetative growth as its Saccharomyces cerevisiae counterpart. This finding contrasts with previous reports where, due to suppressor mutations in the fbh1 gene, rad22 mutants did not display a severe defect. We have analyzed the roles of Rad22 and Rti1, another Rad52 homolog, during meiotic recombination and meiosis in general. Both proteins play an important role in spore viability. During meiotic prophase I, they partially colocalize and partially localize to Rad51 foci and linear elements. Genetic analysis showed that meiotic interchromosomal crossover and conversion events were unexpectedly not much affected by deletion of either or both genes. A strong decrease of intrachromosomal recombination assayed by a gene duplication construct was observed. Therefore, we propose that the most important function of Rad22 and Rti1 in S. pombe meiosis is repair of double-strand breaks with involvement of the sister chromatids. In addition, a novel mating-type-related repair function of Rad22 specific to meiosis and spore germination is described.     

RAD10, an excision repair gene of Saccharomyces cerevisiae, is involved in the RAD1 pathway of mitotic recombination.

The RAD10 gene of Saccharomyces cerevisiae is required for the incision step of excision repair of UV-damaged DNA. We show that the RAD10 gene is also required for mitotic recombination. The rad10 delta mutation lowered the rate of intrachromosomal recombination of a his3 duplication in which one his3 allele has a deletion at the 3' end and the other his3 allele has a deletion at the 5' end (his3 delta 3' his3 delta 5'). The rate of formation of HIS3+ recombinants in the rad10 delta mutant was not affected by the rad1 delta mutation but decreased synergistically in the presence of the rad10 delta mutation in combination with the rad52 delta mutation. These observations indicate that the RAD1 and RAD10 genes function together in a mitotic recombination pathway that is distinct from the RAD52 recombination pathway. The rad10 delta mutation also lowered the efficiency of integration of linear DNA molecules and circular plasmids into homologous genomic sequences. We suggest that the RAD1 and RAD10 gene products act in recombination after the formation of the recombinogenic substrate. The rad1 delta and rad10 delta mutations did not affect meiotic intrachromosomal recombination of the his3 delta 3' his3 delta 5' duplication or mitotic and meiotic recombination of ade2 heteroalleles located on homologous chromosomes.     

Contribution of base excision repair, nucleotide excision repair, and DNA recombination to alkylation resistance of the fission yeast Schizosaccharomyces pombe

DNA damage is unavoidable, and organisms across the evolutionary spectrum possess DNA repair pathways that are critical for cell viability and genomic stability. To understand the role of base excision repair (BER) in protecting eukaryotic cells against alkylating agents, we generated Schizosaccharomyces pombe strains mutant for the mag1 3-methyladenine DNA glycosylase gene. We report that S. pombe mag1 mutants have only a slightly increased sensitivity to methylation damage, suggesting that Mag1-initiated BER plays a surprisingly minor role in alkylation resistance in this organism. We go on to show that other DNA repair pathways play a larger role than BER in alkylation resistance. Mutations in genes involved in nucleotide excision repair (rad13) and recombinational repair (rhp51) are much more alkylation sensitive than mag1 mutants. In addition, S. pombe mutant for the flap endonuclease rad2 gene, whose precise function in DNA repair is unclear, were also more alkylation sensitive than mag1 mutants. Further, mag1 and rad13 interact synergistically for alkylation resistance, and mag1 and rhp51 display a surprisingly complex genetic interaction. A model for the role of BER in the generation of alkylation-induced DNA strand breaks in S. pombe is discussed.     

Schizosaccharomyces pombe rad32 protein: a phosphoprotein with an essential phosphoesterase motif required for repair of DNA double strand breaks.

The Schizosaccharomyces pombe Rad32 protein is required for repair of DNA double strand breaks, minichromosome stability and meiotic recombination. We show here that the Rad32 protein is phosphorylated in a cell cycle-dependent manner and during meiosis. The phosphorylation is not dependent on the checkpoint protein Rad3. Analysis of a partially purified protein preparation indicates that Rad32 is likely to act in a complex. Characterisation of the rad32-1 mutation and site-directed mutagenesis indicate that three aspartate residues in the conserved phosphoesterase motifs are important for both mitotic and meiotic functions, namely response to UV and ionising radiation and spore viability.     

Cloning and characterisation of the Schizosaccharomyces pombe rad8 gene, a member of the SNF2 helicase family.

The Schizosaccharomyces pombe rad8 mutant is sensitive to both UV and gamma irradiation. We have cloned the rad8 gene by complementation of the UV sensitivity of a rad8.190 mutant strain. The gene comprises an open reading frame of 3.4 kb which does not contain any introns and is capable of encoding a 1133 amino acid protein of 129 kDa. Deletion of the gene indicates that it is not essential for cell viability. Recognisable motifs are present for a nuclear localisation signal, a RING finger and helicase domains. The predicted protein is a member of the SNF2 subfamily of proteins and shows particular homology to the Saccharomyces cerevisiae RAD5 protein. Double mutant analysis demonstrated that the rad8 mutant is not epistatic to mutants in the excision repair pathway (rad13) or checkpoint pathway (rad9). Analysis of radiation sensitivity though the cell cycle indicates that, unlike most other rad mutants, rad8 is most sensitive to irradiation during the G1/S period.     

The Schizosaccharomyces pombe rad60 gene is essential for repairing double-strand DNA breaks spontaneously occurring during replication and induced by DNA-damaging agents

To identify novel genes involved in DNA double-strand break (DSB) repair, we previously isolated Schizosaccharomyces pombe mutants which are hypersensitive to methyl methanesulfonate (MMS) and synthetic lethals with rad2. This study characterizes one of these mutants, rad60-1. The gene that complements the MMS sensitivity of this mutant was cloned and designated rad60. rad60 encodes a protein with 406 amino acids which has the conserved ubiquitin-2 motif found in ubiquitin family proteins. rad60-1 is hypersensitive to UV and gamma rays, epistatic to rhp51, and defective in the repair of DSBs caused by gamma-irradiation. The rad60-1 mutant is also temperature sensitive for growth. At the restrictive temperature (37 degrees C), rad60-1 cells grow for several divisions and then arrest with 2C DNA content; the arrested cells accumulate DSBs and have a diffuse and often aberrantly shaped nuclear chromosomal domain. The rad60-1 mutant is a synthetic lethal with rad18-X, and expression of wild-type rad60 from a multicopy plasmid partially suppresses the MMS sensitivity of rad18-X cells. rad18 encodes a conserved protein of the structural maintenance of chromosomes (SMC) family (A. R. Lehmann, M. Walicka, D. J. Griffiths, J. M. Murray, F. Z. Watts, S. McCready, and A. M. Carr, Mol. Cell. Biol. 15:7067-7080, 1995). These results suggest that S. pombe Rad60 is required to repair DSBs, which accumulate during replication, by recombination between sister chromatids. Rad60 may perform this function in concert with the SMC protein Rad18.     

An alternative eukaryotic DNA excision repair pathway.

DNA lesions induced by UV light, cyclobutane pyrimidine dimers, and (6-4)pyrimidine pyrimidones are known to be repaired by the process of nucleotide excision repair (NER). However, in the fission yeast Schizosaccharomyces pombe, studies have demonstrated that at least two mechanisms for excising UV photo-products exist; NER and a second, previously unidentified process. Recently we reported that S. pombe contains a DNA endonuclease, SPDE, which recognizes and cleaves at a position immediately adjacent to cyclobutane pyrimidine dimers and (6-4)pyrimidine pyrimidones. Here we report that the UV-sensitive S. pombe rad12-502 mutant lacks SPDE activity. In addition, extracts prepared from the rad12-502 mutant are deficient in DNA excision repair, as demonstrated in an in vitro excision repair assay. DNA repair activity was restored to wild-type levels in extracts prepared from rad12-502 cells by the addition of partially purified SPDE to in vitro repair reaction mixtures. When the rad12-502 mutant was crossed with the NER rad13-A mutant, the resulting double mutant was much more sensitive to UV radiation than either single mutant, demonstrating that the rad12 gene product functions in a DNA repair pathway distinct from NER. These data directly link SPDE to this alternative excision repair process. We propose that the SPDE-dependent DNA repair pathway is the second DNA excision repair process present in S. pombe.     

A novel allele of fission yeast rad11 that causes defects in DNA repair and telomere length regulation

Replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein involved in DNA replication, recombination and repair. In Saccharomyces cerevisiae, several mutants in the RFA1 gene encoding the large subunit of RPA have been isolated and one of the mutants with a missense allele, rfa1-D228Y, shows a synergistic reduction in telomere length when combined with a yku70 mutation. So far, only one mutant allele of the rad11⁺ gene encoding the large subunit of RPA has been reported in Schizosaccharomyces pombe. To study the role of S.pombe RPA in DNA repair and possibly in telomere maintenance, we constructed a rad11-D223Y mutant, which corresponds to the S.cerevisiae rfa1-D228Y mutant. rad11-D223Y cells were methylmethane sulfonate, hydroxyurea, UV and γ-ray sensitive, suggesting that rad11-D223Y cells have a defect in DNA repair activity. Unlike the S.cerevisiae rfa1-D228Y mutation, the rad11-D223Y mutation itself caused telomere shortening. Moreover, Rad11-Myc bound to telomere in a ChIP assay. These results strongly suggest that RPA is directly involved in telomere maintenance.