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Hiroshi Iwasaki,1,
Antony M. Carr,2 and
Hideo Shinagawa1*
Department of Molecular Microbiology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan,1 Genome Damage and Stability Centre, School of Biological Sciences, University of Sussex, Brighton, United Kingdom2
Received 9 April 2004/ Returned for modification 3 May 2004/ Accepted 27 July 2004
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and cloned the genes by complementation of the MMS sensitivity. Genes identified in this way include rhp57 (44), rad60 (35), rad32, nbs1 (45), and fdh1, which encodes an F-box DNA helicase (our unpublished data). A recent genome-wide search for mutations synthetically lethal with rad27 in S. cerevisiae identified mutations in all of the well-studied recombination repair genes including RAD52, RAD50, MRE11, XRS2 RAD51, RAD55, RAD57, RAD54, SGS1, MUS81, and MMS4 (43). The screen also identified genes involved in checkpoint regulation (RAD9, RAD17, RAD24, and DDC1) and those related to regulation of chromatin structure (CAC2, ESC2, HST1, and HST3). We report here a novel gene, rad62, which was identified by isolating mutants that were hypersensitive to MMS and synthetically lethal with rad2 mutation and cloning the gene that complemented the MMS sensitivity of such a mutant. rad62 mutants show very similar phenotypes to those of rad60 mutants (7, 35) and rad18 mutants (30, 47). They are hypersensitive to UV, MMS, and gamma rays, epistatic with rhp51 with respect to the damage sensitivity, required for chromosome integrity during replication, and essential for growth. The spr18 and rad18 genes encode proteins whose structures are characteristic of the SMC family of proteins involved in chromosome condensation (Smc2-Smc4) and sister chromatid cohesion (Smc1-Smc3) (13, 21, 41). Since RAD18 is a different gene in budding yeast, and the human homologs of rad18 and spr18 are named hSMC6 and hSMC5, respectively (41), to avoid confusion we have used the names smc6 and smc5, respectively.
SMC proteins share a common structure in that they contain two ATPase motifs composed of Walker A motifs at the N termini and Walker B motifs at the C termini, which are separated by long coiled-coil domains with a hinge region in the middle. The two coiled-coil domains interact and bring two ATPase motifs into close contact to form a globular structure (21). Eukaryotic SMC proteins including Smc5 and Smc6 form tight heterodimers (13). The condensin complex plays an essential role in folding extended chromosomes in an ATP-dependent manner during mitosis. The cohesin complex forms a ring structure which holds sister chromatids in close proximity after replication until they are separated at anaphase in mitosis (16). The Smc5-Smc6 (Smc5-6) complex is proposed to play a role in repairing DSBs by recombination by holding broken DNA together (41). In common with the other eukaryotic SMC complexes, the Smc5-6 heterodimer is associated with non-SMC protein subunits, Nse1 and Nse2, and interacts with another essential protein, Rad60 (7, 19, 33, 35). Like smc5 and smc6, rad60, nse1, and nse2 are essential for growth, and the hypomorphic mutants are sensitive to DNA damaging agents.
Analysis of the genetic interactions between rad60 and smc6 mutants suggests that the products of these genes play a role in a common pathway for repairing DSBs and maintaining chromosome integrity (35). A recent study demonstrated that substoichiometric amounts of Rad60 were associated with Smc5-6 (7). Chromosomes were fragmented when a temperature-sensitive rad60-1 mutant was grown at nonpermissive temperature, suggesting that the Rad60 protein is required to prevent chromosome breakage during replication (35). Like smc6 and rad60 mutants (35, 47), the rad62-1 mutant is defective in joining DSBs produced by ionizing radiation and rad62 is essential for growth. From the phenotypic similarities and analysis of genetic and physical interactions, we propose that Rad62 plays a role as a non-SMC component of the Smc5-6 complex in maintaining chromosome integrity and repairing DSBs.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Strains and plasmids. The strains and plasmids used in this study are shown in Tables 1 and 2. pUR19 is a multicopy plasmid which contains the ars1 and ura4 genes (3). pREP41 is a plasmid that contains a weak nmt promoter, which is inducible by thiamine depletion (39).
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Cloning of the rad62 gene. rad62-1 mutant cells were transformed with an S. pombe genomic DNA library (3), and MMS-resistant transformants were selected on plates containing 0.004% MMS (44). The plasmids that conferred MMS resistance to the mutant were isolated, and the region responsible for the recovery of the repair activity was identified (Fig. 1A) and sequenced.
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Based on the above results, the rad62 open reading frame encoding 300 amino acids was identified (Fig. 1B). The cDNA for the rad62 coding region was amplified with primers S4NB (5'-GCGGATCCCATATGTCCTCCATTGATAAAC-3'), corresponding to the region for translational initiation, and S4CB (5'-GCGGATCCTCAGCCATACCAAGTATTAC-3'), corresponding to the region for translational termination by using the LA PCR kit, version 2. The resulting reverse transcription-PCR product was digested with BamHI and ligated to the BamHI site of pUC118. Nucleotide sequence analysis of the cDNA confirmed the existence of six introns.
Construction and characterization of rad62 deletion strain. A rad62 deletion mutant was constructed essentially as described previously (15). pSLR4, a derivative of pUC118 carrying the 2.7-kb BamHI-BamHI region containing the rad62 gene (Fig. 1A), was used to generate a plasmid construct for gene disruption. The 0.9-kb HpaI-XhoI region of the rad62 gene on the plasmid was removed, and the staggered ends were converted to blunt ends by filling them in with E. coli DNA polymerase I. The ends of the 1.8-kb HindIII fragment containing the ura4 gene from pREP2 (31) were converted to blunt ends, and the fragment was ligated with the plasmid to generate pSLR4D (Fig. 1A). pSLR4D was linearized by BamHI digestion and transformed into the diploid S. pombe strain MPD1. The Ura+ transformed colonies were isolated and checked for the rad62 deletion by genomic Southern analysis. They were sporulated and subjected to tetrad analysis. For cytological observation, the diploid cells were incubated on ME at 26°C for 2 days for sporulation. The spores were treated with 0.5% glusulase, washed in water three times, and then spread onto an EMM plate lacking uracil. After 2 to 3 days, germinated spores of the rad62::ura4+ strain were collected and washed in water three times. The cells were then fixed in 70% ethanol and stained with 1.5 µg of 4, 6-diamidino-2-phenylindole (DAPI)/ml. The stained cells were observed under an epifluorescent microscope.
PFGE. To examine the ability of the rad62-1 mutant to repair DSBs, the cells were irradiated with gamma rays at 600 Gy and the repair of DSBs was followed by pulsed-field gel electrophoresis (PFGE) as described previously (35), with slight modifications in the PFGE conditions: voltage gradient, 2 V/cm; pulse time, 30 min; angle, 120°; time, 48 h; temperature, 16°C.
Measurement of mitotic homologous recombination frequency. The mitotic homologous recombination of rad62-1 was measured by the efficiency of integration of a linear DNA carrying the leu1+ gene into the leu1-32 locus on the chromosome as described previously (44).
Spontaneous mitotic intrachromosomal recombination frequencies of rad62-1, rhp51, and wild-type cells were measured by using the strains PS3, MPS401, and MPS501, containing a nontandem direct repeat of ade6– heteroalleles flanking ura4+ as described previously (17, 38).
Construction of epitope-tagged genes. For immunoprecipitation experiments and protein complex purification, an epitope-tagged rad62 gene generated as described previously (27a by the PCR-based method to place a FLAG-His epitope at the C terminus of the protein and mark the allele with the kanMx6 gene (27a). The rad62-1 strain containing the tagged rad62 gene was not sensitive to MMS and grew as normally as the wild-type strain, indicating that the tagged gene is functional.
Immunoprecipitation. The cells carrying the epitope-tagged gene were grown in YES medium. Mid-log-phase cells from a 50-ml culture were collected, resuspended in 500 µl of buffer A (10 mM HEPES [pH 8.0], 0.1% NP-40, 25 mM KCl, 1 mM phenylmethylsulfonyl fluoride) containing protease inhibitor cocktail, and lysed by acid-washed glass beads. The lysate was clarified by centrifugation (12,000 x g, 15 min). Ten microliters of protein G-Sepharose 4FF (Amersham Pharmacia Biotech) prewashed in buffer A was added to adsorb nonspecific binding proteins to protein G. The suspension was incubated for 1 h at 4°C, and the supernatant was collected by centrifugation. Anti-Myc antibody (PL14; MBL) with protein G-Sepharose 4FF or anti-FLAG M2 affinity gel (Sigma) was added to the supernatant, and the suspensions were rocked for 1 h at 4°C. Immunoprecipitates were washed three times with buffer A, resuspended in 40 µl of 5% sodium dodecyl sulfate (SDS), and incubated for 10 min at 37°C. After centrifugation, the supernatants were mixed with 10 µl of SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer, boiled for 10 min, separated by SDS-PAGE, and analyzed by Western blotting with the ECL Advance Western blotting detection kit (Amersham Bioscience).
Nucleotide sequence accession number. Nucleotide sequence data, including the exon and intron information, have been deposited with the DDBJ under accession number AB158548.
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RESULTS |
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The rad62 coding region was analyzed by the 5' and 3' RACE and reverse transcription-PCR methods, and the existence of seven exons in the rad62 gene was shown by sequence analysis of the cDNA (Fig. 1B). The gene is unusual in having at least as many as four transcription initiation sites and such a long untranslated upstream exon. The open reading frame of rad62 was determined from these analyses and is shown in Fig. 1B. It was different from the one described in the DNA database as SPBC20F10.04c. The gene encodes a protein with 300 amino acids with no apparent functional sequence motifs, as analyzed by using databases (http://motif.genome.ad.jp/). Database search revealed that the Rad62 homologs exist ubiquitously throughout eukaryotes, as shown in Fig. 2. The nucleotide sequence covering the rad62 region of the rad62-1 allele and its flanking genomic regions were amplified by PCR, and the nucleotide sequence was analyzed directly. A single transition mutation from A to G was found at the first base of codon 223, altering the AAA codon for lysine to a GAA codon for glutamic acid. Codon 223 is likely to be important for function, as the corresponding sites are conserved as positive residues, including Lys and Arg, and the mutation changing Lys to an acidic residue, Glu, caused the functional defect in the rad62-1 mutant.
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mutant, but it was less sensitive at higher doses. rhp51 partially suppresses the UV sensitivity of rad62-1 at the lower doses, and the rad62-1 rhp51 double mutant showed a similar UV survival curve to that of the rhp51 single mutant. This result suggests that, at low doses of UV, the Rhp51 protein converts DNA damage to a toxic recombination intermediate in the rad62-1 mutant. The rad62-1 mutant is also hypersensitive to gamma rays, but it is less sensitive than the rhp51 mutant at all doses tested. The double mutant showed the same sensitivity as the rhp51 single mutant. These results suggest that rhp51 is epistatic to rad62 with respect to the DNA repair function. rhp57 is also epistatic to rad62-1 (data not shown). rhp51 is epistatic to rhp57, and both of them are involved in recombination repair (25, 44). rad60-1 and smc6-X (rad18-X) mutants are hypersensitive to UV and gamma rays and show a similar epistasis with rhp51 (30, 35).
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The synthetic lethality of rad62-1 with rad2 was examined by constructing a diploid strain by mating the two mutant strains rad62-1 (MMP7) and rad2 (MMP5) and analyzing the resultant tetrads. The double mutants formed extremely small colonies, and when they were streaked on a YES plate, they showed little sign of growth, confirming that the two mutations are synthetically lethal (data not shown).
rad62-1 is not defective in Rhp51-dependent genetic recombination.
Since the efficiency of homology-dependent genetic recombination is reduced in rhp51 and rhp57 mutants, we examined the ability of the rad62-1 mutant to carry out the recombination. The efficiency of integrating the leu1+ gene into the leu1-32 locus of the rad62-1 mutant (MMPX6) was not reduced, whereas it was reduced threefold (34%) in the rhp57 mutant (MMPX5). This shows that the rad62-1 mutant is not defective in recombination during mitosis, as examined by the integration of linear homologous DNA into the chromosome. rad60-1 was also not defective in such recombination (our unpublished data).
Next we examined spontaneous mitotic intrachromosomal recombination frequencies in the rad62-1 mutant containing a nontandem direct repeat of ade6– heteroalleles flanking ura4+, and intrachromosomal mitotic recombination was assayed by the formation of Ade+ recombinants. Two main classes of Ade6+ recombinants could be distinguished: Ade+ Ura– deletion type recombinants and Ade+ Ura+ conversion type recombinants.
The frequency of conversion type of recombination was much reduced in the rhp51 mutant (0.16 ± 0.07 per viable 104 cells) compared to wild-type cells (1.58 ± 0.30), but it was not reduced in rad62-1 cells (1.42 ± 0.72 per 104 viable cells). This result suggests that the rad62-1 mutant is not defective in Rhp51-dependent spontaneous intrachromosomal recombination such as the gene conversion type of recombination. On the other hand, deletion type recombination was significantly increased in the rhp51 mutant (9.68 ± 1.44 per 104 viable cells) compared to wild-type cells (1.29 ± 0.85 per 104 viable cells and it was not affected in the rad62-1 mutant (1.30 ± 0.59 per 104 viable cells). Therefore, the rad62-1 mutant is similar to the wild-type strain and different from rhp51 and rhp57 in regard to the recombination phenotypes examined by these methods.
rad62 is essential for growth. To study the phenotype of the rad62 null mutant, 70% of the rad62 coding region of one of the chromosomes in a diploid strain was replaced with the DNA fragment containing the ura4 gene by homologous recombination. The heterozygous diploid cells (MMPD1) were sporulated and subjected to tetrad analysis. Thirty asci were dissected and allowed to germinate on YES medium at 30°C, and only two viable segregants arose from each of the 30 asci (Fig. 4A). All 60 segregants required uracil for growth, indicating that these segregants did not carry the rad62::ura4+ allele and that the rad62+ gene is essential for growth. Microcolonies consisting of a few 10s of elongated cells were formed from the rad62::ura4+ segregants. The terminal morphology of the mutant cells grown for 2 days on a minimal plate lacking uracil was examined under an epifluorescent microscope after staining with DAPI (Fig. 4B). They were elongated, and the nuclei were mostly abnormal, sometimes extended, shrunk, fragmented, bisected by a septum (cut phenotype), or biased in position. The terminal morphology was very similar to that of rad60 and smc5 (spr18) deletion cells (13, 35).
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strain (MMP8). The cells were fixed, DNA stained with DAPI, and observed under an epifluorescent microscope (Fig. 4C). Rad62 colocalized with DNA, suggesting that it localizes to the nucleus. rad62 genetically interacts with rad60, smc6, and brc1. Since the phenotypes of rad62 mutants were very similar to those of rad60 and smc6 mutants, we suspected that these genes may be involved in a common pathway for maintaining chromosome integrity and repairing DNA damage by recombination. The brc1 gene was isolated as an allele-specific multicopy suppressor of the smc6-74 (rad18-74) mutation, and the deletion mutant, which is viable, was synthetically lethal with smc6 mutants, indicating close functional interaction with smc6 (47). The genetic interactions among the above genes were examined by constructing double mutants between rad62-1, rad60-1, smc6-X (rad18-X), and brc1. The mutants were crossed between each other, and the spores were subjected to tetrad analysis at 26°C. The double mutants combining any of the above mutations were inviable (Fig. 5).
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To examine whether Rad62 was included in the Smc5-6 complex, we performed coimmunoprecipitation experiments with FLAG-His-tagged Rad62 and Myc-tagged Smc5 expressed from their cognate promoters. From the cells expressing FLAG-His-tagged Rad62 and Myc-tagged Smc5, the tagged Rad62 was coimmunoprecipitated with the tagged Smc5 by the anti-Myc antibody and the tagged Smc5 was coimmunoprecipitated with the tagged Rd62 by the anti-FLAG M2 antibody (Fig. 7). As a negative control experiment, Rad62 was not immunoprecipitated by the anti-Myc antibody from the cells expressing FLAG-His-tagged Rad62 and untagged Smc5. The tagged Smc5 was not immunoprecipitated by the anti-FLAG M2 antibody from the cells expressing Myc-tagged Smc5 and untagged Rad62. From the comparison of the amounts of Rad62 and Smc5 in the extracts, supernatants, and immunoprecipitates, we conclude that large majorities of Rad62 and Smc5 were physically associated with each other (Fig. 7). These results suggest that Rad62 forms a protein complex with Smc5 in vivo.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We made an extensive analysis of the genetic interactions of rad62 with other genes involved in resolving the replication fork block in various ways (Fig. 8 and Table 1). The rad62-1 mutant was synthetically lethal with mutations in the rqh1, srs2, mus81, and rhp18 genes, all of which are required for repairing DNA damage or bypassing blocks to replication (26). To our surprise, it was also synthetically lethal with mutations in rad16 and nearly so with mutations in rad13, which encodes nucleases required for making incisions at the sites flanking large DNA adducts, such as pyrimidine dimers in nucleotide excision repair (29). These results are consistent with Rad62 being required for viability when replication is blocked by spontaneous damage in the absence of the functions that repair the damage or override the block by translesion DNA synthesis. The rad62 gene seems to possess at least two discrete functions, one essential for growth and the other for repair. The latter function is epistatic with rhp51, which plays a dominant role in homologous recombination and recombination repair. The rhp51 mutation could not suppress the lethality of a rad62 deletion mutant. So lethality is not due to the accumulation of recombination intermediates. Both the rad62-1 and rad60-1 mutants were not defective in homology-dependent recombination, as examined by integration of homologous linear DNA into the chromosome and by formation of spontaneous intrachromosomal conversion type recombinants. Preliminary results suggest that rad60-1 is not defective in meiotic recombination (data not shown). However, rad62 and rad60 belong to the same epistasis group with rhp51 and rhp57 with respect to repair function. The apparent discrepancies could be resolved if we assume that they function for recombination repair only at the replication fork but are not required for recombination in nonreplicating regions of chromosome. However, we cannot rule out a role for rad60 and rad62 in genetic recombination, since they are essential genes and these hypomorphic alleles may not give a strong change in recombination rates.
The Smc5-Smc6-Nse1-Nse2-Rad62 complex together with Rad60 may be a specific SMC complex functioning only at the replication fork (Fig. 9). Human Smc6 localizes to the nuclear region during the interphase and is excluded from condensed mitotic chromosomes (41). The Smc5-6 complex may provide a scaffold for the recombination reaction by holding the newly replicated sister chromatids in close proximity so that DSBs produced by replication block can be efficiently repaired by homologous recombination. The essential role of this SMC complex may be to maintain proper structure of the chromosome at the fork during replication. Spontaneous fragmentation of chromosomes and the diffuse nuclear structure observed in rad60-1 cells grown at the nonpermissive temperature may be caused by the defect of this function. The fact that the rhp51 mutation partially suppresses UV sensitivity at lower doses than the rad62-1 mutant (Fig. 3B) may suggest that Rhp51 bound to the UV-damaged fork may be toxic rather than beneficial when the newly replicated chromatids are not properly held. Since Rad60 seems transiently associated with Smc5-6 in contrast to other non-SMC components, which are more tightly associated, and is phosphorylated in response to a replication block in a manner dependent on Cds1 kinase (7), Rad60 may regulate the function of the Smc5-6 complex in response to the replication block. Further experiments are required to prove that the Smc5-6 complex functions at the replication fork.
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and swi5. rad50 has been proposed to play a role in promoting recombination between sister chromatids in collaboration with the cohesin complex (18). Swi5 has dual roles, one in mating type switching and the other in recombination repair in a novel pathway dependent on rhp51 but independent of rhp55-57 (1). Since the swi5 rhp57 double mutants are additive in UV sensitivity and equally as sensitive as rhp51, the recombination repair pathway involving the Smc5-6 complex function is likely to be the same as the one involving the rhp55-57 functions. The additive UV sensitivity in the rad62-1 uve1 double mutant indicates that rad62 may function in a pathway different from the UV nucleotide excision repair pathway (32).
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ACKNOWLEDGMENTS |
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This work is supported by the Human Frontier Science Program (A.M.C. and H.S.) and Grants-in-Aid for Scientific Research of Priority Areas from Ministry of Education, Culture, Sports, Science, and Technology of Japan to H.S.
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FOOTNOTES |
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Present address: Tsukuba Research Institute, Novartis Pharma, Tsukuba, Ibaraki 300-2611, Japan.
Present address: Graduate School of Integrated Science, Yokohama City University, Tsurumi-ku, Yokohama 230-0045, Japan.
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REFERENCES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2. Aono, N., T. Sutani, T. Tomonaga, S. Mochida, and M. Yanagida. 2002. Cnd2 has dual roles in mitotic condensation and interphase. Nature 417:197-202.[CrossRef][Medline]
3. Barbet, N., W. J. Muriel, and A. M. Carr. 1992. Versatile shuttle vectors and genomic libraries for use with Schizosaccharomyces pombe. Gene 114:59-66.[CrossRef][Medline]
4. Birkenbihl, R. P., and S. Subramani. 1992. Cloning and characterization of rad21 an essential gene of Schizosaccharomyces pombe involved in DNA double-strand-break repair. Nucleic Acids Res. 20:6605-6611.
5. Boddy, M. N., P. H. Gaillard, W. H. McDonald, P. Shanahan, J. R. Yates III, and P. Russell. 2001. Mus81-Eme1 are essential components of a Holliday junction resolvase. Cell 107:537-548.[CrossRef][Medline]
6. Boddy, M. N., A. Lopez-Girona, P. Shanahan, H. Interthal, W. D. Heyer, and P. Russell. 2000. Damage tolerance protein Mus81 associates with the FHA1 domain of checkpoint kinase Cds1. Mol. Cell. Biol. 20:8758-8766.
7. Boddy, M. N., P. Shanahan, W. H. McDonald, A. Lopez-Girona, E. Noguchi, I. J. Yates, and P. Russell. 2003. Replication checkpoint kinase Cds1 regulates recombinational repair protein Rad60. Mol. Cell. Biol. 23:5939-5946.
8. Cao, Y., and T. Kogoma. 1995. The mechanism of recA polA lethality: suppression by RecA-independent recombination repair activated by the lexA(Def) mutation in Escherichia coli. Genetics 139:1483-1494.[Abstract]
9. Craven, R. A., D. J. Griffiths, K. S. Sheldrick, R. E. Randall, I. M. Hagan, and A. M. Carr. 1998. Vectors for the expression of tagged proteins in Schizosaccharomyces pombe. Gene 221:59-68.[CrossRef][Medline]
10. Cuperus, G., and D. Shore. 2002. Restoration of silencing in Saccharomyces cerevisiae by tethering of a novel Sir2-interacting protein, Esc8. Genetics 162:633-645.
11. Debrauwere, H., S. Loeillet, W. Lin, J. Lopes, and A. Nicolas. 2001. Links between replication and recombination in Saccharomyces cerevisiae: a hypersensitive requirement for homologous recombination in the absence of Rad27 activity. Proc. Natl. Acad. Sci. USA 98:8263-8269.
12. Forsburg, S. L. 1993. Comparison of Schizosaccharomyces pombe expression systems. Nucleic Acids Res. 21:2955-2956.
13. Fousteri, M. I., and A. R. Lehmann. 2000. A novel SMC protein complex in Schizosaccharomyces pombe contains the Rad18 DNA repair protein. EMBO J. 19:1691-1702.[CrossRef][Medline]
14. Fujioka, Y., Y. Kimata, K. Nomaguchi, K. Watanabe, and K. Kohno. 2002. Identification of a novel non-structural maintenance of chromosomes (SMC) component of the SMC5-SMC6 complex involved in DNA repair. J. Biol. Chem. 277:21585-21591.
15. Grimm, C., J. Kohli, J. Murray, and K. Maundrell. 1988. Genetic engineering of Schizosaccharomyces pombe: a system for gene disruption and replacement using the ura4 gene as a selectable marker. Mol. Gen. Genet. 215:81-86.[CrossRef][Medline]
16. Gruber, S., C. H. Haering, and K. Nasmyth. 2003. Chromosomal cohesin forms a ring. Cell 112:765-777.[CrossRef][Medline]
17. Gysler-Junker, A., Z. Bodi, and J. Kohli. 1991. Isolation and characterization of Schizosaccharomyces pombe mutants affected in mitotic recombination. Genetics 128:495-504.[Abstract]
18. Hartsuiker, E., E. Vaessen, A. M. Carr, and J. Kohli. 2001. Fission yeast Rad50 stimulates sister chromatid recombination and links cohesion with repair. EMBO J. 20:6660-6671.[CrossRef][Medline]
19. Harvey, S. H., D. M. Sheedy, A. R. Cuddihy, and M. J. O'Connell. 2004. Coordination of DNA damage responses via the Smc5/Smc6 complex. Mol. Biol. Cell 24:662-674.
20. Hazbun, T. R., L. Malmstrom, S. Anderson, B. J. Graczyk, B. Fox, M. Riffle, B. A. Sundin, J. D. Aranda, W. H. McDonald, C. H. Chiu, B. E. Snydsman, P. Bradley, E. G. Muller, S. Fields, D. Baker, J. R. Yates III, and T. N. Davis. 2003. Assigning function to yeast proteins by integration of technologies. Mol. Cell 12:1353-1365.[CrossRef][Medline]
21. Hirano, T. 2002. The ABCs of SMC proteins: two-armed ATPases for chromosome condensation, cohesion, and repair. Genes Dev. 16:399-414.
22. Hollingsworth, N. M., and S. J. Brill. 2004. The Mus81 solution to resolution: generating meiotic crossovers without Holliday junctions. Genes Dev. 18:117-125.
23. Ishioka, K., A. Fukuoh, H. Iwasaki, A. Nakata, and H. Shinagawa. 1998. Abortive recombination in Escherichia coli ruv mutants blocks chromosome partitioning. Genes Cells 3:209-220.[Abstract]
24. Ishioka, K., H. Iwasaki, and H. Shinagawa. 1997. Roles of the recG gene product of Escherichia coli in recombination repair: effects of the delta recG mutation on cell division and chromosome partition. Genes Genet. Syst. 72:91-99.[CrossRef][Medline]
25. Jang, Y. K., Y. H. Jin, Y. S. Shim, M. J. Kim, E. J. Yoo, I. S. Choi, J. S. Lee, R. H. Seong, S. H. Hong, and S. D. Park. 1996. Identification of the DNA damage-responsive elements of the rhp51+ gene, a recA and RAD51 homolog from the fission yeast Schizosaccharomyces pombe. Mol. Gen. Genet. 251:167-175.[Medline]
26. Kai, M., and T. S. Wang. 2003. Checkpoint responses to replication stalling: inducing tolerance and preventing mutagenesis. Mutat. Res. 532:59-73.[Medline]
27. Kostrub, C. F., F. al-Khodairy, H. Ghazizadeh, A. M. Carr, and T. Enoch. 1997. Molecular analysis of hus1+, a fission yeast gene required for S-M and DNA damage checkpoints. Mol. Gen. Genet. 254:389-399.[Medline]
27. Krawchuk, M. D., and W. P. Wahls. 1999. High-efficiency gene targeting in Schizosaccharcmyces pombe using a modular, PCR-based approach with long tracts of flanking homology. Yeast 15:1419-1427.
28. Krejci, L., S. Van Komen, Y. Li, J. Villemain, M. S. Reddy, H. Klein, T. Ellenberger, and P. Sung. 2003. DNA helicase Srs2 disrupts the Rad51 presynaptic filament. Nature 423:305-309.[CrossRef][Medline]
29. Lehmann, A. R. 1996. Molecular biology of DNA repair in the fission yeast Schizosaccharomyces pombe. Mutat. Res. 363:147-161.[CrossRef][Medline]
30. Lehmann, A. R., M. Walicka, D. J. Griffiths, J. M. Murray, F. Z. Watts, S. McCready, and A. M. Carr. 1995. The rad18 gene of Schizosaccharomyces pombe defines a new subgroup of the SMC superfamily involved in DNA repair. Mol. Cell. Biol. 15:7067-7080.[Abstract]
31. Maundrell, K. 1993. Thiamine-repressible expression vectors pREP and pRIP for fission yeast. Gene 123:127-130.[CrossRef][Medline]
32. McCready, S. J., F. Osman, and A. Yasui. 2000. Repair of UV damage in the fission yeast Schizosaccharomyces pombe. Mutat. Res. 451:197-210.[Medline]
33. McDonald, W. H., Y. Pavlova, J. R. Yates III, and M. N. Boddy. 2003. Novel essential DNA repair proteins Nse1 and Nse2 are subunits of the fission yeast Smc5-Smc6 complex. J. Biol. Chem. 278:45460-45467.
34. Moreno, S., A. Klar, and P. Nurse. 1991. Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol. 194:795-823.[Medline]
35. Morishita, T., Y. Tsutsui, H. Iwasaki, and H. Shinagawa. 2002. The Schizosaccharomyces pombe rad60 gene is essential for repairing double-strand DNA breaks spontaneously occurring during replication and induced by DNA-damaging agents. Mol. Cell. Biol. 22:3537-3548.
36. Murray, J. M., H. D. Lindsay, C. A. Munday, and A. M. Carr. 1997. Role of Schizosaccharomyces pombe RecQ homolog, recombination, and checkpoint genes in UV damage tolerance. Mol. Cell. Biol. 17:6868-6875.[Abstract]
37. Murray, J. M., M. Tavassoli, R. al-Harithy, K. S. Sheldrick, A. R. Lehmann, A. M. Carr, and F. Z. Watts. 1994. 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. Mol. Cell. Biol. 14:4878-4888.
38. Osman, F., M. Adriance, and S. McCready. 2000. The genetic control of spontaneous and UV-induced mitotic intrachromosomal recombination in the fission yeast Schizosaccharomyces pombe. Curr. Genet. 38:113-125.[CrossRef][Medline]
39. Osman, F., E. A. Fortunato, and S. Subramani. 1996. Double-strand break-induced mitotic intrachromosomal recombination in the fission yeast Schizosaccharomyces pombe. Genetics 142:341-357.[Abstract]
40. Symington, L. S. 2002. Role of RAD52 epistasis group genes in homologous recombination and double-strand break repair. Microbiol. Mol. Biol. Rev. 66:630-670.
41. Taylor, E. M., J. S. Moghraby, J. H. Lees, B. Smit, P. B. Moens, and A. R. Lehmann. 2001. Characterization of a novel human SMC heterodimer homologous to the Schizosaccharomyces pombe Rad18/Spr18 complex. Mol. Biol. Cell 12:1583-1594.
42. Tomita, K., A. Matsuura, T. Caspari, A. M. Carr, Y. Akamatsu, H. Iwasaki, K. Mizuno, K. Ohta, M. Uritani, T. Ushimaru, K. Yoshinaga, and M. Ueno. 2003. Competition between the Rad50 complex and the Ku heterodimer reveals a role for Exo1 in processing double-strand breaks but not telomeres. Mol. Cell. Biol. 23:5186-5197.
43. Tong, A. H., M. Evangelista, A. B. Parsons, H. Xu, G. D. Bader, N. Page, M. Robinson, S. Raghibizadeh, C. W. Hogue, H. Bussey, B. Andrews, M. Tyers, and C. Boone. 2001. Systematic genetic analysis with ordered arrays of yeast deletion mutants. Science 294:2364-2368.
44. Tsutsui, Y., T. Morishita, H. Iwasaki, H. Toh, and H. Shinagawa. 2000. 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. Genetics 154:1451-1461.
45. Ueno, M., T. Nakazaki, Y. Akamatsu, K. Watanabe, K. Tomita, H. D. Lindsay, H. Shinagawa, and H. Iwasaki. 2003. Molecular characterization of the Schizosaccharomyces pombe nbs1+ gene involved in DNA repair and telomere maintenance. Mol. Cell. Biol. 23:6553-6563.
46. Van Komen, S., M. S. Reddy, L. Krejci, H. Klein, and P. Sung. 2003. ATPase and DNA helicase activities of the Saccharomyces cerevisiae anti-recombinase Srs2. J. Biol. Chem. 278:44331-44337.
47. Verkade, H. M., S. J. Bugg, H. D. Lindsay, A. M. Carr, and M. J. O'Connell. 1999. Rad18 is required for DNA repair and checkpoint responses in fission yeast. Mol. Biol. Cell 10:2905-2918.
48. Verkade, H. M., T. Teli, L. V. Laursen, J. M. Murray, and M. J. O'Connell. 2001. A homologue of the Rad18 postreplication repair gene is required for DNA damage responses throughout the fission yeast cell cycle. Mol. Genet. Genomics 265:993-1003.[CrossRef][Medline]
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