A new Schizosaccharomyces pombe base excision repair mutant,nth1, reveals overlapping pathways for repair of DNA base damage

Endonuclease III (Nth) enzyme from Escherichia coli is involved in base excision repair of oxidised pyrimidine residues in DNA. The Schizosaccharomyces pombe Nth1 protein is a sequence and functional homologue of E. coli Nth, possessing both DNA glycosylase and apurinic/apyrimidinic (AP) lyase activity. Here, we report the construction and characterization of the S. pombe nth1 mutant. The nth1 mutant exhibited no enhanced sensitivity to oxidising agents, UV or gamma-irradiation, but was hypersensitive to the alkylating agent methyl methanesulphonate (MMS). Analysis of base excision from DNA exposed to [3H]methyl-N-nitrosourea showed that the purified Nth1 enzyme did not remove alkylated bases such as 3-methyladenine and 7-methylguanine whereas methyl-formamidopyrimidine was excised efficiently. The repair of AP sites in S. pombe has previously been shown to be independent of Apn1-like AP endonuclease activity, and the main reason for the MMS sensitivity of nth1 cells appears to be their lack of AP lyase activity. The nth1 mutant also exhibited elevated frequencies of spontaneous mitotic intrachromosomal recombination, which is a phenotype shared by the MMS-hypersensitive DNA repair mutants rad2, rhp55 and NER repair mutants rad16, rhp14, rad13 and swi10. Epistasis analyses of nth1 and these DNA repair mutants suggest that several DNA damage repair/tolerance pathways participate in the processing of alkylation and spontaneous DNA damage in S. pombe.     

Involvement of 3-methyladenine DNA glycosylases Mag1p and Mag2p in base excision repair of methyl methanesulfonate-damaged DNA in the fission yeast Schizosaccharomyces pombe

Schizosaccharomyces pombe has two paralogues of 3-methyladenine DNA glycosylase, Mag1p and Mag2p, which share homology with Escherichia coli AlkA. To clarify the function of these redundant enzymes in base excision repair (BER) of alkylation damage, we performed several genetic analyses. The mag1 and mag2 single mutants as well as the double mutant showed no obvious methyl methanesulfonate (MMS) sensitivity. Deletion of mag1 or mag2 from an nth1 mutant resulted in tolerance to MMS damage, indicating that both enzymes generate AP sites in vivo by removal of methylated bases. A rad16 mutant that is deficient in nucleotide excision repair (NER) exhibited moderate MMS sensitivity. Deletion of mag1 from the rad16 mutant greatly enhanced MMS sensitivity, and the mag2 deletion also weakened the resistance to MMS of the rad16 mutant. A mag1/mag2/rad16 triple mutant was most sensitive to MMS. These results suggest that the NER pathway obscures the mag1 and mag2 functions in MMS resistance and that both paralogues initiate the BER pathway of MMS-induced DNA damage at the same level in NER-deficient cells or that Mag2p tends to make a little lower contribution than Mag1p. Mag1p and Mag2p functioned additively in vivo. Expression of mag1 and mag2 in the triple mutant confirmed the contribution of Mag1p and Mag2p to BER of MMS resistance.     

Biochemical characterization and DNA repair pathway interactions of Mag1-mediated base excision repair in Schizosaccharomyces pombe

The Schizosaccharomyces pombe mag1 gene encodes a DNA repair enzyme with sequence similarity to the AlkA family of DNA glycosylases, which are essential for the removal of cytotoxic alkylation products, the premutagenic deamination product hypoxanthine and certain cyclic ethenoadducts such as ethenoadenine. In this paper, we have purified the Mag1 protein and characterized its substrate specificity. It appears that the substrate range of Mag1 is limited to the major alkylation products, such as 3-mA, 3-mG and 7-mG, whereas no significant activity was found towards deamination products, ethenoadducts or oxidation products. The efficiency of 3-mA and 3-mG removal was 5–10 times slower for Mag1 than for Escherichia coli AlkA whereas the rate of 7-mG removal was similar to the two enzymes. The relatively low efficiency for the removal of cytotoxic 3-methylpurines is consistent with the moderate sensitivity of the mag1 mutant to methylating agents. Furthermore, we studied the initial steps of Mag1-dependent base excision repair (BER) and genetic interactions with other repair pathways by mutant analysis. The double mutants mag1 nth1, mag1 apn2 and mag1 rad2 displayed increased resistance to methyl methanesulfonate (MMS) compared with the single mutants nth1, apn2 and rad2, respectively, indicating that Mag1 initiates both short-patch (Nth1-dependent) and long-patch (Rad2-dependent) BER of MMS-induced damage. Spontaneous intrachromosomal recombination frequencies increased 3-fold in the mag1 mutant suggesting that Mag1 and recombinational repair (RR) are both involved in repair of alkylated bases. Finally, we show that the deletion of mag1 in the background of rad16, nth1 and rad2 single mutants reduced the total recombination frequencies of all three double mutants, indicating that abasic sites formed as a result of Mag1 removal of spontaneous base lesions are substrates for nucleotide excision repair, long- and short-patch BER and RR.     

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.     

Excision repair of nitrogen mustard-DNA adducts in Saccharomyces cerevisiae.

The bifunctional alkylating anticancer drug nitrogen mustard forms a variety of DNA lesions, including monoadducts and intrastrand and interstrand crosslinks. Although it is known that nucleotide excision repair (NER) is important in processing these adducts, the role of the other principal excision repair pathway, base excision repair (BER) is less well defined. Using isogenic Saccharomyces cerevisiae strains disrupted for a variety of NER and BER genes we have examined the relative importance of the two pathways in the repair of nitrogen mustard adducts. As expected, NER defective cells (rad4 and rad14 strains) are extremely sensitive to the drug. One of the BER mutants, a 3-methyladenine glycosylase defective (mag1) strain also shows significant hypersensitivity. Using a rad4/mag1 double mutant it is shown that the two excision repair pathways are epistatic to each other for nitrogen mustard sensitivity. Furthermore, both rad14 and mag1 disruptants show elevated levels of nitrogen mustard-induced forward mutation. Measurements of repair rates of nitrogen mustard N-alkylpurine adducts in the highly transcribed RPB2 gene demonstrate defects in the processing of mono-adducts in rad4, rad14 and mag1 strains. However, there are differences in the kinetics of adduct removal in the NER mutants compared to the mag1 strain. In the mag1 strain significant repair occurs within 1 h with evidence of enhanced repair on the transcribed strand. Adducts however accumulate at later times in this strain. In contrast, in the NER mutants repair is only evident at times greater than 1 h. In a mag1/rad4 double mutant damage accumulates with no evidence of repair. Comparison of the rates of repair in this gene with those in a different genomic region indicate that the contributions of NER and BER to the repair of nitrogen mustard adducts may not be the same genome wide.     

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.     

Polynucleotide kinase/phosphatase, Pnk1, is involved in base excision repair in Schizosaccharomyces pombe

We previously reported that Schizosaccharomyces pombe pnk1 cells are more sensitive than wild-type cells to γ-radiation and camptothecin, indicating that Pnk1 is required for DNA repair. Here, we report that pnk1pku70 and pnk1rhp51 double mutants are more sensitive to γ-radiation than single mutants, from which we infer that Pnk1's primary role is independent of either homologous recombination or non-homologous end joining mechanisms. We also report that pnk1 cells are more sensitive than wild-type cells to oxidizing and alkylating agents, suggesting that Pnk1 is involved in base excision repair. Mutational analysis of Pnk1 revealed that the DNA 3'-phosphatase activity is necessary for repair of DNA damage, whereas the 5'-kinase activity is dispensable. A role for Pnk1 in base excision repair is supported by genetic analyses which revealed that pnk1apn2 is synthetically lethal, suggesting that Pnk1 and Apn2 may function in parallel pathways essential for the repair of endogenous DNA damage. Furthermore, the nth1pnk1apn2 and tdp1pnk1apn2 triple mutants are viable, implying that single-strand breaks with 3'-blocked termini produced by Nth1 and Tdp1 contribute to synthetic lethality. We also examined the sensitivity to methyl methanesulfonate of all single and double mutant combinations of nth1, apn2, tdp1 and pnk1. Together, our results support a model where Tdp1 and Pnk1 act in concert in an Apn2-independent base excision repair pathway to repair 3'-blocked termini produced by Nth1; and they also provide evidence that Pnk1 has additional roles in base excision repair.     

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.     

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.     

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.     

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.     

The Schizosaccharomyces pombe rhp3+ gene required for DNA repair and cell viability is functionally interchangeable with the RAD3 gene of Saccharomyces cerevisiae.

The RAD3 gene of Saccharomyces cerevisiae is required for excision repair and is essential for cell viability. RAD3 encoded protein possesses a single stranded DNA-dependent ATPase and DNA and DNA.RNA helicase activities. Mutational studies have indicated a requirement for the RAD3 helicase activities in excision repair. To examine the extent of conservation of structure and function of RAD3 during eukaryotic evolution, we have cloned the RAD3 homolog, rhp3+, from the distantly related yeast Schizosaccharomyces pombe. RAD3 and rhp3+ encoded proteins are highly similar, sharing 67% identical amino acids. We show that like RAD3, rhp3+ is indispensable for excision repair and cell viability, and our studies indicate a requirement of the putative rhp3+ DNA helicase activity in DNA repair. We find that the RAD3 and rhp3+ genes can functionally substitute for one another. The level of complementation provided by the rhp3+ gene in S.cerevisiae rad3 mutants or by the RAD3 gene in S.pombe rhp3 mutants is remarkable in that both the excision repair and viability defects in both yeasts are restored to wild type levels. These observations suggest a parallel evolutionary conservation of other protein components with which RAD3 interacts in mediating its DNA repair and viability functions.     

Characterization of the rhp7(+) and rhp16(+) genes in Schizosaccharomyces pombe.

The global genome repair (GGR) subpathway of nucleotide excision repair (NER) is capable of removing lesions throughout the genome. In Saccharomyces cerevisiae the RAD7 and RAD16 genes are essential for GGR. Here we identify rhp7 (+), the RAD7 homolog in Schizosaccharomyces pombe. Surprisingly, rhp7 (+)and the previously cloned rhp16 (+)are located very close together and are transcribed in opposite directions. Upon UV irradiation both genes are induced, reaching a maximum level after 45-60 min. These observations suggest that the genes are co-regulated. Schizo-saccharomyces pombe rhp7 or rhp16 deficient cells are, in contrast to S.cerevisiae rad7 and rad16 mutants, not sensitive to UV irradiation. In S.pombe an alternative repair mechanism, UV damage repair (UVDR), is capable of efficiently removing photolesions from DNA. In the absence of this UVDR pathway both rhp7 and rhp16 deficient cells display an enhanced UV sensitivity. Epistatic analyses show that rhp7 (+)and rhp16 (+)are only involved in NER. Repair analyses at nucleotide resolution demonstrate that both Rhp7 and Rhp16, probably acting in a complex, are essential for GGR in S.pombe.     

Binding and repair of mismatched DNA mediated by Rhp14, the fission yeast homologue of human XPA.

Rhp14 of Schizosaccharomyces pombe is homologous to human XPA and Saccharomyces cerevisiae Rad14, which act in nucleotide excision repair of DNA damages induced by ultraviolet light and chemical agents. Cells with disrupted rhp14 were highly sensitive to ultraviolet light, and epistasis analysis with swi10 (nucleotide excision repair) and rad2 (Uve1-dependent ultraviolet light damage repair pathway) revealed that Rhp14 is an important component of nucleotide excision repair for ultraviolet light-induced damages. Moreover, defective rhp14 caused instability of a GT repeat, similar to swi10 and synergistically with msh2 and exo1. Recombinant Rhp14 with an N-terminal hexahistidine tag was purified from Escherichia coli. Complementation studies with a rhp14 mutant demonstrated that the tagged Rhp14 is functional in repair of ultraviolet radiation-induced damages and in mitotic mutation avoidance. In bandshift assays, Rhp14 showed a preference to substrates with mismatched and unpaired nucleotides. Similarly, XPA bound more efficiently to C/C, A/C, and T/C mismatches than to homoduplex DNA. Our data show that mismatches and loops in DNA are substrates of nucleotide excision repair. Rhp14 is likely part of the recognition complex but alone is not sufficient for the high discrimination of nucleotide excision repair for modified DNA.     

Synergism between base excision repair,mediated by the DNA glycosylases Ntg1 and Ntg2, and nucleotide excision repair in the removal of oxidatively damaged DNA bases in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, inactivation of the two DNA N-glycosylases Ntg1p and Ntg2p does not result in a spontaneous mutator phenotype, whereas simultaneous inactivation of Ntglp, Ntg2p and Radlp or Rad14p, both of which are involved in nucleotide excision repair (NER), does. The triple mutants rad1 ntg1 ntg2 and rad14 ntg1 ntg2 show 15- and 22-fold increases, respectively, in spontaneous forward mutation to canavanine resistance (CanR) relative to the wild-type strain (WT). In contrast, neither of these triple mutants shows an increase in the incidence of Lys+ revertants of the lys1-1 ochre allele. Furthermore, the rad1 ntg1 ntg2 mutant is hypersensitive to the lethal effect of H2O2 relative to WT, rad1 and ntg1 ntg2 mutant strains. Moreover, the rad1 ntg1 ntg2 strain is hypermutable (CanR and Lys+) upon exposure to H2O2, relative to WT, rad1 and ntg1 ntg2 strains. Mutagen sensitivity and enhanced mutagenesis in the rad1 ntg1 ntg2 triple mutant, relative to the other strains tested, were also observed upon exposure to oxidizing agents such as tertbutylhydroperoxide and menadione. In contrast, the sensitivity of the rad1 ntg1 ntg2 triple mutant to gamma-irradiation does not differ from that of the WT. However, the triple mutant shows an increase in the frequency of Lys+ revertants recovered after gamma-irradiation. The results reported in this study demonstrate that base excision repair (BER) mediated by Ntglp and Ntg2p acts synergistically with NER to repair endogenous or induced lethal and mutagenic oxidative DNA damage in yeast. The substrate specificity of Ntg1 p and Ntg2p, and the spectrum of lesions induced by the DNA-damaging agents used, strongly suggest that oxidized DNA bases, presumably oxidized pyrimidines, represent the major targets of this repair pathway.     

Role of the DNA repair nucleases Rad13, Rad2 and Uve1 of Schizosaccharomyces pombe in mismatch correction.

Repair of mismatched DNA occurs mainly by the long-patch mismatch repair (MMR) pathway, requiring Msh2 and Pms1. In Schizosaccharomyces pombe mismatches can be repaired by a short-patch repair system, containing nucleotide excision repair (NER) factors. We studied mismatch correction efficiency in cells with inactivated DNA repair nucleases Rad13, Rad2 or Uve1 in MMR proficient and deficient background. Rad13 incises 3' of damaged DNA during NER. Rad2 has a function in the Uve1-dependent repair of DNA damages and in replication. Loss of Rad13 caused a strong reduction of short-patch processing of mismatches formed during meiotic recombination. Mitotic mutation rates were increased, but not to the same extent as in the NER mutant swi10, which is defective in 5' incision. The difference might be caused by an additional role of Rad13 in base excision repair or due to partial redundancy with other 3' endonucleases. Meiotic mismatch repair was not or only slightly affected in rad2 and uve1 mutants. In addition, inactivation of uve1 caused only weak effects on mutation avoidance. Mutation rates were elevated when rad2 was mutated, but not further increased in swi10 rad2 and rad13 rad2 double mutants, indicating an epistatic relationship. However, the mutation spectra of rad2 were different from that of swi10 and rad13. Thus, the function of Rad2 in mutation avoidance is rather independent of NER. rad13, swi10 and rad2, but not uve1 mutants were sensitive to the DNA-damaging agent methyl methane sulphonate. Cell survival was further reduced in the double mutants swi10 rad2, rad13 rad2 and, surprisingly, swi10 rad13. These data confirm that NER and Rad2 act in distinct damage repair pathways and further indicate that the function of Rad13 in repair of alkylated bases is partially independent of NER.     

Two budding yeast RAD4 homologs in fission yeast play different roles in the repair of UV-induced DNA damage

We have identified two fission yeast homologs of budding yeast Rad4 and human xeroderma pigmentosum complementation group C (XP-C) correcting protein, designated Rhp4A and Rhp4B. Here we show that the rhp4 genes encode NER factors that are required for UV-induced DNA damage repair in fission yeast. The rhp4A-deficient cells but not the rhp4B-deficient cells are sensitive to UV irradiation. However, the disruption of both rhp4A and rhp4B resulted in UV sensitivity that was greater than that of the rhp4A-deficient cells, revealing that Rhp4B plays a role in DNA repair on its own. Fission yeast has two pathways to repair photolesions on DNA, namely, nucleotide excision repair (NER) and UV-damaged DNA endonuclease-dependent excision repair (UVER). Studies with the NER-deficient rad13 and the UVER-deficient (Delta)uvde mutants showed the two rhp4 genes are involved in NER and not UVER. Assessment of the ability of the various mutants to remove cyclobutane pyrimidine dimers (CPDs) from the rbp2 gene locus indicated that Rhp4A is involved in the preferential repair of lesions on the transcribed DNA strand and plays the major role in fission yeast NER. Rhp4B in contrast acts as an accessory protein in non-transcribed strand (NTS) repair.