Relationships between yeast Rad27 and Apn1 in response to apurinic/apyrimidinic (AP) sites in DNA.

Yeast Rad27 is a 5'-->3' exonuclease and a flap endo-nuclease. Apn1 is the major apurinic/apyrimidinic (AP) endonuclease in yeast. The rad27 deletion mutants are highly sensitive to methylmethane sulfonate (MMS). By examining the role of Rad27 in different modes of DNA excision repair, we wish to understand why the cytotoxic effect of MMS is dramatically enhanced in the absence of Rad27. Base excision repair (BER) of uracil-containing DNA was deficient in rad27 mutant extracts in that (i) the Apn1 activity was reduced, and (ii) after DNA incision by Apn1, hydrolysis of 1-5 nucleotides 3' to the baseless sugar phosphate was deficient. Thus, some AP sites may lead to unprocessed DNA strand breaks in rad27 mutant cells. The severe MMS sensitivity of rad27 mutants is not caused by a reduction of the Apn1 activity. Surprisingly, we found that Apn1 endonuclease sensitizes rad27 mutant cells to MMS. Deleting the APN1 gene largely restored the resistance of rad27 mutants to MMS. These results suggest that unprocessed DNA strand breaks at AP sites are mainly responsible for the MMS sensitivity of rad27 mutants. In contrast, nucleotide excision repair and BER of oxidative damage were not affected in rad27 mutant extracts, indicating that Rad27 is specifically required for BER of AP sites in DNA.     

Complementary functions of the Saccharomyces cerevisiae Rad2 family nucleases in Okazaki fragment maturation,mutation avoidance,and chromosome stability

Rad2 family nucleases, identified by sequence similarity within their catalytic domains, function in multiple pathways of DNA metabolism. Three members of the Saccharomyces cerevisiae Rad2 family, Rad2, Rad27, and exonuclease 1 (Exo1), exhibit both 5' exonuclease and flap endonuclease activities. Deletion of RAD27 results in defective Okazaki fragment maturation, DNA repair, and subsequent defects in mutation avoidance and chromosomal stability. However, strains lacking Rad27 are viable. The expression profile of EXO1 during the cell cycle is similar to that of RAD27 and other genes encoding proteins that function in DNA replication and repair, suggesting Exo1 may function as a back up nuclease for Rad27 in DNA replication. We show that overexpression of EXO1 suppresses multiple rad27 null mutation-associated phenotypes derived from DNA replication defects, including temperature sensitivity, Okazaki fragment accumulation, the rate of minichromosome loss, and an elevated mutation frequency. While generally similar findings were observed with RAD2, overexpression of RAD2, but not EXO1, suppressed the MMS sensitivity of the rad27 null mutant cells. This suggests that Rad2 can uniquely complement Rad27 in base excision repair (BER). Furthermore, Rad2 and Exo1 complemented the mutator phenotypes and cell cycle defects of rad27 mutant strains to differing extents, suggesting distinct in vivo nucleic acid substrates.     

Requirement for ATP by the DNA damage checkpoint clamp loader

The DNA damage clamp loader replication factor C (RFC-Rad24) consists of the Rad24 protein and the four small Rfc2-5 subunits of RFC. This complex loads the heterotrimeric DNA damage clamp consisting of Rad17, Mec3, and Ddc1 (Rad17/3/1) onto partial duplex DNA in an ATP-dependent manner. Interactions between the clamp loader and the clamp have been proposed to mirror those of the replication clamp loader RFC and the sliding clamp proliferating cell nuclear antigen (PCNA). In that system, three ATP molecules bound to the Rfc2, Rfc3, and Rfc4 subunits are necessary and sufficient for efficient loading of PCNA, whereas ATP binding to Rfc1 is not required. In contrast, in this study, we show that mutant RFC-Rad24 with a rad24-K115E mutation in the ATP-binding domain of Rad24 shows defects in the ATPase of the complex and is defective for interaction with Rad17/3/1 and for loading of the checkpoint clamp. A similar defect was measured with a mutant RFC-Rad24 clamp loader carrying a rfc4K55R ATP-binding mutation, whereas the rfc4K55E clamp loader showed partial loading activity, in agreement with genetic studies of these mutants. These studies show that ATP utilization by the checkpoint clamp/clamp loader system is effectively different from that by the structurally analogous replication system.     

Genetic network interactions among replication, repair and nuclear pore deficiencies in yeast

The yeast RAD27 gene encodes a functional homolog of the mammalian FEN1 protein, a structure-specific endo/exonuclease which plays an important role in DNA replication and repair. Previous genetic interaction studies, including a synthetic genetic array (SGA) analysis, showed that the survival of rad27Delta cells requires several DNA metabolic processes, in particular those mediated by all members of the Rad52-dependent recombinational repair pathway. Here, we report the results of our SGA analysis of the collection of non-essential yeast genes against the rad27Delta mutation, which resulted in the identification of a novel synthetic lethal interaction conferred by mutations affecting the Nup84 nuclear pore subcomplex (nup133Delta, nup120Delta and nup84Delta). Additional screens showed that all Rad52 group genes are required for the survival of the nup133Delta and nup120Delta mutants, which are defective in nuclear pore distribution and mRNA export, but not of the nup133DeltaN mutant, which is solely defective in pore distribution. This requirement for the DNA double-strand break (DSB) repair pathway is consistent with the observation that, like rad27Delta, the nup133Delta, nup120Delta and nup84Delta mutants are sensitive to methyl methanesulfonate (MMS). Furthermore, nup133Delta cells exhibit an increased number of spontaneous DNA repair foci containing Rad52. Altogether, these data suggest that the pathological interactions between the rad27Delta and specific nupDelta mutations result from the accumulation of unrepaired DNA damages.     

The PCNA Interaction Protein Box Sequence in Rad54 Is an Integral Part of Its ATPase Domain and Is Required for Efficient DNA Repair and Recombination

Rad54 is an ATP-driven translocase involved in the genome maintenance pathway of homologous recombination (HR). Although its activity has been implicated in several steps of HR, its exact role(s) at each step are still not fully understood. We have identified a new interaction between Rad54 and the replicative DNA clamp, proliferating cell nuclear antigen (PCNA). This interaction was only mildly weakened by the mutation of two key hydrophobic residues in the highly-conserved PCNA interaction motif (PIP-box) of Rad54 (Rad54-AA). Intriguingly, the rad54-AA mutant cells displayed sensitivity to DNA damage and showed HR defects similar to the null mutant, despite retaining its ability to interact with HR proteins and to be recruited to HR foci in vivo. We therefore surmised that the PCNA interaction might be impaired in vivo and was unable to promote repair synthesis during HR. Indeed, the Rad54-AA mutant was defective in primer extension at the MAT locus as well as in vitro, but additional biochemical analysis revealed that this mutant also had diminished ATPase activity and an inability to promote D-loop formation. Further mutational analysis of the putative PIP-box uncovered that other phenotypically relevant mutants in this domain also resulted in a loss of ATPase activity. Therefore, we have found that although Rad54 interacts with PCNA, the PIP-box motif likely plays only a minor role in stabilizing the PCNA interaction, and rather, this conserved domain is probably an extension of the ATPase domain III.     

Interaction of the yeast Pso5/Rad16 and Sgs1 proteins: influences on DNA repair and aging.

The interaction trap method was used to isolate putative binding partners of Rad16/Pso5, a protein responsible for repair of silent DNA. One of the interactors found was Sgs1, a DNA helicase influencing the life span of Saccharomyces cerevisiae, with homology to the human BLM, WRN and RECQL4 proteins. Using the same fusion proteins from the two-hybrid screening, we show evidence that both proteins also interact in vitro. We tested isogenic strains, containing mutant alleles of the two genes in single and double mutant combination, for phenotypic similarity. Life span in sgs1Delta single and sgs1Delta rad16Delta double mutants is about 40% of that of WT, and the rad16/pso5Delta single mutant also had its life span reduced to 75%. Sensitivity to different mutagens, whose lesions are poorly repaired in rad16/pso5Delta mutants, was tested in sgs1Delta mutants. The sgs1Delta conferred sensitivity to MMS, H2O2 and was moderately sensitive to UV(254nm) (UVC) and 4-NQO. An epistatic interaction between rad16 and sgs1 mutations after UVC, 4-NQO and H2O2 was observed. Moreover, we found that in a top3 background, functional Sgs1p and Rad16p apparently channel MMS, 4-NQO and H2O2 induced lesions into aberrant DNA repair. Our results demonstrate that Sgs1 is not only involved in genome stability, somatic recombination and aging, but is also implicated, together with Rad16/Pso5, in the repair of specific DNA damage.     

Characterization of the hyperrecombination phenotype of the pol3-t mutation of Saccharomyces cerevisiae

The DNA polymerase delta (Pol3p/Cdc2p) allele pol3-t of Saccharomyces cerevisiae has previously been shown to increase the frequency of deletions between short repeats (several base pairs), between homologous DNA sequences separated by long inverted repeats, and between distant short repeats, increasing the frequency of genomic deletions. We found that the pol3-t mutation increased intrachromosomal recombination events between direct DNA repeats up to 36-fold and interchromosomal recombination 14-fold. The hyperrecombination phenotype of pol3-t was partially dependent on the Rad52p function but much more so on Rad1p. However, in the double-mutant rad1 Delta rad52 Delta, the pol3-t mutation still increased spontaneous intrachromosomal recombination frequencies, suggesting that a Rad1p Rad52p-independent single-strand annealing pathway is involved. UV and gamma-rays were less potent inducers of recombination in the pol3-t mutant, indicating that Pol3p is partly involved in DNA-damage-induced recombination. In contrast, while UV- and gamma-ray-induced intrachromosomal recombination was almost completely abolished in the rad52 or the rad1 rad52 mutant, there was still good induction in those mutants in the pol3-t background, indicating channeling of lesions into the above-mentioned Rad1p Rad52p-independent pathway. Finally, a heterozygous pol3-t/POL3 mutant also showed an increased frequency of deletions and MMS sensitivity at the restrictive temperature, indicating that even a heterozygous polymerase delta mutation might increase the frequency of genetic instability.     

Intrinsic 5'-deoxyribose-5-phosphate lyase activity in Saccharomyces cerevisiae Trf4 protein with a possible role in base excision DNA repair

In Saccharomyces cerevisiae, the base excision DNA repair (BER) pathway has been thought to involve only a multinucleotide (long-patch) mechanism (LP-BER), in contrast to most known cases that include a major single-nucleotide pathway (SN-BER). The key step in mammalian SN-BER, removal of the 5'-terminal abasic residue generated by AP endonuclease incision, is effected by DNA polymerase beta (Polbeta). Computational analysis indicates that yeast Trf4 protein, with roles in sister chromatin cohesion and RNA quality control, is a new member of the X family of DNA polymerases that includes Polbeta. Previous studies of yeast trf4Delta mutants revealed hypersensitivity to methylmethane sulfonate (MMS) but not UV light, a characteristic of BER mutants in other organisms. We found that, like mammalian Polbeta, Trf4 is able to form a Schiff base intermediate with a 5'-deoxyribose-5-phosphate substrate and to excise the abasic residue through a dRP lyase activity. Also like Polbeta, Trf4 forms stable cross-links in vitro to 5'-incised 2-deoxyribonolactone residues in DNA. We determined the sensitivity to MMS of strains with a trf4Delta mutation in a rad27Delta background, in an AP lyase-deficient background (ogg1 ntg1 ntg2), or in a pol4Delta background. Only a RAD27 genetic interaction was detected: there was higher sensitivity for strains mutated in both TRF4 and RAD27 than either single mutant, and overexpression of Trf4 in a rad27Delta background partially suppressed MMS sensitivity. The data strongly suggest a role for Trf4 in a pathway parallel to the Rad27-dependent LP-BER in yeast. Finally, we demonstrate that Trf5 significantly affects MMS sensitivity and thus probably BER efficiency in cells expressing either wild-type Trf4 or a C-terminus-deleted form.     

Yeast Rad52 and Rad51 recombination proteins define a second pathway of DNA damage assessment in response to a single double-strand break

Saccharomyces cells with a single unrepaired double-strand break adapt after checkpoint-mediated G(2)/M arrest. We have found that both Rad51 and Rad52 recombination proteins play key roles in adaptation. Cells lacking Rad51p fail to adapt, but deleting RAD52 suppresses rad51Delta. rad52Delta also suppresses adaptation defects of srs2Delta mutants but not those of yku70Delta or tid1Delta mutants. Neither rad54Delta nor rad55Delta affects adaptation. A Rad51 mutant that fails to interact with Rad52p is adaptation defective; conversely, a C-terminal truncation mutant of Rad52p, impaired in interaction with Rad51p, is also adaptation defective. In contrast, rad51-K191A, a mutation that abolishes recombination and results in a protein that does not bind to single-stranded DNA (ssDNA), supports adaptation, as do Rad51 mutants impaired in interaction with Rad54p or Rad55p. An rfa1-t11 mutation in the ssDNA binding complex RPA partially restores adaptation in rad51Delta mutants and fully restores adaptation in yku70Delta and tid1Delta mutants. Surprisingly, although neither rfa1-t11 nor rad52Delta mutants are adaptation defective, the rad52Delta rfa1-t11 double mutant fails to adapt and exhibits the persistent hyperphosphorylation of the DNA damage checkpoint protein Rad53 after HO induction. We suggest that monitoring of the extent of DNA damage depends on independent binding of RPA and Rad52p to ssDNA, with Rad52p's activity modulated by Rad51p whereas RPA's action depends on Tid1p.     

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.     

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.     

Competition between the Rad50 complex and the Ku heterodimer reveals a role for Exo1 in processing double-strand breaks but not telomeres

The Mre11-Rad50-Nbs1(Xrs2) complex and the Ku70-Ku80 heterodimer are thought to compete with each other for binding to DNA ends. To investigate the mechanism underlying this competition, we analyzed both DNA damage sensitivity and telomere overhangs in Schizosaccharomyces pombe rad50-d, rad50-d pku70-d, rad50-d exo1-d, and pku70-d rad50-d exo1-d cells. We found that rad50 exo1 double mutants are more methyl methanesulfonate (MMS) sensitive than the respective single mutants. The MMS sensitivity of rad50-d cells was suppressed by concomitant deletion of pku70+. However, the MMS sensitivity of the rad50 exo1 double mutant was not suppressed by the deletion of pku70+. The G-rich overhang at telomere ends in taz1-d cells disappeared upon deletion of rad50+, but the overhang reappeared following concomitant deletion of pku70+. Our data suggest that the Rad50 complex can process DSB ends and telomere ends in the presence of the Ku heterodimer. However, the Ku heterodimer inhibits processing of DSB ends and telomere ends by alternative nucleases in the absence of the Rad50-Rad32 protein complex. While we have identified Exo1 as the alternative nuclease targeting DNA break sites, the identity of the nuclease acting on the telomere ends remains elusive.     

Role of budding yeast Rad18 in repair of HO-induced double-strand breaks

The Rad6-Rad18 complex mono-ubiquitinates proliferating cell nuclear antigen (PCNA) at the lysine 164 residue after DNA damage and promotes DNA polymerase eta (Poleta)- and Polzeta/Rev1-dependent DNA synthesis. Double-strand breaks (DSBs) of DNA can be repaired by homologous recombination (HR) or non-homologous end-joining (NHEJ), both of which require new DNA synthesis. HO endonuclease introduces DSBs into specific DNA sequences. We have shown that Polzeta and Rev1 localize to HO-induced DSBs in a Mec1-dependent manner and promote Ku-dependent DSB repair. However, Polzeta and Rev1 localize to DSBs independently of PCNA ubiquitination. Here we provide evidence indicating that Rad18-mediated PCNA ubiquitination stimulates DNA synthesis by Polzeta and Rev1 in repair of HO-induced DSBs. Ubiquitination defective PCNA mutation or rad18Delta mutation confers the same DSB repair defect as rev1Delta mutation. Consistent with a role in DSB repair, Rad18 localizes to HO-induced DSBs in a Rad6-dependent manner. Unlike Polzeta or Rev1, Poleta is dispensable for repair of HO-induced DSBs. Ku and DNA ligase IV constitute a central NHEJ pathway. We also show that Polzeta and Rev1 act in the same pathway as DNA ligase IV, suggesting that Polzeta and Rev1 are involved in DNA synthesis during NHEJ. Our results suggest that Polzeta-Rev1 accumulates at regions near DSBs independently of PCNA ubiquitination and then interacts with ubiquitinated PCNA to facilitate DNA synthesis.     

Interaction with Rad51 is indispensable for recombination mediator function of Rad52

In the yeast Saccharomyces cerevisiae, the RAD52 gene is indispensable for homologous recombination and DNA repair. Rad52 protein binds DNA, anneals complementary ssDNA strands, and self-associates to form multimeric complexes. Moreover, Rad52 physically interacts with the Rad51 recombinase and serves as a mediator in the Rad51-catalyzed DNA strand exchange reaction. Here, we examine the functional significance of the Rad51/Rad52 interaction. Through a series of deletions, we have identified residues 409-420 of Rad52 as being indispensable and likely sufficient for its interaction with Rad51. We have constructed a four-amino acid deletion mutation within this region of Rad52 to ablate its interaction with Rad51. We show that the rad52delta409-412 mutant protein is defective in the mediator function in vitro even though none of the other Rad52 activities, namely, DNA binding, ssDNA annealing, and protein oligomerization, are affected. We also show that the sensitivity of the rad52delta409-412 mutant to ionizing radiation can be complemented by overexpression of Rad51. These results thus demonstrate the significance of the Rad51-Rad52 interaction in homologous recombination.     

Saccharomyces cerevisiae RAD27 complements its Escherichia coli homolog in damage repair but not mutation avoidance

In eukaryotes, the flap endonuclease of Rad27/Fen-1 is thought to play a critical role in lagging-strand DNA replication by removing ribonucleotides present at the 5' ends of Okazaki fragments, and in base excision repair by cleaving a 5' flap structure that may result during base excision repair. Saccharomyces cerevisiae rad27Delta mutants further display a repeat tract instability phenotype and a high rate of forward mutations to canavanine resistance that result from duplications of DNA sequence, indicating a role in mutation avoidance. Two conserved motifs in Rad27/Fen-1 show homology to the 5' --> 3' exonuclease domain of Escherichia coli DNA polymerase I. The strain defective in the 5' --> 3' exonuclease domain in DNA polymerase I shows essentially the same phenotype as the yeast rad27Delta strain. In this study, we expressed the yeast RAD27 gene in an E. coli strain lacking the 5' --> 3' exonuclease domain in DNA polymerase I in order to test whether eukaryotic RAD27/FEN-1 can complement the defect of its bacterial homolog. We found that the yeast Rad27 protein complements sensitivity to methyl methanesulfonate in an E. coli mutant. On the other hand, Rad27 protein did not reduce the high rate of spontaneous mutagenesis in the E. coli tonB gene which results from duplication of DNA. These results indicate that the yeast Rad27 and E. coli 5' --> 3' exonuclease act on the same substrate. We argue that the lack of mutation avoidance of yeast RAD27 in E. coli results from a lack of interaction between the yeast Rad27 protein and the E. coli replication clamp (beta-clamp).     

The roles of PCNA SUMOylation, Mms2-Ubc13 and Rad5 in translesion DNA synthesis in Saccharomyces cerevisiae

Mms2, in concert with Ubc13 and Rad5, is responsible for polyubiquitination of replication processivity factor PCNA. This modification activates recombination-like DNA damage-avoidance mechanisms, which function in an error-free manner. Cells deprived of Mms2, Ubc13 or Rad5 exhibit mutator phenotypes as a result of the channelling of premutational DNA lesions to often error-prone translesion DNA synthesis (TLS). Here we show that Siz1-mediated PCNA SUMOylation is required for the stimulation of this TLS, despite the presence of PCNA monoubiquitination. The stimulation of spontaneous mutagenesis by Siz1 in cells carrying rad5 and/or mms2 mutations is connected with the known role of PCNA SUMOylation in the inhibition of Rad52-mediated recombination. However, following UV irradiation, Siz1 is engaged in additional, as yet undefined, mechanisms controlling genetic stability at the replication fork. We also demonstrate that in the absence of PCNA SUMOylation, Mms2-Ubc13 and Rad5 may, independently of each other, function in the stimulation of TLS. Based on this finding and on an analysis of the epistatic relationships between SIZ1, MMS2 and RAD5, with respect to UV sensitivity, we conclude that PCNA SUMOylation is responsible for the functional differences between the Mms2 and Rad5 homologues of Saccharomyces cerevisiae and Schizosaccharomyces pombe.     

Analysis of the Tolerance to DNA Alkylating Damage in MEC1 and RAD53 Checkpoint Mutants of Saccharomyces cerevisiae

Checkpoint response, tolerance and repair are three major pathways that eukaryotic cells evolved independently to maintain genome stability and integrity. Here, we studied the sensitivity to DNA damage in checkpoint-deficient budding yeast cells and found that checkpoint kinases Mec1 and Rad53 may modulate the balance between error-free and error-prone branches of the tolerance pathway. We have consistently observed that mutation of the RAD53 counterbalances error-free and error-prone branches upon exposure of cells to DNA damage induced either by MMS alkylation or by UV-radiation. We have also found that the potential Mec1/Rad53 balance modulation is independent from Rad6/Rad18-mediated PCNA ubiquitylation, as mec1Δ or rad53Δ mutants show no defects in the modification of the sliding clamp, therefore, we infer that it is likely exerted by acting on TLS polymerases and/or template switching targets.     

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.     

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.     

Genetic and functional interactions between Mus81–Mms4 and Rad27

The two endonucleases, Rad27 (yeast Fen1) and Dna2, jointly participate in the processing of Okazaki fragments in yeasts. Mus81–Mms4 is a structure-specific endonuclease that can resolve stalled replication forks as well as toxic recombination intermediates. In this study, we show that Mus81–Mms4 can suppress dna2 mutational defects by virtue of its functional and physical interaction with Rad27. Mus81–Mms4 stimulated Rad27 activity significantly, accounting for its ability to restore the growth defects caused by the dna2 mutation. Interestingly, Rad27 stimulated the rate of Mus81–Mms4 catalyzed cleavage of various substrates, including regressed replication fork substrates. The ability of Rad27 to stimulate Mus81–Mms4 did not depend on the catalytic activity of Rad27, but required the C-terminal 64 amino acid fragment of Rad27. This indicates that the stimulation was mediated by a specific protein–protein interaction between the two proteins. Our in vitro data indicate that Mus81–Mms4 and Rad27 act together during DNA replication and resolve various structures that can impede normal DNA replication. This conclusion was further strengthened by the fact that rad27 mus81 or rad27 mms4 double mutants were synergistically lethal. We discuss the significance of the interactions between Rad27, Dna2 and Mus81–Mms4 in context of DNA replication.