Characterization of Insertion Mutations in the Saccharomyces Cerevisiae Msh1 and Msh2 Genes: Evidence for Separate Mitochondrial and Nuclear Functions

The MSH1 and MSH2 genes of Saccharomyces cerevisiae are predicted to encode proteins that are homologous to the Escherichia coli MutS and Streptococcus pneumoniae HexA proteins and their homologs. Disruption of the MSH1 gene caused a petite phenotype which was established rapidly. A functional MSH1 gene present on a single-copy centromere plasmid was incapable of rescuing the established msh1 petite phenotype. Analysis of msh1 strains demonstrated that mutagenesis and large-scale rearrangement of mitochondrial DNA had occurred. 4',6-Diamidino-2-phenylindole (DAPI) staining of msh1 yeast revealed an aberrant distribution of mtDNA. Haploid msh2 mutants displayed an increase of 85-fold in the rate of spontaneous mutation to canavanine resistance. Sporulation of homozygous msh2/msh2 diploids gave rise to a high level of lethality which was compounded during increased vegetative growth prior to sporulation. msh2 mutations also affected gene conversion of two HIS4 alleles. The his4x mutation, lying near the 5' end of the gene, was converted with equal frequency in both wild-type and msh2 strains. However, many of the events in the msh2 background were post-meiotic segregation (PMS) events (46.4%) while none (< 0.25%) of the aberrant segregations in wild type were PMS events. The his4b allele, lying 1.6 kb downstream of his4x, was converted at a 10-fold higher frequency in the msh2 background than in the corresponding wild-type strain. Like the his4x allele, his4b showed a high level of PMS (30%) in the msh2 background compared to the corresponding wild-type strain where no (< 0.26%) PMS events were observed. These results indicate that MSH1 plays a role in repair or stability of mtDNA and MSH2 plays a role in repair of 4-bp insertion/deletion mispairs in the nucleus.     

Msh1p counteracts oxidative lesion-induced instability of mtDNA and stimulates mitochondrial recombination in Saccharomyces cerevisiae

The proximity of the mitochondrial genome to the respiratory chain, a major source of ROS (radical oxygen species), makes mtDNA more vulnerable to oxidative damage than nuclear DNA. Mitochondrial BER (base excision repair) is generally considered to be the main pathway involved in the prevention of oxidative lesion-induced mutations in mtDNA. However, we previously demonstrated that the increased frequency of mitochondrial Oli^r mutants in an ogg1Δ strain, lacking the activity of a crucial mtBER glycosylase, is reduced in the presence of plasmids encoding Msh1p, the mitochondrial homologue of the bacterial mismatch protein MutS. This finding suggested that Msh1p might be involved in the prevention of mitochondrial mutagenesis induced by oxidative stress. Here we show that a double mutant carrying the msh1-R813W allele, encoding a variant of the protein defective in the ATP hydrolysis activity, combined with deletion of SOD2, encoding the mitochondrial superoxide dismutase, displays a synergistic effect on the frequency of Oli^r mutants, indicating that Msh1p prevents generation of oxidative lesion-induced mitochondrial mutations. We also show that double mutants carrying the msh1-R813W allele, combined with deletion of either OGG1 or APN1, the latter resulting in deficiency of the Apn1 endonuclease, exhibit a synergistic effect on the frequency of respiration-defective mutants having gross rearrangements of the mitochondrial genome. This suggests that Msh1p, Ogg1p and Apn1p play overlapping functions in maintaining the stability of mtDNA. In addition, we demonstrate, using a novel ARG8^m recombination assay, that a surplus of Msh1p results in enhanced mitochondrial recombination. Interestingly, the mutant forms of the protein, msh1p-R813W and msh1p-G776D, fail to stimulate recombination. We postulate that the Msh1p-enhanced homologous recombination may play an important role in the prevention of oxidative lesion-induced rearrangements of the mitochondrial genome.     

A dominant mitochondrial mutator phenotype of Saccharomyces cerevisiae conferred by msh1 alleles altered in the sequence encoding the ATP-binding domain

In order to improve our understanding of the role of the yeast MSH1 gene in error avoidance in mitochondrial DNA, two msh1 alleles were constructed, which encode proteins with amino acid substitutions in an ATP-binding domain that is highly conserved among MutS homologs. Here, we report that moderate overexpression of the msh1-R813W or msh1-G776D allele, in strains which also carry the wild-type MSH1 allele, slightly increases the frequency of mutations conferring resistance to erythromycin (E(r)) and elevates the frequency of alterations within a polyGT tract present in mitochondrial DNA (mtDNA). This result indicates that the mutant alleles confer a dominant mitochondrial mutator phenotype and strongly suggests that the ATP-binding domain plays a crucial role in the in vivo function of Msh1p. Interestingly, we have found that overexpression of wild-type MSH1 has opposite effects on the stability of polyGT vs. polyAT tracts present in mtDNA; excess of Msh1p slightly increases the stability of polyGT tracts, whereas the stability of polyAT tracts is dramatically decreased. We show that although overexpression of msh1-R813W or msh1-G776D also results in a marked overall increase in the frequency of alterations in polyAT tracts, the spectrum of alterations differs from that found in cells overexpressing MSH1; large deletions predominate in the latter case, while 2-bp deletions are generated in cells that overproduce the mutant msh1p. This result strongly suggests that the mutations in the ATP binding domain change the specificity of the protein with respect to the recognition of potentially mutagenic structures in mtDNA.     

Oxidative DNA damage causes mitochondrial genomic instability in Saccharomyces cerevisiae

Mitochondria contain their own genome, the integrity of which is required for normal cellular energy metabolism. Reactive oxygen species (ROS) produced by normal mitochondrial respiration can damage cellular macromolecules, including mitochondrial DNA (mtDNA), and have been implicated in degenerative diseases, cancer, and aging. We developed strategies to elevate mitochondrial oxidative stress by exposure to antimycin and H(2)O(2) or utilizing mutants lacking mitochondrial superoxide dismutase (sod2Delta). Experiments were conducted with strains compromised in mitochondrial base excision repair (ntg1Delta) and oxidative damage resistance (pif1Delta) in order to delineate the relationship between these pathways. We observed enhanced ROS production, resulting in a direct increase in oxidative mtDNA damage and mutagenesis. Repair-deficient mutants exposed to oxidative stress conditions exhibited profound genomic instability. Elimination of Ntg1p and Pif1p resulted in a synergistic corruption of respiratory competency upon exposure to antimycin and H(2)O(2). Mitochondrial genomic integrity was substantially compromised in ntg1Delta pif1Delta sod2Delta strains, since these cells exhibit a total loss of mtDNA. A stable respiration-defective strain, possessing a normal complement of mtDNA damage resistance pathways, exhibited a complete loss of mtDNA upon exposure to antimycin and H(2)O(2). This loss was preventable by Sod2p overexpression. These results provide direct evidence that oxidative mtDNA damage can be a major contributor to mitochondrial genomic instability and demonstrate cooperation of Ntg1p and Pif1p to resist the introduction of lesions into the mitochondrial genome.     

Antimutator alleles of yeast DNA polymerase gamma modulate the balance between DNA synthesis and excision

Mutations in mitochondrial DNA (mtDNA) are an important cause of disease and perhaps aging in human. DNA polymerase gamma (pol γ), the unique replicase inside mitochondria, plays a key role in the fidelity of mtDNA replication through selection of the correct nucleotide and 3'-5' exonuclease proofreading. For the first time, we have isolated and characterized antimutator alleles in the yeast pol γ (Mip1). These mip1 mutations, localised in the 3'-5' exonuclease and polymerase domains, elicit a 2-15 fold decrease in the frequency of mtDNA point mutations in an msh1-1 strain which is partially deficient in mtDNA mismatch-repair. In vitro experiments show that in all mutants the balance between DNA synthesis and exonucleolysis is shifted towards excision when compared to wild-type, suggesting that in vivo more opportunity is given to the editing function for removing the replicative errors. This results in partial compensation for the mismatch-repair defects and a decrease in mtDNA point mutation rate. However, in all mutants but one the antimutator trait is lost in the wild-type MSH1 background. Accordingly, the polymerases of selected mutants show reduced oligonucleotide primed M13 ssDNA synthesis and to a lesser extent DNA binding affinity, suggesting that in mismatch-repair proficient cells efficient DNA synthesis is required to reach optimal accuracy. In contrast, the Mip1-A256T polymerase, which displays wild-type like DNA synthesis activity, increases mtDNA replication fidelity in both MSH1 and msh1-1 backgrounds. Altogether, our data show that accuracy of wild-type Mip1 is probably not optimal and can be improved by specific (often conservative) amino acid substitutions that define a pol γ area including a loop of the palm subdomain, two residues near the ExoII motif and an exonuclease helix-coil-helix module in close vicinity to the polymerase domain. These elements modulate in a subtle manner the balance between DNA polymerization and excision.     

Increases in mitochondrial DNA content and 4977-bp deletion upon ATM/Chk2 checkpoint activation in HeLa cells

Activation of the Mec1/Rad53 damage checkpoint pathway influences mitochondrial DNA (mtDNA) content and point mutagenesis in Saccharomyces cerevisiae. The effects of this conserved checkpoint pathway on mitochondrial genomes in human cells remain largely unknown. Here, we report that knockdown of the human DNA helicase RRM3 enhances phosphorylation of the cell cycle arrest kinase Chk2, indicating activation of the checkpoint via the ATM/Chk2 pathway, and increases mtDNA content independently of TFAM, a regulator of mtDNA copy number. Cell-cycle arrest did not have a consistent effect on mtDNA level: knockdown of cell cycle regulators PLK1 (polo-like kinase), MCM2, or MCM3 gave rise, respectively, to decreased, increased, or almost unchanged mtDNA levels. Therefore, we concluded that the mtDNA content increase upon RRM3 knockdown is not a response to delay of cell cycle progression. Also, we observed that RRM3 knockdown increased the levels of reactive oxygen species (ROS); two ROS scavengers, N-acetyl cysteine and vitamin C, suppressed the mtDNA content increase. On the other hand, in RRM3 knockdown cells, we detected an increase in the frequency of the common 4977-bp mtDNA deletion, a major mtDNA deletion that can be induced by abnormal ROS generation, and is associated with a decline in mitochondrial genome integrity, aging, and various mtDNA-related disorders in humans. These results suggest that increase of the mitochondrial genome by TFAM-independent mtDNA replication is connected, via oxidative stress, with the ATM/Chk2 checkpoint activation in response to DNA damage, and is accompanied by generation of the common 4977-bp deletion.     

Different DNA repair strategies to combat the threat from 8-oxoguanine

Oxidative DNA damage is one of the most common threats to genome stability and DNA repair enzymes provide protection from the effects of oxidized DNA bases. In mammalian cells, base excision repair (BER) mediated by the OGG1 and MYH DNA glycosylases prevents the accumulation of 8-oxoguanine (8-oxoG) in DNA. When steady-state levels of DNA 8-oxoG were measured in myh(-/-) and myh(-/-)/ogg1(-/-) mice, an age-dependent accumulation of the oxidized purine was found in lung and small intestine of doubly defective myh(-/-)/ogg1(-/-) mice. Since there is an increased incidence of lung and small intestinal cancer in myh(-/-)/ogg1(-/-) mice, these findings are consistent with a causal role for unrepaired oxidized DNA bases in cancer development. We previously presented in vitro evidence that mismatch repair (MMR) participates in the repair of oxidative DNA damage and msh2(-/-) mouse embryo fibroblasts also have increased steady state levels of DNA 8-oxoG. To investigate whether DNA 8-oxoG also accumulates in vivo, basal levels were measured in several organs of 4-month-old msh2(-/-) mice and their wild-type counterparts. Msh2(-/-) mice had significantly increased levels of DNA 8-oxoG in spleen, heart, liver, lung, kidney and possibly small intestine but not in bone marrow, thymus or brain. The tissue-specificity of DNA 8-oxoG accumulation in msh2(-/-) and other DNA repair defective mice suggests that DNA protection of different organs is mediated by different combinations of repair pathways.     

Interaction between Mismatch Repair and Genetic Recombination in Saccharomyces Cerevisiae

The yeast Saccharomyces cerevisiae encodes a set of genes that show strong amino acid sequence similarity to MutS and MutL, proteins required for mismatch repair in Escherichia coli. We examined the role of MSH2 and PMS1, yeast homologs of mutS and mutL, respectively, in the repair of base pair mismatches formed during meiotic recombination. By using specifically marked HIS4 and ARG4 alleles, we showed that msh2 mutants displayed a severe defect in the repair of all base pair mismatches as well as 1-, 2- and 4-bp insertion/deletion mispairs. The msh2 and pms1 phenotypes were indistinguishable, suggesting that the wild-type gene products act in the same repair pathway. A comparison of gene conversion events in wild-type and msh2 mutants indicated that mismatch repair plays an important role in genetic recombination. (1) Tetrad analysis at five different loci revealed that, in msh2 mutants, the majority of aberrant segregants displayed a sectored phenotype, consistent with a failure to repair mismatches created during heteroduplex formation. In wild type, base pair mismatches were almost exclusively repaired toward conversion rather than restoration. (2) In msh2 strains 10-19% of the aberrant tetrads were Ab4:4. (3) Polarity gradients at HIS4 and ARG4 were nearly abolished in msh2 mutants. The frequency of gene conversion at the 3' end of these genes was increased and was nearly the frequency observed at the 5' end. (4) Co-conversion studies were consistent with mismatch repair acting to regulate heteroduplex DNA tract length. We favor a model proposing that recombination events occur through the formation and resolution of heteroduplex intermediates and that mismatch repair proteins specifically interact with recombination enzymes to regulate the length of symmetric heteroduplex DNA.     

Mitochondrial Changes in Ageing Caenorhabditis elegans – What Do We Learn from Superoxide Dismutase Knockouts?

One of the most popular damage accumulation theories of ageing is the mitochondrial free radical theory of ageing (mFRTA). The mFRTA proposes that ageing is due to the accumulation of unrepaired oxidative damage, in particular damage to mitochondrial DNA (mtDNA). Within the mFRTA, the "vicious cycle" theory further proposes that reactive oxygen species (ROS) promote mtDNA mutations, which then lead to a further increase in ROS production. Recently, data have been published on Caenorhabditis elegans mutants deficient in one or both forms of mitochondrial superoxide dismutase (SOD). Surprisingly, even double mutants, lacking both mitochondrial forms of SOD, show no reduction in lifespan. This has been interpreted as evidence against the mFRTA because it is assumed that these mutants suffer from significantly elevated oxidative damage to their mitochondria. Here, using a novel mtDNA damage assay in conjunction with related, well established damage and metabolic markers, we first investigate the age-dependent mitochondrial decline in a cohort of ageing wild-type nematodes, in particular testing the plausibility of the "vicious cycle" theory. We then apply the methods and insights gained from this investigation to a mutant strain for C. elegans that lacks both forms of mitochondrial SOD. While we show a clear age-dependent, linear increase in oxidative damage in WT nematodes, we find no evidence for autocatalytic damage amplification as proposed by the "vicious cycle" theory. Comparing the SOD mutants with wild-type animals, we further show that oxidative damage levels in the mtDNA of SOD mutants are not significantly different from those in wild-type animals, i.e. even the total loss of mitochondrial SOD did not significantly increase oxidative damage to mtDNA. Possible reasons for this unexpected result and some implications for the mFRTA are discussed.     

Mitochondrial DNA damage Is associated with reduced mitochondrial bioenergetics in Huntington's disease

Oxidative stress and mitochondrial dysfunction have been implicated in the pathology of HD; however, the precise mechanisms by which mutant huntingtin modulates levels of oxidative damage in turn resulting in mitochondrial dysfunction are not known. We hypothesize that mutant huntingtin increases oxidative mtDNA damage leading to mitochondrial dysfunction. We measured nuclear and mitochondrial DNA lesions and mitochondrial bioenergetics in the STHdhQ7 and STHdhQ111 in vitro striatal model of HD. Striatal cells expressing mutant huntingtin show higher basal levels of mitochondrial-generated ROS and mtDNA lesions and a lower spare respiratory capacity. Silencing of APE1, the major mammalian apurinic/apyrimidinic (AP) endonuclease that participates in the base excision repair (BER) pathway, caused further reductions of spare respiratory capacity in the mutant huntingtin-expressing cells. Localization experiments show that APE1 increases in the mitochondria of wild-type Q7 cells but not in the mutant huntingtin Q111 cells after treatment with hydrogen peroxide. Moreover, these results are recapitulated in human HD striata and HD skin fibroblasts that show significant mtDNA damage (increased lesion frequency and mtDNA depletion) and significant decreases in spare respiratory capacity, respectively. These data suggest that mtDNA is a major target of mutant huntingtin-associated oxidative stress and may contribute to subsequent mitochondrial dysfunction and that APE1 (and, by extension, BER) is an important target in the maintenance of mitochondrial function in HD.     

Mitochondria-targeted Ogg1 and Aconitase-2 Prevent Oxidant-induced Mitochondrial DNA Damage in Alveolar Epithelial Cells

Mitochondria-targeted human 8-oxoguanine DNA glycosylase (mt-hOgg1) and aconitase-2 (Aco-2) each reduce oxidant-induced alveolar epithelial cell (AEC) apoptosis, but it is unclear whether protection occurs by preventing AEC mitochondrial DNA (mtDNA) damage. Using quantitative PCR-based measurements of mitochondrial and nuclear DNA damage, mtDNA damage was preferentially noted in AEC after exposure to oxidative stress (e.g. amosite asbestos (5–25 μg/cm²) or H₂O₂ (100–250 μm)) for 24 h. Overexpression of wild-type mt-hOgg1 or mt-long α/β 317–323 hOgg1 mutant incapable of DNA repair (mt-hOgg1-Mut) each blocked A549 cell oxidant-induced mtDNA damage, mitochondrial p53 translocation, and intrinsic apoptosis as assessed by DNA fragmentation and cleaved caspase-9. In contrast, compared with controls, knockdown of Ogg1 (using Ogg1 shRNA in A549 cells or primary alveolar type 2 cells from ogg1^−/− mice) augmented mtDNA lesions and intrinsic apoptosis at base line, and these effects were increased further after exposure to oxidative stress. Notably, overexpression of Aco-2 reduced oxidant-induced mtDNA lesions, mitochondrial p53 translocation, and apoptosis, whereas siRNA for Aco-2 (siAco-2) enhanced mtDNA damage, mitochondrial p53 translocation, and apoptosis. Finally, siAco-2 attenuated the protective effects of mt-hOgg1-Mut but not wild-type mt-hOgg1 against oxidant-induced mtDNA damage and apoptosis. Collectively, these data demonstrate a novel role for mt-hOgg1 and Aco-2 in preserving AEC mtDNA integrity, thereby preventing oxidant-induced mitochondrial dysfunction, p53 mitochondrial translocation, and intrinsic apoptosis. Furthermore, mt-hOgg1 chaperoning of Aco-2 in preventing oxidant-mediated mtDNA damage and apoptosis may afford an innovative target for the molecular events underlying oxidant-induced toxicity.     

Mitochondrial dysfunction due to oxidative mitochondrial DNA damage is reduced through cooperative actions of diverse proteins

The mitochondrial genome is a significant target of exogenous and endogenous genotoxic agents; however, the determinants that govern this susceptibility and the pathways available to resist mitochondrial DNA (mtDNA) damage are not well characterized. Here we report that oxidative mtDNA damage is elevated in strains lacking Ntg1p, providing the first direct functional evidence that this mitochondrion-localized, base excision repair enzyme functions to protect mtDNA. However, ntg1 null strains did not exhibit a mitochondrial respiration-deficient (petite) phenotype, suggesting that mtDNA damage is negotiated by the cooperative actions of multiple damage resistance pathways. Null mutations in ABF2 or PIF1, two genes implicated in mtDNA maintenance and recombination, exhibit a synthetic-petite phenotype in combination with ntg1 null mutations that is accompanied by enhanced mtDNA point mutagenesis in the corresponding double-mutant strains. This phenotype was partially rescued by malonic acid, indicating that reactive oxygen species generated by the electron transport chain contribute to mitochondrial dysfunction in abf2 Delta strains. In contrast, when two other genes involved in mtDNA recombination, CCE1 and NUC1, were inactivated a strong synthetic-petite phenotype was not observed, suggesting that the effects mediated by Abf2p and Pif1p are due to novel activities of these proteins other than recombination. These results document the existence of recombination-independent mechanisms in addition to base excision repair to cope with oxidative mtDNA damage in Saccharomyces cerevisiae. Such systems are likely relevant to those operating in human cells where mtDNA recombination is less prevalent, validating yeast as a model system in which to study these important issues.     

The basal levels of 8-oxoG and other oxidative modifications in intact mitochondrial DNA are low even in repair-deficient (Ogg1(-/-)/Csb(-/-)) mice

Mitochondrial DNA (mtDNA) is assumed to be highly prone to damage by reactive oxygen species (ROS) because of its location in close proximity to the mitochondrial electron transport chain. Accordingly, mitochondrial oxidative DNA damage has been hypothesized to be responsible for various neurological diseases, ageing and cancer. Since 7,8-dihydro-8-oxoguanine (8-oxoG), one of the most frequent oxidative base modifications, is removed from the mitochondrial genome by the glycosylase OGG1, the basal levels of this lesion are expected to be highly elevated in Ogg1^−/− mice. To investigate this hypothesis, we have used a mtDNA relaxation assay in combination with various repair enzymes (Fpg, MutY, endonuclease III, endonuclease IV) to determine the average steady-state number of oxidative DNA modifications within intact (supercoiled) mtDNA from the livers of wild-type mice and those deficient in OGG1 and/or the Cockayne syndrome B (CSB) protein for mice aged up to 23 months. The levels of all types of oxidative modifications were found to be less than 12 per million base pairs, and the difference between wild-type and repair-deficient (Ogg1^−/−/Csb^−/−) mice was not significant. Thus, the increase of 8-oxoG caused by the repair deficiency in intact mtDNA is not much higher than in the nuclear DNA, i.e., not more than a few modifications per million base pairs. Based on these data, it is hypothesized that the load of oxidative base modifications in mtDNA is efficiently reduced during replication even in the absence of excision repair.     

Lack of the DNA glycosylases MYH and OGG1 in the cancer prone double mutant mouse does not increase mitochondrial DNA mutagenesis

Reactive oxygen species (ROS) are formed as natural byproducts during aerobic metabolism and readily induce premutagenic base lesions in the DNA. The 8-oxoguanine DNA glycosylase (OGG1) and MutY homolog 1 (MYH) synergistically prevent mutagenesis and cancer formation in mice. Their localization in the mitochondria as well as in the nucleus suggests that mutations in mitochondrial DNA (mtDNA) contribute to the carcinogenesis in the myh^?/?/ogg1^?/? double knockout mouse.In order to test this hypothesis, we analyzed mtDNA mutagenesis and mitochondrial function in young (1 month) and adult (6 months) wt and myh^?/?/ogg1^?/? mice. To our surprise, the absence of OGG1 and MYH had no impact on mtDNA mutation rates in these mice, even at the onset of cancer. This indicates that mtDNA mutagenesis is not responsible for the carcinogenesis of myh^?/?/ogg1^?/? mice. In line with these results, mitochondrial function was unaffected in the cancerous tissues liver and lung, whereas a significant reduction in respiration capacity was observed in brain mitochondria from the adult myh^?/?/ogg1^?/? mouse. The reduced respiration capacity correlated with a specific reduction (?25%) in complex I biochemical activity in brain mitochondria.Our results demonstrate that mtDNA mutations are not associated with cancer development in myh^?/?/ogg1^?/? mice, and that impairment of mitochondrial function in brain could be linked to nuclear DNA mutations in this strain. OGG1 and MYH appear to be dispensable for antimutator function in mitochondria.     

Evidence for a role of FEN1 in maintaining mitochondrial DNA integrity

Although the nuclear processes responsible for genomic DNA replication and repair are well characterized, the pathways involved in mitochondrial DNA (mtDNA) replication and repair remain unclear. DNA repair has been identified as being particularly important within the mitochondrial compartment due to the organelle's high propensity to accumulate oxidative DNA damage. It has been postulated that continual accumulation of mtDNA damage and subsequent mutagenesis may function in cellular aging. Mitochondrial base excision repair (mtBER) plays a major role in combating mtDNA oxidative damage; however, the proteins involved in mtBER have yet to be fully characterized. It has been established that during nuclear long-patch (LP) BER, FEN1 is responsible for cleavage of 5′ flap structures generated during DNA synthesis. Furthermore, removal of 5′ flaps has been observed in mitochondrial extracts of mammalian cell lines; yet, the mitochondrial localization of FEN1 has not been clearly demonstrated. In this study, we analyzed the effects of deleting the yeast FEN1 homolog, RAD27, on mtDNA stability in Saccharomyces cerevisiae. Our findings demonstrate that Rad27p/FEN1 is localized in the mitochondrial compartment of both yeast and mice and that Rad27p has a significant role in maintaining mtDNA integrity.     

Analysis of in vivo correction of defined mismatches in the DNA mismatch repair mutants msh2, msh3 and msh6 of Saccharomyces cerevisiae

We have analysed the correction of defined mismatches in wild-type and msh2, msh3, msh6 and msh3 msh6 mutants of Saccharomyces cerevisiae in two different yeast strain backgrounds by transformation with plasmid heteroduplex DNA constructs. Ten different base/base mismatches, two single-nucleotide loops and a 38-nucleotide loop were tested. Repair of all types of mismatches was severely impaired in msh2 and msh3 msh6 mutants. In msh6 mutants, repair efficiency of most base/base mismatches was reduced to a similar extent as in msh3 msh6 double mutants. G/T and A/C mismatches, however, displayed residual repair in msh6 mutants in one strain background, implying a role for Msh3p in recognition of base/base mismatches. Furthermore, the efficiency of repair of base/base mismatches was considerably reduced in msh3 mutants in one strain background, indicating a requirement for MSH3 for fully efficient mismatch correction. Also the efficiency of repair of the 38-nucleotide loop was reduced in msh3 mutants, and to a lesser extent in msh6 mutants. The single-nucleotide loop with an unpaired A was less efficiently repaired in msh3 mutants and that with an unpaired T was less efficiently corrected in msh6 mutants, indicating non-redundant functions for the two proteins in the recognition of single-nucleotide loops.     

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.     

Evidence for preferential mismatch repair of lagging strand DNA replication errors in yeast

Duplex DNA is replicated in the 5'-3' direction by coordinated copying of leading and lagging strand templates with somewhat different proteins and mechanics, providing the potential for differences in the fidelity of replication of the two strands. We previously showed that in Saccharomyces cerevisiae, active replication origins establish a strand bias in the rate of base substitutions resulting from replication of unrepaired 8-oxo-guanine (GO) in DNA. Lower mutagenesis was associated with replicating lagging strand templates. Here, we test the hypothesis that this bias is due to more efficient repair of lagging stand mismatches by measuring mutation rates in ogg1 strains with a reporter allele in two orientations at loci on opposite sides of a replication origin on chromosome III. We compare a MMR-proficient strain to strains deleted for the MMR genes MSH2, MSH6, MLH1, or EXOI. Loss of MMR reduces the strand bias by preferentially increasing mutagenesis for lagging strand replication. We conclude that GO-A mismatches generated during lagging strand replication are more efficiently repaired. This is consistent with the hypothesis that 5' ends of Okazaki fragments and PCNA, present at high density during lagging strand replication, are used as strand discrimination signals for mismatch repair in vivo.     

Msh2 separation of function mutations confer defects in the initiation steps of mismatch repair

In eukaryotes the MSH2-MSH3 and MSH2-MSH6 heterodimers initiate mismatch repair (MMR) by recognizing and binding to DNA mismatches. The MLH1-PMS1 heterodimer then interacts with the MSH proteins at or near the mismatch site and is thought to act as a mediator to recruit downstream repair proteins. Here we analyzed five msh2 mutants that are functional in removing 3' non-homologous tails during double-strand break repair but are completely defective in MMR. Because non-homologous tail removal does not require MSH6, MLH1, or PMS1 functions, a characterization of the msh2 separation of function alleles should provide insights into early steps in MMR. Using the Taq MutS crystal structure as a model, three of the msh2 mutations, msh2-S561P, msh2-K564E, msh2-G566D, were found to map to a domain in MutS involved in stabilizing mismatch binding. Gel mobility shift and DNase I footprinting assays showed that two of these mutations conferred strong defects on MSH2-MSH6 mismatch binding. The other two mutations, msh2-S656P and msh2-R730W, mapped to the ATPase domain. DNase I footprinting, ATP hydrolysis, ATP binding, and MLH1-PMS1 interaction assays indicated that the msh2-S656P mutation caused defects in ATP-dependent dissociation of MSH2-MSH6 from mismatch DNA and in interactions between MSH2-MSH6 and MLH1-PMS1. In contrast, the msh2-R730W mutation disrupted MSH2-MSH6 ATPase activity but did not strongly affect ATP binding or interactions with MLH1-PMS1. These results support a model in which MMR can be dissected into discrete steps: stable mismatch binding and sensing, MLH1-PMS1 recruitment, and recycling of MMR components.