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

DNA Repair 8 (2009) 318–329

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Msh1p counteracts oxidative lesion-induced instability of mtDNA and stimulates mitochondrial recombination in Saccharomyces cerevisiae Aneta Kaniak ∗,1 , Piotr Dzierzbicki 1 , Agata T. Rogowska, Ewa Malc, Marta Fikus, Zygmunt Ciesla ∗ Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02-106 Warsaw, Poland

a r t i c l e

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Article history: Received 3 July 2008 Received in revised form 2 November 2008 Accepted 5 November 2008 Available online 18 December 2008 Keywords: mtDNA Oxidative lesions Msh1p Recombination Mitochondria

a b s t r a c t 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 Olir mutants in an ogg1 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 Olir 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 ARG8m 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. © 2008 Elsevier B.V. All rights reserved.

1. Introduction The mitochondrial genome is constantly exposed to endogenous reactive oxygen species (ROS) generated in the electron transport chain. The proximity of the mitochondrial DNA (mtDNA) to the electron transport chain makes mtDNA more vulnerable to damage by ROS than nuclear DNA. Consistently, several groups have demonstrated that the levels of oxidized bases in mtDNA are 2–3 times greater than in nuclear DNA [1,2]. The efficient repair of oxidative lesions that arise in mtDNA plays a central role in maintaining the stability of the mitochondrial genome [3–5]. If not repaired, lesions in mtDNA may result in mutations, which

Abbreviations: BER, base excision repair; MMR, mismatch repair; mtDNA, mitochondrial DNA; ROS, reactive oxygen species; 8-oxoG, 8-oxo-7,8-dihydroguanine. ∗ Corresponding authors. Tel.: +48 22 658 4734; fax: +48 22 658 4636. E-mail addresses: [email protected] (A. Kaniak), [email protected] (Z. Ciesla). 1 These authors contributed equally to this work. 1568-7864/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.dnarep.2008.11.004

in humans may cause a variety of hereditary diseases such as mitochondrial encephalomyopathies and neuropathies, and are associated with the pathogenesis of a variety of complex diseases including heart disease, neurodegenerative diseases such as Parkinson’s, Alzheimer’s and Huntington’s, and other neurological disorders [6,7]. Oxidative damage to mitochondria and accumulation of somatic mutations in mtDNA also appear to be a feature of the normal aging process [3,4]. The yeast Saccharomyces cerevisiae has provided an excellent model system to study mtDNA repair. Unfortunately, our current knowledge of mtDNA repair mechanisms is rather limited. The only relatively well-documented DNA repair pathway that occurs in mitochondria of S. cerevisiae is base excision repair, BER [3,4]. An important enzyme functioning in mtBER is the Ogg1 glycosylase that excises 8-oxoG, an abundant oxidative lesion in DNA, opposite cytosine, but that acts poorly on 8-oxoG opposite adenine [8]. Another substrate of Ogg1 is the DNA replication-blocking lesion FapyG [8]. The yeast Ogg1 protein has a bipartite nuclear localization signal at the carboxy terminus and a mitochondrial targeting

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sequence at the amino terminus [9]. It has been demonstrated that inactivation of OGG1 leads to a 2-fold increase in the production of spontaneous respiration-deficient petite mutants compared with the wild-type strain [9], and to an approximately 10-fold enhancement of mitochondrial point mutations that confer resistance to oligomycin [10]. Another DNA glycosylase which seems to function in the mitochondria is the Ntg1 protein. This enzyme of broad specificity excises several oxidized pyrimidines [11,12]. It has been reported that deletion of NTG1 causes only a very weak increase in the frequency of mitochondrial Eryr point mutations conferring resistance to erythromycin [13]. Another BER enzyme functioning in mitochondria is the Apn1 endonuclease transported into mitochondria after physical interaction with Pir1p [14]. It is noteworthy that AP sites generated by the action of either Ogg1p or Ntg1p can be further processed by these enzymes, since they have an associated AP lyase activity. Alternatively, AP sites in mtDNA, frequently generated by a direct action of ROS, can be cleaved by the Apn1 endonuclease Several lines of evidence suggest that other pathways may also participate in the repair of oxidative lesions in mtDNA. It has been shown that an S. cerevisiae mhr1-1 mutant, which is partially defective in mitochondrial recombination at 30 ◦ C, the temperature permissive for growth of the strain on respiratory carbon sources, accumulates at a non-permissive temperature more oxidative lesions in mtDNA than the wild-type strain, suggesting that recombination processes may be involved in the repair [15]. Biochemical studies of the isolated Mhr1 protein revealed that it catalyses homologous pairing essential to homologous DNA recombination [16]. Furthermore, a synergistic effect on the generation of respiration-deficient petite mutants has been observed in a strain deleted for both NTG1 and PIF1 [13,17]. PIF1 encodes a DNA helicase implicated in mtDNA recombination [18]. However, the involvement of PIF1 in mtDNA repair was proposed to be mediated by a recombination-independent mechanism [17]. Another pathway which may counteract the damaging effects of oxidative lesions in mtDNA was suggested by our finding that the presence of a plasmid carrying an additional copy of MSH1, encoding a homologue of the bacterial mismatch protein MutS [19,20], markedly reduced the increased frequency of Olir mutants in an ogg1 null strain [10]. Msh1p is a mitochondrial protein that preferentially binds to heteroduplex DNA and has an ATPase activity [20]. Deletion of the

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MSH1 gene leads to a high instability of mtDNA, resulting in the loss of the wild-type mitochondrial genome. Vanderstraeten et al. [21] described msh1 mutants that exhibited a strong mtDNA mutator phenotype, but still maintained some functional mitochondrial DNA under selective pressure, suggesting that Msh1p may have a dual role in the maintenance of mtDNA. This suggestion was recently confirmed by Mookerjee and co-workers [22,23] who analyzed the phenotypes of several msh1 point mutants and proposed that, in addition to the mitochondrial mismatch repair (mtMMR), Msh1p may be involved in the mtDNA recombination surveillance. The suppression of the mitochondrial mutator phenotype in the ogg1 strain by the pRS416-MSH1 plasmid has led us to suggest that Msh1p may play an important role in the prevention of oxidative lesion-induced mutagenesis. In the present study we investigated this hypothesis further. We present data indicating that, on the one hand, a surplus of Msh1p suppresses oxidative lesion-induced mutagenesis, and, on the other hand, that a partial dysfunction of Msh1p, particularly when combined with deletion of OGG1 or APN1, significantly decreases the stability of the mitochondrial genome. Furthermore, we show that a surplus of Msh1p results in enhanced mitochondrial recombination. We hypothesize that the Msh1p-stimulated recombination plays an important role in the prevention of oxidative lesion-induced instability of the mitochondrial genome. 2. Materials and methods 2.1. Yeast strains and plasmids Strains of S. cerevisiae used in this study are listed in Table 1. MF121 (sod2::kanMX) was constructed with the use of the kanMX cassette amplified on the template of genomic DNA from a Euroscarf sod2 strain with the primers A and D from the Saccharomyces Genome Deletion Project (sequences available at http://wwwsequence.stanford.edu/group/yeast deletion project) [24,25]. The PCR product was transformed into FF18733 and the G418r strain was verified by PCR analysis using the same primers. YAK151 (msh1 msh1-R813W) was obtained as follows. The msh1-R813W allele was cloned in pRS416 by replacement of the wild-type MSH1 intragenic 2 kb BsrGI fragment from pRS416-MSH1 with the corresponding fragment from pPK12 (pYES2-msh1-

Table 1 S. cerevisiae strains used in this study. Strain

Relevant genotype

Reference

FF18733 BJ2168 CD138 CAB183-1 DFS160 GW22 MCC259 TF236 MF121 AR29 AR30 AR33 AR34 AR39 AR40 AR41 AR42 PD13 PD14 YAK29/1 YAK47 YAK149 YAK151

MATa leu2-3,112 trp1-289 ura3-52 his7-2 lys1-1 ura3-52 leu2-3 trp-289 prbl-1122 prc1-407 pep4-3 ogg1::TRP1 MATa ura3-52 leu2-3 lys2 his3 arg8::hisG/+ cox3::arg8m (AT)16 + 1 MAT˛ ade2-101 leu2 ura3-52 arg8::URA3 kar1-1/0 MATa lys2/+ cox3-421 MAT˛ ade2-101 ura3-52 kar1-1/− [COX3] MATa ino1::HIS3 arg8::hisG pet9 ura3-52 lys2/+ cox3::arg8m -1 sod2::kanMX4 ogg1::TRP1 msh1::kanMX ura3::URA3-MSH1 ogg1::TRP1 msh1::kanMX ura3::URA3-msh1-R813W msh1::natMX ura3::URA3-MSH1 msh1::natMX ura3::URA3-msh1-R813W apn1::kanMX msh1::natMX ura3::URA3-MSH1 apn1::kanMX msh1::natMX ura3::URA3-msh1-R813W sod2::kanMX4 msh1::natMX ura3::URA3-MSH1 sod2::kanMX4 msh1::natMX ura3::URA3-msh1-R813W ntg1::kanMX msh1::natMX ura3::URA3-MSH1 ntg1::kanMX msh1::natMX ura3::URA3-msh1-R813W MAT˛ ade2-101 leu2 ura3-52 arg8::URA3 kar1-1/+ cox3::arg8m -1 MATa ura3-52 leu2-3 lys2 his3 arg8::hisG/0 msh1::kanMX ura3::URA3-MSH1 msh1::kanMX ura3::URA3-msh1-R813W

F. Fabre K. Drotschmann S. Boiteux E.A. Sia T.D. Fox T.D. Fox E.A. Sia T.D. Fox This study This study This study This study This study This study This study This study This study This study This study This study This study This study This study

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R813W) [26]. The plasmid was named pMF17. To integrate the mutant allele into the yeast genome, the msh1-R813W allele was recloned from pMF17 on a 3.7 kb KpnI–BamHI fragment into the integrative pRS306 plasmid (pCK14). As a control, in the same way we also cloned the wild-type MSH1 gene from pRS416-MSH1 in pRS306 (pCK13). Both the wild-type and mutant alleles were introduced into the genome of FF18733 msh1::kanMX/pMF28 (pRS315-MSH1), a strain designated as YAK143, by transformation with plasmids pCK13 and pCK14, respectively, linearized with NcoI to direct integration to the ura3 locus. Upon isolation of respiring transformant Ura+ Leu+ clones, the LEU2 plasmid covering the msh1 deletion, pMF28, was segregated out from transformant cells by growing them in a leucine-containing synthetic medium (YNB + casamino acids + tryptophan + adenine). Finally, respiring Ura+ Leu− clones were isolated and tested for oligomycin-resistance mutagenesis and maintenance of the mitochondrial genome. A wild-type integrant strain was designated as YAK149, and an msh1R813W integrant as strain YAK151. AR29 and AR30 were obtained by transformation of YAK149 and YAK151, respectively, with an ogg1::TRP1 disruption cassette amplified from the genomic DNA of CD138. Primers used for the amplification were described by Thomas et al. [27]. The PCR product was transformed into YAK149 and YAK151 and Trp+ colonies selected. Correct integration of the ogg1::TRP1 cassette in the genome of transformant clones was verified by PCR analysis. AR33 and AR34 are derivatives of YAK149 and YAK151, respectively, in which the kanMX cassette was replaced by the natMX cassette by transformation with the p4339 plasmid (a kanMX to natMX marker switcher plasmid [28]) cut with EcoRI. AR39 and AR40 are derivatives of AR33 and AR34, respectively, in which APN1 was deleted using the kanMX cassette. AR41 and AR42 are derivatives of AR33 and AR34, respectively, in which SOD2 was deleted using the Euroscarf kanMX cassette, as described above for the MF121 strain. PD13 and PD14 were obtained from AR33 and AR34, respectively, by deletion of NTG1 using the Euroscarf kanMX cassette. YAK29/1 (MAT˛ ade2-101 leu2 ura3-52 arg8::URA3 kar1-1/␳+ cox3::arg8m -1) was constructed by cytoduction of mitochondria from TF236 (MATa ino1::HIS3 arg8::hisG pet9 (op1) ura3-52 lys2/␳+ cox3:: arg8m -1) [29] to DFS160 (MAT˛ ade2-101 leu2 ura352 arg8::URA3 kar1-1 [rho0 ]; [30]). Description of the procedure used to select the cytoductant strain, with the nuclear genome of DFS160 and the mit− mitochondrial genome from TF236, will be provided upon request. YAK47 is a rho0 version of CAB183-1 obtained by treatment of the latter strain with ethidium bromide as described by Fox et al. [31]. The plasmid pRS416-MSH1 was a kind gift from R. Butow. Construction of pMF17 is described above. The pMF28 plasmid was generated by recloning MSH1 from pRS416-MSH1 as an ApaI-BamHI fragment into pRS315 cut with the same restriction enzymes. Construction of pPK11–pPK13 was described previously [26]. Plasmids used in the study were basically the same as pYES2, pPK11, pPK12 and pPK13, except that the URA3 marker in those plasmids was replaced by LEU2 using the “marker swap” pUL9 plasmid [32]. Thus, in our study the LEU2 equivalent of pYES2 is pCK19, the equivalent of pPK11 (pYES2-MSH1) is pCK20, the equivalent of pPK12 (pYES2-msh1-R813W) is pCK22 or pCK23, and the equivalent of pPK13 (pYES2-msh1-G776D) is pCK24. The pPK15 plasmid, carrying the MSH1 gene under control of the GAL promoter and tagged at 3 end with the sequence coding for 6× His epitope under the control of the GAL1 promoter, was constructed as pPK11 [26], but with the use of the MSH16HIS LW primer (5 -TTAGTGATGGTGATGGTGATGTCCCAATATTTCGCGGGCTTCTT-3 ) instead of the MSH1 LW primer. The plasmids pCK27, pCK28 and pCK29 were derived from pCK20 (pMSH1+ ), pCK23 (pmsh1R813W) and pCK24 (pmsh1-G776D), respectively, by C-terminal

TAP tagging of the MSH1 alleles on the plasmids. The TAP tagging was accomplished using a PCR product amplified from p2623 (a generous gift from Andrzej Dziembowski; an unpublished plasmid construction for generating a TAP tagging cassette with the kanMX marker) using primer pair OA1 (5 -CCGGATTTCCAATGGAAGCGTTAAAAGAAGCCCGCGAAATATTGGGATCCATGGAAAAGAGAAG-3 ) and altOA2 (5 -TATCTGCAGAATTCCAGCACACTGGCGGCCGTTACTAGTGGATCCTACGACTCACTATAGGG-3 ), according to Puig et al. [33]. 2.2. Growth media and growth conditions YP medium contained 1% yeast extract, 1% Bacto peptone and 2% dextrose (YPD) or 2% glycerol (YPG). YNBD medium contained 0.67% yeast nitrogen base and 2% dextrose. YNBD media were supplemented with casamino acids, adenine, uracil and tryptophan. To select plasmids with the LEU2 marker we used SCD-LEU or SCD5-LEU media that were YNB medium supplemented with 2% or 5% dextrose, respectively, and a drop-out mix of auxotrophic requirements without leucine, according to [34]. Solid media contained 2% Bacto agar. Non-respiring (petite) colonies were scored on YPG medium supplemented with 0.1% glucose (YPGD). Oligomycin resistant mutants (Olir ) were scored on plates with YPG medium, buffered at pH 6, containing 3 ␮g/ml oligomycin; erythromycin resistant mutants (Eryr ) were selected on plates with pH-buffered YPG medium (pH 6) supplemented with 4 mg/ml of erythromycin. 2.3. Analysis of mitochondrial genomes by restriction digestion Total cellular DNA was isolated, stained with bisbenzimide, and mtDNA separated from nuclear DNA by centrifugation in CsCl gradients as described previously [35]. Mitochondrial DNA was digested with AccI and separated by agarose gel electrophoresis. 2.4. Purification of the Msh1 protein S. cerevisiae strain BJ2168 harbouring the pPK15 plasmid, which carries the MSH1 sequence fused at its C-terminus to the hexahistidine-tag sequence and whose expression is under the control of the GAL1 promoter control, was grown in 500 ml of YPD medium to OD600 = 1.0. Subsequently, the culture was diluted with 4 l of YPGal medium and the cells were grown for 12 h. The cells were collected by centrifugation, washed once with water, and then resuspended in cell breakage buffer, pH 7.8 (50 mM Na phosphate, 300 mM NaCl, 10% glycerol, 2 mM imidazole, 1 mM DTT, 1 mM PMSF). All the purification steps were performed at 4 ◦ C and 1 mM PMSF was added to all the buffers. Cells (8 g) were disrupted with glass beads. The cell debris was removed by centrifugation at 15 000 rpm for 45 min. A total of 0.5 ml of Ni-NTA agarose (Qiagen) was added to 10 ml of the resulting supernatant. After 45 min the resin was used to form a column. Proteins bound to Ni-NTA were eluted with a step gradient of imidazole. Under the experimental conditions used, a considerable portion of Msh1p was eluted with 70 mM imidazole. For the nicking assay the fractions were concentrated with Vivaspin 4 ml concentrator 50 000 MW (Viva Science). 2.5. Assay for nicking activity at 8-oxoG A 40mer containing a single 8-oxoG (5 -GCTACCTACCTAGCGACCTGOCGACTGTCCCACTGCTCGAA-3 ) was kindly provided by B. Tudek. Oligos with complementary sequences that, after annealing with the 8-oxoG oligo or the control oligo, provide C or A opposite 8oxoG, or A opposite G, were also synthesized. 20 pmol of the 40mer containing 8-oxoG, or G at the same position (the control oligo), were labeled at 5 end using 25 ␮Ci of (␥32 )ATP and T4 polynucleotide kinase. The reaction was carried out at 37 ◦ C for 1 h. Then,

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30 ␮l of STE buffer (10 mM Tris pH 8, 1 mM EDTA, 50 mM NaCl) were added to the reaction mixture (20 ␮l) and the product purified using a BIO-RAD G-30 column. The labeled oligo was hybridized to unlabeled oligos with complementary sequences yielding either 8-oxoG/C, 8-oxoG/A, G/C or G/A duplexes. 20 pmol of the labeled oligonucleotide and 40 pmol of a complementary oligonucleotide were mixed in a tube, which was then put in boiling water and left to cool down slowly for about 2 h at room temperature. The assay for the nicking activity was performed as described by Maynard et al. [36]. The reaction mixture (30 ␮l) contained 20 mM Hepes-KOH pH 7.6, 1 mM EDTA, 1 mM DTT, 50 mM KCl, 10% glycerol, 300 fmol oligonucleotide and either the purified Msh1 protein, or the Fpg protein. After 30 min at 30 ◦ C, an equal volume of the buffer containing 90% formamide, 1 mM EDTA, 0.1% bromophenol blue was added and samples were analyzed by 20% polyacrylamide/7 M urea gel electrophoresis. Reaction products were visualized using the Molecular Dynamics Storm imaging system (GE Healthcare) and computer program ImageQuant 5.2 (GE Healthcare). 2.6. Determination of mutant frequencies To measure the frequency of petite formation, single colonies from YPG plates were inoculated into 4 ml YNBD supplemented with casamino acids, tryptophan and uracil (or without uracil if selection for the presence of a URA3 plasmid was needed, or the strain to be tested was Ura+ ) and grown to stationary phase at 30 ◦ C. Appropriate dilutions were plated on YPGD plates and the percentage of petites was scored after growth for 3 days at 30 ◦ C. Colonies were scored as ␳+ or ␳− /␳0 by the tetrazolium overlay method [37]. The median value from each set of at least 10 cultures was used to determine the percentage of petite colonies. To measure the frequency of Olir and Eryr mutants, single colonies from YPG plates were inoculated into YNBD liquid medium supplemented with casamino acids, tryptophan and uracil, as above, and grown to stationary phase at 30 ◦ C. Cells were then harvested by centrifugation, washed with water and plated either on YPG + oligomycin or YPG + erythromycin. Appropriate dilutions of the cultures were also spread on YPGD plates to calculate the number of petite and grande cells in tested cultures. The median value derived from at least 10 independent cultures was used to determine the frequency of mutants resistant to oligomycin or erythromycin (calculated per number of grande cells). P values for statistical significance of any differences in mutant frequencies between pairs of strains were determined by applying the nonparametric Mann-Whitney criterion using the program STATISTICA (Statsoft). 2.7. Assay of mtDNA recombination To assess levels of mitochondrial recombination within the ARG8m gene in cells harbouring either the vector plasmid or plasmids with additional copies of the MSH1 gene, parental strains carrying either the cox3::polyAT(+1)-arg8m (CAB183) or cox3::arg8m -1 (YAK29/1) allele were crossed, resulting diploids selected and the frequency of arginine prototrophs scored in final diploid cultures. In brief, 100 ␮l of a fresh 2 day preculture of each parent in the medium selective for the presence of plasmids, SCD5LEU, were mixed in a tube, spun down and resuspended in 1 ml of fresh YPD. Since we have noticed that transformants of the YAK29/1 strain tend to grow in the selective medium in the form of cellular aggregates that are not easily dispersed by vortexing, before mixing of parental cells we added EDTA at 20 mM to YAK29/1 transformant cultures. This pretreatment often inhibits cation-dependent flocculation of yeast cells [38] and, in our hands, has proved to efficiently disperse cellular aggregates also in YAK29/1 cultures. Cells were

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pelleted and incubated in the pellet for 1 h at 30 ◦ C. Afterwards, the mating mixture was briefly vortexed and incubated for 2 h at 27 ◦ C with shaking. An aliquot of 100 ␮l from the resulting mating culture was added to 5 ml of YNB medium supplemented with 2% glucose and arginine at 40 mg/l (leucine, another requirement of the expected diploids, was omitted to sustain the selection for the presence of plasmids being examined). In parallel, titres of diploid cells in the same mating cultures at the moment of inoculation were determined by plating appropriate dilutions on YNBD plates with arginine. After 3 days of incubation at 27 ◦ C with shaking, appropriate dilutions of diploid cultures were plated on YNB medium with 2% glucose, arginine (as above) and leucine (218 mg/l; after Styles [39]) to establish the final titre of selected diploid cells, and on the same medium without arginine to determine the frequency of Arg+ cells. Since an even moderate overexpression of the wild-type MSH1 or msh1-R813W in yeast cells may destabilize mtDNA [10], we established the frequency of mitochondrial genome retention in three independent cultures of each parent by a test cross. Cells from a tested parental culture were plated and grown for single colonies on SCD-LEU plates. Usually 52 transformant colonies per culture were picked up (thus, in all, 156 colonies per parental strain per plasmid) and mated with an appropriate tester strain. For YAK29/1 transformant clones, the tester strain was GW22 (MATa lys2 [␳ + cox3-421]) after Steele et al. [30]. For CAB183-1 transformants, the tester strain was MCC259 (MAT␣ ade2-101 ura3-52 kar1-1 [␳− COX3]) after Sia et al. [40]. The principle of the test crosses is explained in the cited references. 2.8. Western blot analysis Transformant clones of the FF18733 strain carrying the plasmids pCK27 (GAL1-MSH1+ -TAP), pCK28 (GAL1-msh1-R813W-TAP) or pCK29 (GAL1-msh1-G776D-TAP) were grown to OD600 ∼1.0 in YP medium supplemented with 2% galactose and G418 (at 200 mg/l). Cells were harvested and whole cell protein extracts were prepared as described by Knop et al. [41]. Aliquots of the resulting protein extracts, corresponding to 0.5 OD600 of cells, were separated by SDS-PAGE (8% polyacrylamide gel) and proteins transferred to a Hybond nitrocellulose membrane. Blots were blocked overnight in 5% dried non-fat milk solution in TBST (25 mM Tris–HCl [pH 7.5], 137 mM NaCl, 27 mM KCl, 0.05% Tween 20). Actin was detected during primary antibody incubation of the membrane with a mouse monoclonal antibody (C4, CHEMICON) in TBST with 5% milk (as above). Secondary antibody incubation was performed with goat anti-mouse IgG conjugated to horseradish peroxidase (Promega) combined with an antibody probing for TAP-tagged proteins, rabbit peroxidase-anti-peroxidase (PAP, Sigma–Aldrich). Immunoreactive proteins were visualized using home-made chemiluminescent substrates for HRP (luminol, coumaric acid, hydrogen peroxide; Sigma–Aldrich) and images were acquired on a cooled chargecoupled (CCD) camera. 3. Results 3.1. The mitochondrial mutator phenotype of sod2 mutant is abolished by pRS416-MSH1 We reported previously that deletion of the OGG1 gene, encoding a glycosylase that excises 8-oxoG, an abundant oxidative lesion in DNA [8], results in an increased frequency of mitochondrial Olir mutants [10]. Resistance to oligomycin is acquired through specific point mutations in the mitochondrial OLI1 and OLI2 genes, encoding subunits 9 and 6, respectively, of the mitochondrial ATPase complex [42,43]. The sequence analysis of mutations arising in the

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tor were grown in YNBD synthetic medium, supplemented with casamino acids and tryptophan, but not with uracil to select for cells hosting the plasmids, and the frequency of mutants resistant to oligomycin (Olir ), as well as the frequency of mutants resistant to erythromycin (Eryr ) was measured. Resistance to erythromycin is acquired through specific point mutations in the mitochondrial 21S rRNA gene and acquisition of resistance to the drug is a standard method of measuring mtDNA point mutagenesis [13,46]. The sod2 strain harbouring the vector plasmid displayed a 3.9-fold increase (P < 0.001) in the frequency of Olir mutants and a slight, but significant (P < 0.01), 2.3-fold elevation in the incidence of Eryr mutants compared to that in the wild-type strain. This increased frequency of both Olir and Eryr mutants in sod2 was suppressed to the wildtype level by the presence of pRS416-MSH1. These results indicate that under oxidative stress conditions a surplus of Msh1p can effectively prevent enhanced mitochondrial mutagenesis induced by endogenous oxidative stress. 3.2. The sod2 msh1-R813W double mutant displays a synergistic increase in mitochondrial mutagenesis

Fig. 1. Effect of a moderate overproduction of Msh1p on mitochondrial mutagenesis of sod2. Cultures of FF18733/pRS416, FF18733/pRS416MSH1, MF121 sod2/pRS416, MF121 sod2/pRS416MSH1, were grown in YNBD medium supplemented with casamino acids and tryptophan. The frequencies of Olir mutants (A) and of Eryr mutants (B) were determined. Each bar represents the average of the median values obtained from two separate experiments in which at least 10 independent cultures of each strain was used. Error bars indicate standard deviations.

mitochondrial OLI1 gene of the ogg1 strain revealed that a significant portion of these mutations were G:C to T:A transversions [10], a signature of 8-oxoG [8] lesions. Interestingly, the mitochondrial mutator phenotype of the ogg1 strain was markedly reduced by the presence of plasmids encoding Msh1p [10], a homologue of the bacterial mismatch protein MutS, which specifically functions in mitochondria [19–23,26]]. This implicates an Msh1p-dependent pathway as an additional mechanism preventing oxidative lesioninduced mutagenesis in yeast mtDNA. Furthermore, since the suppression of the mutator phenotype was observed in a strain deleted for OGG1, this suggested that the Ogg1 glycosylase and Msh1p may act independently in this process. To further address the putative role of Msh1p in the prevention of oxidative lesion-induced mitochondrial mutagenesis, we examined whether the introduction of an additional copy of MSH1, present on the pRS416-MSH1 plasmid, might affect the level of mitochondrial mutagenesis in a strain competent for mtBER, but lacking the activity of the mitochondrial superoxide dismutase (Mn-SOD) encoded by SOD2. This enzyme plays an important role in protecting mitochondrial targets against superoxide [44,45]. It has previously been reported that a sod2 mutant displays an approximately 3-fold increase of the intracellular ROS level [13]. Thus, sod2 cells are exposed to a permanent endogenous oxidative stress. In the experiment shown in Fig. 1 we investigated the effect of the pRS416-MSH1 plasmid on mitochondrial mutagenesis in sod2 cells. The sod2 strain harbouring either pRS416-MSH1 or vec-

We reasoned that if Msh1p plays an important role in the prevention of oxidative lesion-induced mitochondrial mutagenesis, then the frequency of mitochondrial mutations should be considerably increased in a strain in which deletion of SOD2 is combined with a mutation partially inactivating Msh1p. To evaluate this assumption, we constructed a sod2 msh1-R813W double mutant. The msh1-R813W allele is altered in the sequence encoding the ATPbinding domain of Msh1p [26]. It has previously been reported that an msh1-R813W strain displays a mitochondrial mutator phenotype [26], as expected for a mutant deficient in mismatch repair of mtDNA. The msh1-R813W strain exhibits also a large increase in the rate of repeat-mediated deletions in mtDNA suggesting that Msh1p may play an additional role in maintaining the stability of mtDNA [23]. The sod2 and msh1-R813W single mutants and the sod2 msh1-R813W double mutant were grown in YPG medium and the frequency of Olir mutants, as well as the frequency of respiration-deficient petite mutants was determined. It is worth noting that intracellular ROS production in sod2 cells growing in YPG medium, as estimated by an assay with the use of 2‘7’dichlorofluoresceine diacetate probe, was approximately 2-fold higher than that of the wild-type strain (data not shown), similar to the results shown by Doudican et al. [13]. As shown in Fig. 2, the msh1-R813W and sod2 single mutations conferred a modest elevation (4.6-fold and 2.6-fold, respectively) in the frequency of Olir mutants as compared to that of the wild-type strain. Strikingly, the sod2 msh1-R813W double mutant displayed a 13.0-fold higher mutation frequency than that observed in wild-type control, indicating that combination of sod2 and msh1-R813W led to a synergistic increase in the frequency of Olir . This result supports the hypothesis that Msh1p is involved in the prevention of oxidative lesion-induced mitochondrial mutagenesis. Unexpectedly, however, although the msh1-R813W mutation resulted in a considerable increase in the frequency of respiration-deficient petite mutants (up to 5.8% of total cell population) this frequency was not further elevated in the sod2 msh1-R813W double mutant. This result suggests that petite mutants are generated by a different mechanism than Olir mutants. To get insight into the nature of alterations within the mitochondrial genomes of the petite strains produced in the msh1-R813W mutant, mtDNA from five independently isolated petite clones was examined by restriction analysis. As shown in Fig. 5B, these isolates displayed gross changes in the restriction pattern indicating to rearrangements in their mitochondrial genomes.

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Fig. 2. Mitochondrial mutagenesis in the sod2 msh1-R813W double mutant. Cultures of AR33 MSH1 SOD2, AR34 msh1-R813W SOD2, AR41 sod2 MSH1 and AR42 sod2 msh1-R813W were grown in YPG medium. The frequencies of Olir mutants and petite mutants were determined. Each bar represents the average of the median values obtained from three separate experiments in which 10 independent cultures of each strain was used. Error bars indicate standard deviations.

3.3. Msh1p fails to cleave DNA containing 8-oxoG To further address the role of Msh1p in the prevention of oxidative lesion-induced instability of mtDNA, we considered the possibility that Msh1p might possess activity which incises DNA at oxidative lesions, such as 8-oxoG. Such an incision function was postulated for the Helicobacter pylori MutS protein [47]. In order to evaluate this possibility we purified Msh1p tagged at the C terminus with a 6× His epitope. Then, we measured the capacity of Msh1p to cleave a 40-mer double-stranded DNA fragment that contains a single 8-oxoG placed either opposite a cytosine (8-oxoG/C) or an adenine (8-oxoG/A). Fig. 3 shows that neither 8-oxoG/C, nor 8-oxoG/A duplex was incised by Msh1p. In contrast, the Fpg protein [48], which was used as a control in the nicking assay, efficiently cleaved the 8-oxoG/C duplex, and with a lower efficiency also the 8oxoG/A duplex. This result indicates that by itself Msh1p does not possess the capacity to cleave DNA containing 8-oxoG. However, this result does not discount the possibility that Msh1p might activate a downstream acting protein possessing the nicking activity at oxidative lesions in DNA.

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pared to that of the wild-type strain (Fig. 4A). The frequency of Olir in ogg1 was 6.4 times higher than in OGG1. This increase in mtDNA mutagenesis was enhanced, in an additive manner, in the msh1-R813W ogg1 double mutant to 10.8-fold. This is another finding suggesting that Msh1p counteracts the generation of mitochondrial Olir mutations acting in a pathway that is independent of the Ogg1 glycosylase. Interestingly, however, a different pattern of mitochondrial mutagenesis was found in the tested mutants when, instead of Olir , the incidence of respiration-deficient petite mutants was estimated. The frequency of petites in the ogg1 single mutant was similar to that of the wild-type strain. In contrast, the incidence of petites was enhanced 12.5-fold in the msh1-R813W mutant and this increase was further enhanced by a factor of 2.4 (P < 0.001) in the msh1-R813W ogg1 double-mutant strain. This result shows a synergistic effect of ogg1 and msh1-R813W on the generation of petite mutants. To get insight into the nature of alterations within the mitochondrial genomes of the petite strains produced in the ogg1 and msh1-R813W ogg1 mutants, mtDNA from several independently isolated respiration proficient clones and petite clones was examined by restriction analysis. As shown in Fig. 5A, an identical restriction pattern of mtDNA was observed in four independent rho+ isolates indicating that there is no easily detectable natural variation between them with respect to mtDNA profiles. Out of four petite mutants generated in the ogg1 mutant, one was lacking mtDNA (a rho0 strain) and the remaining three isolates displayed gross changes in the restriction pattern indicating rearrangements in their mitochondrial genomes (Fig. 5A, lanes 1–3). Gross changes in the restriction profiles were also found in mtDNA of petite clones isolated from the msh1-R813W ogg1 double mutant strain (Fig. 5C).

3.4. Analysis of the mitochondrial mutator phenotype in the msh1-R813W ogg1, msh1-R813W apn1 and msh1-R813 ntg1 double mutants The only relatively well documented DNA repair pathway that occurs in mitochondria of S. cerevisiae is base excision repair, BER [5,7,49]. Therefore, it was of interest to study the relationship between the roles of Msh1p and mitochondrial BER in the prevention of oxidative lesion-induced mitochondrial mutagenesis. For this purpose, we constructed three double-mutant strains deleted for either OGG1, NTG1 or APN1, encoding enzymes predicted to be involved in mtBER [9,10,13,14], and carrying the msh1-R813W allele integrated into the chromosome. The frequencies of Olir and respiration-deficient petite mutants arising in the msh1-R813W, ogg1, apn1, and ntg1 single mutants, as well as in the msh1R813W ogg1, msh1-R813W apn1, and msh1-R813 ntg1 double mutants were determined. A 4.2-fold increase in the incidence of Olir mutants was observed in the msh1-R813W single mutant com-

Fig. 3. Msh1p does not possess a nicking activity at 8-oxoG. (A) Msh1–6 × His protein was overexpressed in BJ2168 hosting pPK15 (GAL1-MSH1–6 × His) and purified using Ni-NTA resin and analysed on 20% polyacrylamide SDS gel. Lane 1, cell extract; lane 2, purified Msh1p stained with Coomassie blue; lane 3, Western blotting of the purified Msh1p with the use of anti-His-tag antibody as a primary antibody, and anti-mouse IgG AP conjugate as a secondary antibody. (B) Purified Msh1p was used in the DNA nicking assay with the use of G:C, 8-oxoG:C, G:A and 8-oxoG:A duplexes as substrates and either 30 ng of the Fpg protein, or 250 ng of the Msh1 protein. The upper arrow indicates the 40mer oligonucleotide and the lower arrow indicates the cleavage product.

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3.5. An excess of Msh1p enhances recombination of mtDNA

Fig. 4. Mitochondrial mutagenesis in the msh1-R813W ogg1 double mutant. Cultures of YAK149 MSH1, YAK151 msh1-R813W, AR29 ogg1 and AR30 msh1-R813W ogg1 were grown in YNBD medium supplemented with casamino acids and tryptophan. The frequencies of Olir mutants (A) and petite mutants (B) were determined. Each bar represents the average of the median values obtained from two separate experiments in which at least 10 independent cultures of each strain was used. Error bars indicate standard deviations.

A subtle, but statistically significant 2.2-fold increase in the frequency of Olir (P < 0.05) was found in the apn1 single mutant, and this increase was enhanced in a slightly more than additive manner by the msh1-R813W allele (Fig. 6A). On the other hand, although the deletion of APN1 by itself failed to significantly increase the incidence of petites, synergy was observed when apn1 was combined with msh1-R813W. Restriction analysis of mtDNA isolated from six independent petite clones of the msh1-R813W apn1 double mutant revealed gross changes in the restriction profiles as compared to mtDNA derived from respiration-competent cells (data not shown). Taken together, synergy conferred by the msh1-R813W mutation combined with either the ogg1, or apn1 mutation on formation of respiration-deficient petite clones having gross rearrangements in mtDNA suggests that the Ogg1 glycosylase, the Apn1 endonuclease and Msh1p play overlapping functions in maintaining the stability of the mitochondrial genome. These overlapping functions of Ogg1p, Apn1p and Msh1p may explain why a single deletion of OGG1 or APN1, at least in the genetic background of the strains used in this study, does not result in an enhanced frequency of petite formation. In contrast, deletion of NTG1 failed to have a significant effect on the frequency of either Olir or petite mutants (Fig. 6). Moreover, ntg1 when combined with msh1-R813W, failed to enhance further the elevated incidence of petite mutants conferred by the msh1R813W mutation, and led a 2-fold decrease in the frequency of Olir mutants. The latter observation is consistent with recent finding that Ntg1p may generate mutagenic intermediates in yeast mtDNA [50].

We hypothesized that the mechanism underlying the rearrangements of mtDNA observed in the msh1-R813W strain, the frequency of which is enhanced by additional disruption of either OGG1 or APN1, may be related to the efficiency of recombination processes occurring in the yeast mtDNA. To verify this hypothesis, we developed a novel mtDNA recombination assay. Our assay is based on mtDNA recombination after mating of two yeast strains, each with a different defective allele of ARG8m replacing the COX3 gene [30] in mtDNA (Fig. 7A). In one of the two alleles, a tract of 16 AT repeats was inserted out of frame (+1) 20 bp downstream of the start codon of ARG8m , precluding the synthesis of active Arg8m p [40]. In contrast to other microsatellite insertions, this configuration of microsatellite sequence is very stable in yeast mtDNA with the rate of tract changes established at merely 2.0 × 10−10 per cell division [40]. The other arg8m allele is a cox3::arg8m -1 mutant isolated by Fox and collaborators [29]. The sequence of the mutant allele was determined and it uncovered three mutations, one of which, + 1 insertion of A at the 979-base pair position in the arg8m (starting with the ARG8m ATG at the 1-bp position), is sufficient to abrogate the Arg8m p function, leading to the Arg− phenotype [34]. The allele reverts to arginine prototrophy, by -1 frameshift, at a low rate of 2.0 × 10−8 per cell division [51]. Thus, for both arg8m alleles used in our assay reversion events are rather rare. It turned out that in crosses between the cox3::polyAT(+1)arg8m and cox3::arg8m -1 strains, each harbouring the vector plasmid pCK19, approximately 3% of the resulting diploid clones appeared to be Arg+ prototrophs, most likely originating from a homologous recombination event, in the 957 bp interval between the two mutation sites in the arg8m alleles, that restores a functional copy of the cox3::ARG8m gene. This conclusion was confirmed by the results of control experiments, performed in exactly the same way as the rho+ mit− × rho+ mit− crosses mentioned above, in which we crossed each parent, either cox3::polyAT(+1)arg8m /pCK19 or cox3::arg8m -1/pCK19, with a rho0 counterpart of the other parent (DFS160, a rho0 of the cox3::arg8m -1 strain, or YAK47, a rho0 version of the cox3::polyAT(+1)arg8m strain) hosting pCK19. In these control crosses (each cross was performed in 12 repeats), the percentage of Arg+ prototrophs among diploid progeny was hardly detectable, below 0.01%, confirming the prediction that spontaneous reversion events occurring in the arg8m alleles are much too rare to significantly influence final results of arg8m allele recombination. Control crosses, analogous to the described above crosses rho+ mit− × rho0 in the presence of the vector plasmid, were also performed with strains carrying pCK20(GAL1-MSH1) or pCK22(GAL1-msh1-R813W). Additional expression of either variant of Msh1p did not increase the level of detectable spontaneous reversion of either arg8m allele in diploid progeny of these control crosses. However, we found that, in contrast to cells harbouring the vector plasmid, even a relatively low overexpression of Msh1p in cells hosting the plasmid pCK20(GAL1-MSH1) was associated with a significant decrease in the retention of rho+ mit− genomes (see legend to Fig. 7B). Mitochondrial recombination between the two arg8m can obviously take place only in those diploid cells which obtained rho+ mit− mtDNA from both parents. Consequently, the frequency of ARG8m+ recombinants should be referred exclusively to the number of such diploid cells. For this reason, all Arg+ frequencies shown in Fig. 7 are normalized with respect to estimated retention in diploid cells of mtDNA from both parental strains, as described in the legend to Fig. 7B. To examine the effect of Msh1p on recombination of mtDNA, the assay was performed with cox3::polyAT(+1)arg8m and cox3::arg8m 1 cells carrying the plasmid pCK20(GAL1-MSH1), under conditions

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in cells overproducing Msh1p. Significantly, when recombination tests were performed with cells harbouring plasmids encoding mutant alleles of MSH1, either msh1-R813W or msh1-G776D, no significant increase in the frequency of diploid Arg+ prototrophs was detected in comparison to diploid cells harbouring the vector (Fig. 7B). The result suggests that, in contrast to the wild-type Msh1p, the mutant proteins, msh1p-R813W and msh1p-G776D, are not able to significantly enhance mitochondrial homologous recombination in our assay. The comparable effects of increased rho+ mit− genome loss due to low overexpression of Msh1 proteins imply that also levels of all three variants of Msh1p are comparable in cells tested in our experiments. This is further evidenced by an example of Western blot analysis presented in Fig. 7C, showing similar cellular levels of Msh1p and its mutated forms. Thus, the apparent defect of the mutant proteins, msh1p-R813W and msh1pG776D, to enhance mitochondrial recombination in our tests is not due to instability of the mutant proteins, but rather to a weakened activity of the proteins in comparison to that of the wild-type Msh1p. Theoretically, significant differences in initial numbers of diploid cells (that could result, for example, from differences in mating efficiencies between parental strains harbouring different plasmids) and differences in reproductive capacity of diploid cells, carrying either the vector plasmid or any of the three tested plasmids, could lead to the observed differences in Arg+ frequencies

Fig. 5. Analysis of mitochondrial genomes by restriction digestion. Mitochondrial DNA from four independent rho+ isolates and petite isolates generated either in ogg1 (A) lanes 1–3)), msh1-R813W (B), or msh1-R813W ogg1 (C), was digested with AccI and separated by agarose gel electrophoresis. MW; molecular weight standard.

of a moderate overproduction of Msh1p in YNB medium supplemented with 2% glucose. As shown in Fig. 7, the normalized frequency of Arg+ prototrophs in resulting diploids increased 3-fold (P = 0.00003 with the Mann-Whitney statistics) in comparison to diploids issued in matings of control parental cells hosting the vector plasmid. The difference suggests that homologous recombination between mtDNA molecules, carrying either the cox3::polyAT(+1)arg8m or the cox3::arg8m -1 allele, is stimulated

Fig. 6. Mitochondrial mutagenesis in the msh1-R813W apn1 and msh1-R813W ntg1 double mutants. Cultures of AR33 MSH1, AR34 msh1-R813W, AR39 apn1, AR40 msh1-R813W apn1, PD13 ntg1 and PD14 ntg1 msh1-R813W were grown in YNBD medium supplemented with casamino acids and tryptophan. The frequencies of Olir mutants (A) and petites (B) were determined. Each bar represents the average of the median values obtained from two separate experiments in which at least 10 independent cultures of each strain was used. Error bars indicate standard deviations.

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legend to Fig. 7, numbers of generations estimated for cultures of all the crosses performed were similar, and the small differences between particular crosses in the reported experiment do not correlate by any means with the observed differences in Arg+ frequencies in final diploid cultures. Thus, differences between parental strains in mating efficiencies or differences between their diploid progeny in reproductive capacity cannot account for the observed differences in Arg+ percentages in the cultures. The simplest explanation of the results lies in differences in levels of mitochondrial recombination. In conclusion, we surmise that, in contrast to Msh1p, both mutant proteins, msh1p-R813W and msh1p-G776D, are defective in the activity of stimulating mitochondrial homologous recombination. Both alleles are altered in the sequence encoding the ATP-binding domain of Msh1p and have been shown previously to impair the function/s of the protein [23,26]. We have shown [10] that the low overexpression of MSH1, but not of msh1-R813W, suppresses the mitochondrial mutator phenotype of an ogg1 mutant. We conclude, therefore, that ATP binding and/or hydrolysis is essential for Msh1p both to suppress oxidative lesion-induced mutagenesis and to efficiently enhance mtDNA recombination. 4. Discussion

Fig. 7. Effect of a moderate overproduction of Msh1p, msh1p-R813W and msh1pG776D on recombination within the mitochondrial ARG8m gene. Crosses between strains YAK29/1 (cox3::arg8m -1) and CAB183-1 (cox3::arg8m (AT) + 1), each carrying either the vector plasmid pCK19, pCK20(MSH1), pCK22(msh1-R813W) or pCK24(msh1-G776D), were performed as described in Materials and methods. (A) Schematic representation of the two arg8m alleles in mtDNA of the parental strains. Description of the alleles is in the Results section. The interval between the mutational alterations in the two alleles responsible for Arg− phenotypes of the parental strains is indicated. (B) The percentage of Arg+ in diploid cultures. The presented data are average normalized values from one representative experiment comprising 12 crosses for each plasmid tested, each cross with a different transformant clone of each parental strain. The Arg+ frequency values were normalized to estimated retention in diploids of mtDNA from both parents on the basis of frequencies of mitochondrial mit− genome retention established for each parental strain. Those frequencies in cultures of strains to be mated were determined by crosses with appropriate tester strains (Materials and methods). The percentages of rho+ mit− genomes were as follows: YAK29/1/vector – 97, YAK29/1/pMSH1 – 90, YAK29/1/pmsh1-R813W – 78, YAK29/1/pmsh1-G776 – 79, CAB183-1/vector – 95, CAB183-1/pMSH1 – 58, CAB183-1/pmsh1-R813W – 61, CAB183 –1/pmsh1G776D – 71. Thus, the calculated probability of productive mit− × mit− encounters (pmit × mit ) in the crosses with cells carrying the vector plasmid was, 0.92 (=0.97 × 0.95), for cells with pMSH1 was, 0.52 (=0.90 × 0.58), for cells with pmsh1-R813W was, 0.48 (=0.78 × 0.61), and for cells with pmsh1-G776 was, 0.56 (=0.79 × 0.71). For normalization, the observed frequency of Arg+ in a diploid culture (%) was divided by the estimated probability of a productive mit− × mit− encounter, pmit × mit . Error bars represent standard deviations. For each tested diploid culture, the number of generations, denoting the number of times an average diploid cell has divided in the culture during 3 days of growth, was calculated from the equation n = log2 (N/N0 ), where n is the number of generations, N0 is the diploid cell titre at the moment of inoculation, and N is the diploid cell titre after 3 days of growth (as described in Materials and methods). The average number of generations for diploid cultures with the vector plasmid was 17.4 ± 0.9 (± standard deviation, STD), for cultures with pMSH1 was, 16.4 ± 0.6 STD, for cultures with pmsh1-R813W was, 15.3 ± 0.6 STD, and for cultures with pmsh1-G776 was, 15.4 ± 0.7 STD. (C) Western blot analysis of whole cell protein extracts from cells harbouring pMSH1+ -TAP (pCK27), pmsh1-R813W-TAP (pCK28), or pmsh1-G776D-TAP (pCK29) probed with antibodies against the TAP-tag epitope and actin (Act1p) as a reference protein.

in final diploid cultures. As described in Materials and methods, we calculated the number of generations each diploid culture has passed on average, during growth after mating in the medium selective for diploids, on the basis of established initial and final titres of diploid cells in the cultures. However, as shown in the

We have shown previously [10] that the mitochondrial mutator phenotype of an ogg1 strain is markedly reduced by the presence of a plasmid carrying an additional copy of the gene encoding Msh1p. In accordance with this finding, we show in the present work that a surplus of Msh1p abolishes also the mitochondrial mutator phenotype of a sod2 mutant, which is exposed to a permanent endogenous oxidative stress [44,45]. Moreover, we show that a sod2 msh1-R813W double mutant displays a synergistic effect on the frequency of mitochondrial Olir mutants. Thus, the msh1-R813W mutation, which was previously shown to cause the dominant mitochondrial mutator phenotype, consistent with the role of Msh1p in mitochondrial MMR [26], makes mtDNA exceptionally prone to the mutagenic effects of oxidative damage. These findings imply that Msh1p plays an important role in the prevention of mitochondrial mutagenesis induced by endogenous ROS. Interestingly, however, in contrast to mitochondrial mutagenesis estimated by the frequency of Olir mutants, elevated level of respiration-deficient petite mutants, having gross rearrangements in mitochondrial genome, is not further increased in the msh1R813W mutant by the sod2 mutation. Thus, msh1-R813W cells, that are competent for mtBER, can effectively repair oxidative lesions which lead to rearrangements of mtDNA, but are much less efficient in the prevention of oxidative lesion-induced point mutagenesis. In the present study, we attempted also to determine the genetic relationship between the roles of the Msh1p-dependent pathway and mitochondrial BER in maintaining the stability of the mitochondrial genome. The phenotype analysis of double mutants carrying the msh1-R813W allele, combined with either ogg1 or apn1, has revealed that the msh1-R813W ogg1 and msh1-R813W apn1 double mutant strains exhibit a synergistic effect on the frequency of respiration-deficient petite mutants. We show that these mutants exhibit gross rearrangements in mitochondrial genomes. These results suggest that Msh1p, the Ogg1 glycosylase and the Apn1 endonuclease fulfill overlapping functions in processing mtDNA lesions which potentially lead to rearrangements of the mitochondrial genome. What is the mechanism by which Msh1p counteracts rearrangements of mtDNA? Oxidative lesions which cause stalling of replication fork progression, such as thymine glycol, FapyG (2,6-diamino-4-hydroxy-5-formamidopyrimidine) and abasic

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sites, often produced by ROS, possibly belong to this kind of lesions [52,53]. If the replication-blocking lesions are not removed by BER, mtDNA regions at which the replication machinery is arrested could be particularly susceptible to genome rearrangements. For instance, prolonged blocking of mtDNA replication might lead to dissociation of DNA polymerase ␥ from the template, thus generating free DNA ends. Alternatively, blocking DNA polymerase ␥ might lead to uncoupling of DNA unwinding and polymerization and to generation of long single-stranded regions of mtDNA exposed to nucleases. Furthermore, when a replication fork encounters a single strand break, often produced by ROS, this results in a fork collapse which leads to the generation of a double-strand break (DSB). The efficient repair of DSBs is necessary to prevent rearrangements of the mitochondrial genome. Homologous recombination is known to be a major pathway for the repair of DSBs in eukaryotes [54]. Since recombination of mtDNA in S. cerevisiae is a well documented process [55], it is reasonable to assume that it may play an important role in the repair of mtDNA lesions such as DSBs and other oxidation-induced replication fork impediments. This assumption is supported by the finding of Shibata and collaborators showing that a mutant partially deficient in the homologous pairing activity of the Mhr1 protein accumulates at a non-permissive temperature more oxidative lesions in mtDNA than the wild-type strain [15]. It has been proposed that Mhr1p catalyses the heteroduplex joint formation between the single-stranded regions derived from DSB and intact closed circular double-stranded DNA [56]. A major finding which emerged from this study is that a surplus of Msh1p enhances homologous recombination of mtDNA, as determined by the ARG8m assay. Interestingly, such enhanced recombination is not observed when mutant forms of Msh1p, msh1p-R813W or msh1p-G776D, altered in the ATP-binding domain of the protein, are overproduced. This suggests that ATP binding and/or hydrolysis may be essential for Msh1p to stimulate homologous recombination of mtDNA. This result leads to the question about the mechanism underlying the stimulation of mtDNA recombination by Msh1p. The mechanism might be similar to that used during meiosis by other MutS homologues, hMsh4 and hMsh5 [57,58]. The complex of hMsh4 and hMsh5 facilitates recombination between homologous chromosomes during meiosis [57]. It has been shown that purified hMsh4-hMsh5 heterodimers bind uniquely to Holliday junctions [58]. This binding stimulates the Msh4-Msh5 ATP hydrolysis (ATPase) activity and leads to the formation of a sliding clamp that subsequently dissociates from the Holliday junction region. On the other hand, one should also consider the possibility that the mitochondrial recombination may be enhanced by an excess of Msh1p due to an indirect effect. Although our results show that Msh1p is not endowed with the DNA nicking activity at 8-oxoG sites, it is possible that Msh1p may stimulate the activity of an unidentified endonuclease and indirectly induce recombination. It is also possible that a surplus of Msh1p may interfere with replication of mtDNA. Further work is needed to support the hypothesis that Msh1p acts directly to promote mitochondrial recombination. Our finding that Msh1p, but not msh1p-R813W or msh1pG776D, stimulates homologous recombination of mtDNA seems to be important in light of previous reports suggesting a role of Msh1p in mitochondrial recombination. Firstly, it has been shown that msh1 strains display a rapid loss of the mitochondrial genome [19]. Secondly, the putative role of Msh1p in mitochondrial recombination was also based on the observation that both msh1-R813W and msh1-G776D mutations lead to a large increase in repeat-mediated deletion formation in mtDNA [23]. However, the mechanism underlying the deletion formation at short repeats in mtDNA has not been identified and non-reciprocal pathways such as single-strand annealing (SSA) or polymerase slippage

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may be involved. It merits noting that another MutS homologue, Msh6p, was shown to participate in preventing recombination between divergent DNA sequences in a single-strand annealing assay [59]. This finding is consistent with the well documented role of MutS homologues in suppression of recombination between homeologous substrates (heteroduplex rejection) [60]. It is worth mentioning here that an Arabidopsis thaliana MutS homologue, MSH1, has been shown to control the stability of the mitochondrial genome by a mechanism involving suppression of homeologous recombination between short repetitive sequences [61]. Our findings that, on the one hand, the msh1-R813W allele confers a significant instability of mtDNA, which is enhanced by inactivation of Ogg1p or Apn1p, and, on the other hand, that a surplus of Msh1p, but not of msh1p-R813W or msh1p-G776D, stimulates homologous recombination of mtDNA suggests that the Msh1p-stimulated recombination may be used as an important mechanism counteracting deleterious effects of endogenous ROS on the stability of mtDNA. In particular, Msh1p might function in the repair of those oxidative mtDNA lesions that can be processed to DSBs. Whether Msh1p functions in the same recombination pathway as other proteins that have previously been implicated in homologous recombination in mitochondria, Pif1p [18], Mhr1p [16,62], Cce1p [63] and Din7p [64,65], remains to be established. Our previous data and the results presented in this study show that Msh1p counteracts also oxidative lesion-induced mitochondrial point mutagenesis. This raises the question about the mechanism underlying this suppression. A significant fraction of Olir mutants that are produced in an ogg1 mutant are due to G:C to T:A transversions in the mitochondrial OLI1 gene [10]. These transversions are frequently caused by misinsertion of adenine opposite 8-oxoG, an abundant and highly mutagenic oxidatively modified base [8]. However, although 8-oxoG is a kinetic barrier to DNA synthesis, e.g. catalysed by Escherichia coli DNA polymerase I Klenow fragment, the efficiency of translesion synthesis across 8oxoG is much better than that across FapyG or AP site [53]. Thus, the question arises whether a moderate inhibitory effect of 8-oxoG on fork progression may induce an Msh1p-stimulated recombination repair. Theoretically, this could occur if a temporary idling of DNA polymerase ␥ at 8-oxoG sites might lead to its dissociation from the template, thus generating free recombinogenic DNA ends. It is also possible that unrepaired 8-oxoG lesions might be removed during the Msh1p-stimulated recombination acting on adjacent to 8-oxoG oxidation-induced lesions such as DSB. In other words, these 8oxoG lesions in OLI1 or OLI2 may be removed as a by-product of rounds of the Msh1p-enhanced recombination repair of DSBs. On the other hand, several lines of evidence suggest that Msh1p may be a component of the mitochondrial mismatch repair mechanism (mtMMR) [19,21,26]. It has been suggested that in the nucleus postreplication mismatch repair (MMR), in the reaction requiring the Msh2-Msh6 complex, could be a functional analogue of MutY that removes the misincorporated adenine [8,66]. Thus, it is reasonable to assume that an Msh1p-mediated mismatch repair might fulfill a similar role in the mitochondrial compartment and to prevent mitochondrial point mutagenesis induced by oxidative stress. Altogether, previously published data and the results presented in this study indicate that Msh1p plays multiple roles in maintaining the stability of the yeast mitochondrial genome. In addition to its previously postulated involvement in mitochondrial mismatch repair [19,21,26], suppression of short repeat-mediated deletion formation, and a role in mtDNA transmission [23], our data uncover a novel function of Msh1p and show that it is able to stimulate homologous recombination of mtDNA. We postulate that the latter function of Msh1p may be crucial in the repair of those oxidative lesions which, if left unrepaired, lead to rearrangements of mtDNA. On the other hand, mismatch repair activity of Msh1p may play an

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important role in the prevention of oxidative lesion-induced point mutagenesis. Conflict of interest statement None declared. Acknowledgements The authors thank S. Boiteux, C. Boone, F. Cross, A. Dziembowski, F. Fabre, T.D. Fox and E.A. Sia for the strains and plasmids they provided. The authors thank M. Swoboda for her help in performing DNA nicking assays and B. Tudek for supplying the purified Fpg protein. We are very grateful to two anonymous reviewers for their helpful comments and advice. This work was supported by Grants No. N303 050 32/1807 and No. N301 111 32/3905 from the Polish Ministry of Science. References [1] C. Richter, J.W. Park, B.N. Ames, Normal oxidative damage to mitochondrial and nuclear DNA is extensive, Proc. Natl. Acad. Sci. U.S.A. 85 (1988) 6465–6467. [2] E.K. Hudson, B.A. Hogue, N.C. Souza-Pinto, D.L. Croteau, R.M. Anson, V.A. Bohr, R.G. Hansford, Age-associated change in mitochondrial DNA damage, Free Radic. Res. 29 (1998) 573–579. [3] V.A. Bohr, Repair of oxidative DNA damage in nuclear and mitochondrial DNA, and some changes with aging in mammalian cells, Free Radic. Biol. Med. 32 (2002) 804–812. [4] B.S. Mandavilli, J.H. Santos, B. Van Houten, Mitochondrial DNA repair and aging, Mutat. Res. 509 (2002) 127–151. [5] J.A. Stuart, M.F. Brown, Mitochondrial DNA maintenance and bioenergetics, Biochim. Biophys. Acta 1757 (2006) 79–89. [6] D.C. Wallace, A mitochondrial paradigm of metabolic and degenerative diseases, aging, and cancer: a dawn for evolutionary medicine, Annu. Rev. Genet. 39 (2005) 359–407. [7] F. Foury, J. Hu, S. Vanderstraeten, Mitochondrial DNA mutators, Cell. Mol. Life Sci. 61 (2004) 2799–2811. [8] S. Boiteux, L. Gellon, N. Guibourt, Repair of 8-oxoguanine in Saccharomyces cerevisiae: interplay of DNA repair and replication mechanisms, Free Radic. Biol. Med. 32 (2002) 1244–1253. [9] K.K. Singh, B. Sigala, H.A. Sikder, C. Schwimmer, Inactivation of Saccharomyces cerevisiae OGG1 DNA repair gene leads to an increased frequency of mitochondrial mutants, Nucleic Acids Res. 29 (2001) 1381–1388. [10] P. Dzierzbicki, P. Koprowski, M.U. Fikus, E. Malc, Z. Ciesla, Repair of oxidative damage in mitochondrial DNA of Saccharomyces cerevisiae: involvement of the MSH1-dependent pathway, DNA Repair (Amst.) 3 (2004) 403–411. [11] L. Eide, M. Bjoras, M. Pirovano, I. Alseth, K.G. Berdal, E. Seeberg, Base excision of oxidative purine and pyrimidine DNA damage in Saccharomyces cerevisiae by a DNA glycosylase with sequence similarity to endonuclease III from Escherichia coli, Proc. Natl. Acad. Sci. U.S.A 93 (1996) 10735–10740. [12] H.J. You, R.L. Swanson, P.W. Doetsch, Saccharomyces cerevisiae possesses two functional homologues of Escherichia coli endonuclease III, Biochemistry 37 (1998) 6033–6040. [13] N.A. Doudican, B. Song, G.S. Shadel, P.W. Doetsch, Oxidative DNA damage causes mitochondrial genomic instability in Saccharomyces cerevisiae, Mol. Cell. Biol. 25 (2005) 5196–5204. [14] R. Vongsamphanh, P.K. Fortier, D. Ramotar, Pir1p mediates translocation of the yeast Apn1p endonuclease into the mitochondria to maintain genomic stability, Mol. Cell. Biol. 21 (2001) 1647–1655. [15] F. Ling, H. Morioka, E. Ohtsuka, T. Shibata, A role for MHR1, a gene required for mitochondrial genetic recombination, in the repair of damage spontaneously introduced in yeast mtDNA, Nucleic Acids Res. 28 (2000) 4956–4963. [16] F. Ling, T. Shibata, Recombination-dependent mtDNA partitioning: in vivo role of Mhr1p to promote pairing of homologous DNA, EMBO J. 21 (2002) 4730–4740. [17] T.W. O’Rourke, N.A. Doudican, M.D. Mackereth, P.W. Doetsch, G.S. Shadel, Mitochondrial dysfunction due to oxidative mitochondrial DNA damage is reduced through cooperative actions of diverse proteins, Mol. Cell. Biol. 22 (2002) 4086–4093. [18] A. Lahaye, H. Stahl, D. Thines-Sempoux, F. Foury, PIF1: a DNA helicase in yeast mitochondria, EMBO J. 10 (1991) 997–1007. [19] R.A. Reenan, R.D. Kolodner, Characterization of insertion mutations in the Saccharomyces cerevisiae MSH1 and MSH2 genes: evidence for separate mitochondrial and nuclear functions, Genetics 132 (1992) 975–985. [20] N.W. Chi, R.D. Kolodner, Purification and characterization of MSH1, a yeast mitochondrial protein that binds to DNA mismatches, J. Biol. Chem. 269 (1994) 29984–29992.

[21] S. Vanderstraeten, S. Van den Brule, J. Hu, F. Foury, The role of 3’-5’ exonucleolytic proofreading and mismatch repair in yeast mitochondrial DNA error avoidance, J. Biol. Chem. 273 (1998) 23690–23697. [22] S.A. Mookerjee, H.D. Lyon, E.A. Sia, Analysis of the functional domains of the mismatch repair homologue Msh1p and its role in mitochondrial genome maintenance, Curr. Genet. 47 (2005) 84–99. [23] S.A. Mookerjee, E.A. Sia, Overlapping contributions of Msh1p and putative recombination proteins Cce1p, Din7p, and Mhr1p in large-scale recombination and genome sorting events in the mitochondrial genome of Saccharomyces cerevisiae, Mutat. Res. 595 (2006) 91–106. [24] G. Giaever, et al., Functional profiling of the Saccharomyces cerevisiae genome, Nature 418 (2002) 387–391. [25] E.A. Winzeler, et al., Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis, Science 285 (1999) 901–906. [26] P. Koprowski, M.U. Fikus, P. Mieczkowski, Z. Ciesla, A dominant mitochondrial mutator phenotype of Saccharomyces cerevisiae conferred by msh1 alleles altered in the sequence encoding the ATP-binding domain, Mol. Genet. Genomics 266 (2002) 988–994. [27] D. Thomas, A.D. Scot, R. Barbey, M. Padula, S. Boiteux, Inactivation of OGG1 increases the incidence of G. C– > T. A transversions in Saccharomyces cerevisiae: evidence for endogenous oxidative damage to DNA in eukaryotic cells, Mol. Gen. Genet. 254 (1997) 171–178. [28] A.H. Tong, et al., Systematic genetic analysis with ordered arrays of yeast deletion mutants, Science 294 (2001) 2364–2368. [29] N. Bonnefoy, T.D. Fox, In vivo analysis of mutated initiation codons in the mitochondrial COX2 gene of Saccharomyces cerevisiae fused to the reporter gene ARG8m reveals lack of downstream reinitiation, Mol. Gen. Genet. 262 (2000) 1036–1046. [30] D.F. Steele, C.A. Butler, T.D. Fox, Expression of a recoded nuclear gene inserted into yeast mitochondrial DNA is limited by mRNA-specific translational activation, Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 5253–5257. [31] T.D. Fox, L.S. Folley, J.J. Mulero, T.W. McMullin, P.E. Thorsness, L.O. Hedin, M.C. Costanzo, Analysis and manipulation of yeast mitochondrial genes, Methods Enzymol. 194 (1991) 149–165. [32] F.R. Cross, ‘Marker swap’ plasmids: convenient tools for budding yeast molecular genetics, Yeast 13 (1997) 647–653. [33] O. Puig, F. Caspary, G. Rigaut, B. Rutz, E. Bouveret, E. Bragado-Nilsson, M. Wilm, B. Seraphin, The tandem affinity purification (TAP) method: a general procedure of protein complex purification, Methods 24 (2001) 218–229. [34] M.K. Strand, W.C. Copeland, Measuring mtDNA mutation rates in Saccharomyces cerevisiae using the mtArg8 assay, Methods Mol. Biol. 197 (2002) 151– 157. [35] M. Duchniewicz, A. Germaniuk, B. Westermann, W. Neupert, E. Schwarz, J. Marszalek, Dual role of the mitochondrial chaperone Mdj1p in inheritance of mitochondrial DNA in yeast, Mol. Cell. Biol. 19 (1999) 8201–8210. [36] S. Maynard, et al., Human embryonic stem cells have enhanced repair of multiple forms of DNA damage, Stem Cells 26 (2008) 2266–2274. [37] M. Ogur, R. St. John, S. Nagai, Tetrazolium overlay technique for population studies of respiration deficiency in yeast, Science 125 (1957) 928–929. [38] P.N. Lipke, C. Hull-Pillsbury, Flocculation of Saccharomyces cerevisiae tup1 mutants, J. Bacteriol. 159 (1984) 797–799. [39] C. Styles, How to set up a yeast laboratory, Methods Enzymol. 350 (2002) 42– 71. [40] E.A. Sia, C.A. Butler, M. Dominska, P. Greenwell, T.D. Fox, T.D. Petes, Analysis of microsatellite mutations in the mitochondrial DNA of Saccharomyces cerevisiae, Proc. Natl. Acad. Sci. U.S.A. 97 (2000) 250–255. [41] M. Knop, K. Siegers, G. Pereira, W. Zachariae, B. Winsor, K. Nasmyth, E. Schiebel, Epitope tagging of yeast genes using a PCR-based strategy: more tags and improved practical routines, Yeast 15 (1999) 963–972. [42] P. Nagley, R.M. Hall, B.G. Ooi, Amino acid substitutions in mitochondrial ATPase subunit 9 of Saccharomyces cerevisiae leading to oligomycin or venturicidin resistance, FEBS Lett. 195 (1986) 159–163. [43] U.P. John, P. Nagley, Amino acid substitutions in mitochondrial ATPase subunit 6 of Saccharomyces cerevisiae leading to oligomycin resistance, FEBS Lett. 207 (1986) 79–83. [44] I. Fridovich, Superoxide radical and superoxide dismutases, Annu. Rev. Biochem. 64 (1995) 97–112. [45] V.D. Longo, L.L. Liou, J.S. Valentine, E.B. Gralla, Mitochondrial superoxide decreases yeast survival in stationary phase, Arch. Biochem. Biophys. 365 (1999) 131–142. [46] F. Foury, S. Vanderstraeten, Yeast mitochondrial DNA mutators with deficient proofreading exonucleolytic activity, EMBO J. 11 (1992) 2717–2726. [47] G. Wang, P. Alamuri, M.Z. Humayun, D.E. Taylor, R.J. Maier, The Helicobacter pylori MutS protein confers protection from oxidative DNA damage, Mol. Microbiol. 58 (2005) 166–176. [48] H. Czeczot, B. Tudek, B. Lambert, J. Laval, S. Boiteux, Escherichia coli Fpg protein and UvrABC endonuclease repair DNA damage induced by methylene blue plus visible light in vivo and in vitro, J. Bacteriol. 173 (1991) 3419–3424. [49] N.B. Larsen, M. Rasmussen, L.J. Rasmussen, Nuclear and mitochondrial DNA repair: similar pathways? Mitochondrion 5 (2005) 89–108. [50] N. Phadnis, R. Mehta, N. Meednu, E.A. Sia, Ntg1p, the base excision repair protein, generates mutagenic intermediates in yeast mitochondrial DNA, DNA Repair (Amst.) 5 (2006) 829–839. [51] M.K. Strand, G.R. Stuart, M.J. Longley, M.A. Graziewicz, O.C. Dominick, W.C. Copeland, POS5 gene of Saccharomyces cerevisiae encodes a mitochondrial

A. Kaniak et al. / DNA Repair 8 (2009) 318–329

[52] [53]

[54]

[55]

[56] [57]

[58]

NADH kinase required for stability of mitochondrial DNA, Eukaryot. Cell 2 (2003) 809–820. S. Boiteux, M. Guillet, Abasic sites in DNA: repair and biological consequences in Saccharomyces cerevisiae, DNA Repair (Amst.) 3 (2004) 1–12. K. Asagoshi, H. Terato, Y. Ohyama, H. Ide, Effects of a guanine-derived formamidopyrimidine lesion on DNA replication: translesion DNA synthesis, nucleotide insertion, and extension kinetics, J. Biol. Chem. 277 (2002) 14589– 14597. L.S. Symington, Role of RAD52 epistasis group genes in homologous recombination and double-strand break repair, Microbiol. Mol. Biol. Rev. 66 (2002) 630–670. B. Dujon, Mitochondrial genetics and function, in: The molecular biology of the yeast Saccharomyces: life cycle and inheritance, Cold Spring Harb. Lab., N.Y., 1981, pp. 505–635. T. Shibata, F. Ling, DNA recombination protein-dependent mechanism of homoplasmy and its proposed functions, Mitochondrion 7 (2007) 17–23. N.M. Hollingsworth, L. Ponte, C. Halsey, MSH5, a novel MutS homolog, facilitates meiotic reciprocal recombination between homologs in Saccharomyces cerevisiae but not mismatch repair, Genes Dev. 9 (1995) 1728– 1739. T. Snowden, S. Acharya, C. Butz, M. Berardini, R. Fishel, hMSH4-hMSH5 recognizes Holliday Junctions and forms a meiosis-specific sliding clamp that embraces homologous chromosomes, Mol. Cell 15 (2004) 437– 451.

329

[59] T. Goldfarb, E. Alani, Distinct roles for the Saccharomyces cerevisiae mismatch repair proteins in heteroduplex rejection, mismatch repair and nonhomologous tail removal, Genetics 169 (2005) 563–574. [60] S. Stambuk, M. Radman, Mechanism and control of interspecies recombination in Escherichia coli. I. Mismatch repair, methylation, recombination and replication functions, Genetics 150 (1998) 533–542. [61] V. Shedge, M. Arrieta-Montiel, A.C. Christensen, S.A. Mackenzie, Plant mitochondrial recombination surveillance requires unusual RecA and MutS homologs, Plant Cell 19 (2007) 1251–1264. [62] F. Ling, F. Makishima, N. Morishima, T. Shibata, A nuclear mutation defective in mitochondrial recombination in yeast, EMBO J. 14 (1995) 4090–4101. [63] U.R. Ezekiel, H.P. Zassenhaus, Localization of a cruciform cutting endonuclease to yeast mitochondria, Mol. Gen. Genet. 240 (1993) 414–418. [64] M.U. Fikus, P.A. Mieczkowski, P. Koprowski, J. Rytka, E. Sledziewska-Gojska, Z. Ciesla, The product of the DNA damage-inducible gene of Saccharomyces cerevisiae, DIN7, specifically functions in mitochondria, Genetics 154 (2000) 73–81. [65] P. Koprowski, M.U. Fikus, P. Dzierzbicki, P. Mieczkowski, J. Lazowska, Z. Ciesla, Enhanced expression of the DNA damage-inducible gene DIN7 results in increased mutagenesis of mitochondrial DNA in Saccharomyces cerevisiae, Mol. Genet. Genomics 269 (2003) 632–639. [66] T.T. Ni, G.T. Marsischky, R.D. Kolodner, MSH2 and MSH6 are required for removal of adenine misincorporated opposite 8-oxo-guanine in S. cerevisiae, Mol. Cell 4 (1999) 439–444.