A role for MHR1, a gene required for mitochondrial genetic recombination, in the repair of damage spontaneously introduced in yeast mtDNA.

A nuclear recessive mutant in Saccharomyces cerevisiae, mhr1-1, is defective in mitochondrial genetic recombination at 30 degrees C and shows extensive vegetative petite induction by UV irradiation at 30 degrees C or when cultivated at a higher temperature (37 degrees C). It has been postulated that mitochondrial DNA (mtDNA) is oxidatively damaged by by-products of oxidative respiration. Since genetic recombination plays a critical role in DNA repair in various organisms, we tested the possibility that MHR1 plays a role in the repair of oxidatively damaged mtDNA using an enzyme assay. mtDNA isolated from cells grown under standard (aerobic) conditions contained a much higher level of DNA lesions compared with mtDNA isolated from anaerobically grown cells. Soon after a temperature shift from 30 to 37 degrees C the number of mtDNA lesions increased 2-fold in mhr1-1 mutant cells but not in MHR1 cells. Malonic acid, which decreased the oxidative stress in mitochondria, partially suppressed both petite induction and the temperature-induced increase in the amount of mtDNA damage in mhr1-1 cells at 37 degrees C. Thus, functional mitochondria require active MHR1, which keeps the extent of spontaneous oxidative damage in mtDNA within a tolerable level. These observations are consistent with MHR1 having a possible role in mtDNA repair.

[1]  R. Wiegand,et al.  Specificity of the S1 nuclease from Aspergillus oryzae. , 1975, The Journal of biological chemistry.

[2]  C. Smith,et al.  den V gene of bacteriophage T4 determines a DNA glycosylase specific for pyrimidine dimers in DNA , 1980, Journal of virology.

[3]  M. Eilers,et al.  Import of proteins into mitochondria. , 1988, Philosophical transactions of the Royal Society of London. Series B, Biological sciences.

[4]  E. Southern.  Detection of specific sequences among DNA fragments separated by gel electrophoresis. , 1975, Journal of molecular biology.

[5]  P. Philippsen,et al.  DNA of Saccharomyces cerevisiae. , 1991, Methods in enzymology.

[6]  B Sugden,et al.  Detection of two restriction endonuclease activities in Haemophilus parainfluenzae using analytical agarose--ethidium bromide electrophoresis. , 1973, Biochemistry.

[7]  B. Van Houten,et al.  Mitochondrial DNA damage is more extensive and persists longer than nuclear DNA damage in human cells following oxidative stress. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[8]  R. V. Skavaril,et al.  Maintenance of genetic homogeneity in systems with multiple genomes. , 1976, Genetical research.

[9]  F. Foury,et al.  pif mutation blocks recombination between mitochondrial rho+ and rho- genomes having tandemly arrayed repeat units in Saccharomyces cerevisiae. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[10]  J. Thacker,et al.  The role of homologous recombination processes in the repair of severe forms of DNA damage in mammalian cells. , 1999, Biochimie.

[11]  G. Lenaz.  Role of mitochondria in oxidative stress and ageing. , 1998, Biochimica et biophysica acta.

[12]  S. Ledoux,et al.  Repair of alkylation and oxidative damage in mitochondrial DNA. , 1999, Mutation research.

[13]  R. Lloyd,et al.  A novel DNA N-glycosylase activity of E. coli T4 endonuclease V that excises 4,6-diamino-5-formamidopyrimidine from DNA, a UV-radiation- and hydroxyl radical-induced product of adenine. , 1996, Mutation research.

[14]  Sangkot Marzuki,et al.  MITOCHONDRIAL DNA MUTATIONS AS AN IMPORTANT CONTRIBUTOR TO AGEING AND DEGENERATIVE DISEASES , 1989, The Lancet.

[15]  Y. Kwon,et al.  Chronic In Vivo Sodium Azide Infusion Induces Selective and Stable Inhibition of Cytochrome c Oxidase , 1996, Journal of neurochemistry.

[16]  T. Shibata,et al.  A nuclear mutation defective in mitochondrial recombination in yeast. , 1995, The EMBO journal.

[17]  T. Shibata,et al.  A mutation in a mitochondrial ABC transporter results in mitochondrial dysfunction through oxidative damage of mitochondrial DNA , 1999, Molecular and General Genetics MGG.

[18]  B. Ames,et al.  Endogenous oxidative DNA damage, aging, and cancer. , 1989, Free radical research communications.

[19]  D. Wallace.  Mitochondrial diseases in man and mouse. , 1999, Science.

[20]  A. Lehmann,et al.  Conservation of eukaryotic DNA repair mechanisms. , 2001, International journal of radiation biology.

[21]  M. Sekiguchi,et al.  Initial step of excision repair in Escherichia coli: replacement of defective function of uvr mutants by T4 endonuclease V. , 1972, Journal of Molecular Biology.

[22]  V. Bohr,et al.  Mitochondrial DNA Repair Pathways , 1999, Mutation research.

[23]  C. Richter,et al.  Reactive oxygen and DNA damage in mitochondria. , 1992, Mutation research.

[24]  J. Broach,et al.  The Molecular biology of the yeast saccharomyces, life cycle and inheritance , 1981 .

[25]  R. Barbey,et al.  Cloning and expression in Escherichia coli of the OGG1 gene of Saccharomyces cerevisiae, which codes for a DNA glycosylase that excises 7,8-dihydro-8-oxoguanine and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[26]  F. Foury,et al.  PIF1: a DNA helicase in yeast mitochondria. , 1991, The EMBO journal.

[27]  S. Ledoux,et al.  Repair of oxidative damage within the mitochondrial DNA of RINr 38 cells. , 1993, The Journal of biological chemistry.

[28]  E. Gajewski,et al.  Substrate specificity of the Escherichia coli Fpg protein (formamidopyrimidine-DNA glycosylase): excision of purine lesions in DNA produced by ionizing radiation or photosensitization. , 1992, Biochemistry.

[29]  W. Li,et al.  Spectroscopic characterization of the interaction of azide and thiocyanate with the binuclear center of cytochrome oxidase: evidence for multiple ligand sites. , 1993, Biochemistry.

[30]  H. You,et al.  Substrate specificities of the ntg1 and ntg2 proteins of Saccharomyces cerevisiae for oxidized DNA bases are not identical. , 1998, Nucleic acids research.

[31]  E. Ohtsuka,et al.  Affinity of single- or double-stranded oligodeoxyribonucleotides containing a thymine photodimer for T4 endonuclease V. , 1989, The Journal of biological chemistry.

[32]  B. Van Houten,et al.  Repair of DNA damage in mitochondria. , 1999, Mutation research.

[33]  B. Dujon.  Mitochondrial Genetics and Functions , 1981 .

[34]  H. Ebelt,et al.  Alloxan acts as a prooxidant only under reducing conditions: influence of melatonin , 1999, Cellular and Molecular Life Sciences CMLS.

[35]  H. Edenberg,et al.  den V gene of bacteriophage T4 codes for both pyrimidine dimer-DNA glycosylase and apyrimidinic endonuclease activities , 1981, Journal of virology.

[36]  G. Holmquist,et al.  Mapping frequencies of endogenous oxidative damage and the kinetic response to oxidative stress in a region of rat mtDNA. , 1997, Nucleic acids research.

[37]  K. Shishido,et al.  Site-specific fragmentation of bacteriophage T5 DNA by single-strand-specific S1 endonuclease. , 1975, Biochimica et biophysica acta.

[38]  R. Stierum,et al.  Homogenous repair of singlet oxygen-induced DNA damage in differentially transcribed regions and strands of human mitochondrial DNA. , 1998, Nucleic acids research.

[39]  M. Cox,et al.  Recombinational DNA repair in bacteria and the RecA protein. , 1999, Progress in nucleic acid research and molecular biology.

[40]  J. Miller,et al.  Evidence that MutY and MutM combine to prevent mutations by an oxidatively damaged form of guanine in DNA. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[41]  B. Ames,et al.  Oxidants, antioxidants, and the degenerative diseases of aging. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[42]  T. Ozawa,et al.  Age-associated oxygen damage and mutations in mitochondrial DNA in human hearts. , 1992, Biochemical and biophysical research communications.

[43]  M. Dizdaroglu,et al.  Novel substrates of Escherichia coli nth protein and its kinetics for excision of modified bases from DNA damaged by free radicals. , 2000, Biochemistry.

[44]  G. Daum,et al.  Import of proteins into mitochondria. Cytochrome b2 and cytochrome c peroxidase are located in the intermembrane space of yeast mitochondria. , 1982, The Journal of biological chemistry.

[45]  Y. Nakabeppu,et al.  Physical association of pyrimidine dimer DNA glycosylase and apurinic/apyrimidinic DNA endonuclease essential for repair of ultraviolet-damaged DNA. , 1981, Proceedings of the National Academy of Sciences of the United States of America.