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1
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King DE, Copeland WC. DNA repair pathways in the mitochondria. DNA Repair (Amst) 2025; 146:103814. [PMID: 39914164 PMCID: PMC11848857 DOI: 10.1016/j.dnarep.2025.103814] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2024] [Revised: 01/14/2025] [Accepted: 01/28/2025] [Indexed: 02/24/2025]
Abstract
Mitochondria contain their own small, circular genome that is present in high copy number. The mitochondrial genome (mtDNA) encodes essential subunits of the electron transport chain. Mutations in the mitochondrial genome are associated with a wide range of mitochondrial diseases and the maintenance and replication of mtDNA is crucial to cellular health. Despite the importance of maintaining mtDNA genomic integrity, fewer DNA repair pathways exist in the mitochondria than in the nucleus. However, mitochondria have numerous pathways that allow for the removal and degradation of DNA damage that may prevent accumulation of mutations. Here, we briefly review the DNA repair pathways present in the mitochondria, sources of mtDNA mutations, and discuss the passive role that mtDNA mutagenesis may play in cancer progression.
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Affiliation(s)
- Dillon E King
- Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, United States
| | - William C Copeland
- Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, United States.
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2
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Pigula ML, Ban Y, Schultz PG. Toward a Quadruplet Codon Mitochondrial Genetic Code. ACS Synth Biol 2024; 13:4175-4179. [PMID: 39631441 PMCID: PMC11792677 DOI: 10.1021/acssynbio.4c00630] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/07/2024]
Abstract
Nature has evolved to exclusively use a genetic code consisting of triplet nucleotide codons. The translation system, however, is known to be compatible with 4-nucleotide frameshift or quadruplet codons. In this study, we begin to explore the possibility of a genome made up entirely of quadruplet codons using the minimal mitochondrial genome of Saccharomyces cerevisiae as a model system. We demonstrate that mitochondrial tryptophanyl- and tyrosyl-tRNAs with modified anticodons effectively suppress mutant cox3 genes containing a TAG stop or TAGA quadruplet codon, leading to the production of full-length COX3 and a respiratory-competent phenotype. This work provides a method for introducing heterologous tRNAs into the yeast mitochondria for genetic engineering applications and serves as a starting point for the development of a quadruplet codon genetic code.
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Affiliation(s)
| | | | - Peter G. Schultz
- Department of Chemistry, Scripps Research, 10550 North Torrey Pines Rd, La Jolla, CA 92037, USA
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3
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Veloso Ribeiro Franco L, Barros MH. Biolistic transformation of the yeast Saccharomyces cerevisiae mitochondrial DNA. IUBMB Life 2023; 75:972-982. [PMID: 37470229 DOI: 10.1002/iub.2769] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2023] [Accepted: 06/23/2023] [Indexed: 07/21/2023]
Abstract
The insertion of genes into mitochondria by biolistic transformation is currently only possible in the yeast Saccharomyces cerevisiae and the algae Chlamydomonas reinhardtii. The fact that S. cerevisiae mitochondria can exist with partial (ρ- mutants) or complete deletions (ρ0 mutants) of mitochondrial DNA (mtDNA), without requiring a specific origin of replication, enables the propagation of exogenous sequences. Additionally, mtDNA in this organism undergoes efficient homologous recombination, making it well-suited for genetic manipulation. In this review, we present a summarized historical overview of the development of biolistic transformation and discuss iconic applications of the technique. We also provide a detailed example on how to obtain transformants with recombined foreign DNA in their mitochondrial genome.
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Affiliation(s)
| | - Mario H Barros
- Department of Microbiology, Institute of Biomedical Sciences, Universidade de Sao Paulo, Sao Paulo, Brazil
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4
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Bei L, He C, Liu J, Han C, Zhou H, Zhaorigetu, Siqintuya, Li J, Su X, Wang Y, Chen Q, Nashun, Daolema, Meng H. Genome-wide identification and characterization of microsatellite markers in Bactrian Camel. Genomics 2023; 115:110726. [PMID: 37832857 DOI: 10.1016/j.ygeno.2023.110726] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2023] [Revised: 10/05/2023] [Accepted: 10/08/2023] [Indexed: 10/15/2023]
Abstract
Simple sequence repeats (SSRs) have been widely used for parentage testing, marker-assisted selection, and evolutionary studies. The insufficient availability of SSR markers in Bactrian camels partially accounts for the lack of systematic breeding. Therefore, we aimed to establish a comprehensive SSR dataset for the Bactrian camel. Our approach involved genome searching to locate every SSR in the genome, SSR-enriched sequencing to acquire polymorphism information, and literature research to collect published data. The resulting dataset contains 213,711 SSRs and details their characteristics, including genome coordinates, motifs, lengths, annotations, PCR primers, and polymorphism information. The dataset reveals a biased distribution of SSRs in the Bactrian camel genome, reflecting the mutation mechanism and complex evolution of SSRs. In practice, we successfully demonstrated the utility of the dataset through parentage testing using 15 randomly selected SSRs. This comprehensive dataset can facilitate systematic breeding and enable QTL mapping and GWAS of the Bactrian camel.
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Affiliation(s)
- Lanxin Bei
- Shanghai Key Laboratory of Veterinary Biotechnology, School of Agriculture and Biology, Shanghai Jiao Tong University, 200240 Shanghai, China
| | - Chuan He
- Shanghai Key Laboratory of Veterinary Biotechnology, School of Agriculture and Biology, Shanghai Jiao Tong University, 200240 Shanghai, China
| | - Jiajia Liu
- Shanghai Key Laboratory of Veterinary Biotechnology, School of Agriculture and Biology, Shanghai Jiao Tong University, 200240 Shanghai, China
| | - Chengxiao Han
- Shanghai Key Laboratory of Veterinary Biotechnology, School of Agriculture and Biology, Shanghai Jiao Tong University, 200240 Shanghai, China
| | - Hao Zhou
- Shanghai Key Laboratory of Veterinary Biotechnology, School of Agriculture and Biology, Shanghai Jiao Tong University, 200240 Shanghai, China
| | - Zhaorigetu
- Animal Husbandry Institute of Alxa League, 750306, Inner Mongolia, China
| | - Siqintuya
- Animal Husbandry Institute of Alxa League, 750306, Inner Mongolia, China
| | - Jing Li
- Animal Husbandry Institute of Alxa League, 750306, Inner Mongolia, China
| | - Xue Su
- Animal Husbandry Institute of Alxa League, 750306, Inner Mongolia, China
| | - Yunfei Wang
- Bayannur Institute of Agriculture & Animal Husbandry Science, 015000, Inner Mongolia, China
| | - Qiujv Chen
- Bayannur Institute of Agriculture & Animal Husbandry Science, 015000, Inner Mongolia, China
| | - Nashun
- Alxa Left Banner Agriculture and Animal Husbandry Comprehensive Administrative Law Enforcement Brigade, 735499, Inner Mongolia, China
| | - Daolema
- Animal Husbandry Institute of Alxa League, 750306, Inner Mongolia, China.
| | - He Meng
- Shanghai Key Laboratory of Veterinary Biotechnology, School of Agriculture and Biology, Shanghai Jiao Tong University, 200240 Shanghai, China.
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5
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Melde RH, Bao K, Sharp NP. Recent insights into the evolution of mutation rates in yeast. Curr Opin Genet Dev 2022; 76:101953. [PMID: 35834945 PMCID: PMC9491374 DOI: 10.1016/j.gde.2022.101953] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/24/2022] [Revised: 05/25/2022] [Accepted: 06/13/2022] [Indexed: 02/08/2023]
Abstract
Mutation is the origin of all genetic variation, good and bad. The mutation process can evolve in response to mutations, positive or negative selection, and genetic drift, but how these forces contribute to mutation-rate variation is an unsolved problem at the heart of genetics research. Mutations can be challenging to measure, but genome sequencing and other tools have allowed for the collection of larger and more detailed datasets, particularly in the yeast-model system. We review key hypotheses for the evolution of mutation rates and describe recent advances in understanding variation in mutational properties within and among yeast species. The multidimensional spectrum of mutations is increasingly recognized as holding valuable clues about how this important process evolves.
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Affiliation(s)
- Robert H Melde
- Department of Genetics, University of Wisconsin-Madison, USA.
| | - Kevin Bao
- Department of Genetics, University of Wisconsin-Madison, USA
| | - Nathaniel P Sharp
- Department of Genetics, University of Wisconsin-Madison, USA. https://twitter.com/@sharpnath
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6
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DNA Repair in Haploid Context. Int J Mol Sci 2021; 22:ijms222212418. [PMID: 34830299 PMCID: PMC8620282 DOI: 10.3390/ijms222212418] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2021] [Revised: 11/08/2021] [Accepted: 11/14/2021] [Indexed: 12/15/2022] Open
Abstract
DNA repair is a well-covered topic as alteration of genetic integrity underlies many pathological conditions and important transgenerational consequences. Surprisingly, the ploidy status is rarely considered although the presence of homologous chromosomes dramatically impacts the repair capacities of cells. This is especially important for the haploid gametes as they must transfer genetic information to the offspring. An understanding of the different mechanisms monitoring genetic integrity in this context is, therefore, essential as differences in repair pathways exist that differentiate the gamete’s role in transgenerational inheritance. Hence, the oocyte must have the most reliable repair capacity while sperm, produced in large numbers and from many differentiation steps, are expected to carry de novo variations. This review describes the main DNA repair pathways with a special emphasis on ploidy. Differences between Saccharomyces cerevisiae and Schizosaccharomyces pombe are especially useful to this aim as they can maintain a diploid and haploid life cycle respectively.
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7
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Mitochondrial genome stability in human: understanding the role of DNA repair pathways. Biochem J 2021; 478:1179-1197. [DOI: 10.1042/bcj20200920] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2020] [Revised: 02/15/2021] [Accepted: 02/17/2021] [Indexed: 11/17/2022]
Abstract
Mitochondria are semiautonomous organelles in eukaryotic cells and possess their own genome that replicates independently. Mitochondria play a major role in oxidative phosphorylation due to which its genome is frequently exposed to oxidative stress. Factors including ionizing radiation, radiomimetic drugs and replication fork stalling can also result in different types of mutations in mitochondrial DNA (mtDNA) leading to genome fragility. Mitochondria from myopathies, dystonia, cancer patient samples show frequent mtDNA mutations such as point mutations, insertions and large-scale deletions that could account for mitochondria-associated disease pathogenesis. The mechanism by which such mutations arise following exposure to various DNA-damaging agents is not well understood. One of the well-studied repair pathways in mitochondria is base excision repair. Other repair pathways such as mismatch repair, homologous recombination and microhomology-mediated end joining have also been reported. Interestingly, nucleotide excision repair and classical nonhomologous DNA end joining are not detected in mitochondria. In this review, we summarize the potential causes of mitochondrial genome fragility, their implications as well as various DNA repair pathways that operate in mitochondria.
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8
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Du L, Liu Q, Zhao K, Tang J, Zhang X, Yue B, Fan Z. PSMD: An extensive database for pan-species microsatellite investigation and marker development. Mol Ecol Resour 2019; 20:283-291. [PMID: 31599098 DOI: 10.1111/1755-0998.13098] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2019] [Revised: 09/18/2019] [Accepted: 09/24/2019] [Indexed: 12/21/2022]
Abstract
Microsatellites are widely distributed throughout nearly all genomes which have been extensively exploited as powerful genetic markers for diverse applications due to their high polymorphisms. Their length variations are involved in gene regulation and implicated in numerous genetic diseases even in cancers. Although much effort has been devoted in microsatellite database construction, the existing microsatellite databases still had some drawbacks, such as limited number of species, unfriendly export format, missing marker development, lack of compound microsatellites and absence of gene annotation, which seriously restricted researchers to perform downstream analysis. In order to overcome the above limitations, we developed PSMD (Pan-Species Microsatellite Database, http://big.cdu.edu.cn/psmd/) as a web-based database to facilitate researchers to easily identify microsatellites, exploit reliable molecular markers and compare microsatellite distribution pattern on genome-wide scale. In current release, PSMD comprises 678,106,741 perfect microsatellites and 43,848,943 compound microsatellites from 18,408 organisms, which covered almost all species with available genomic data. In addition to interactive browse interface, PSMD also offers a flexible filter function for users to quickly gain desired microsatellites from large data sets. PSMD allows users to export GFF3 formatted file and CSV formatted statistical file for downstream analysis. We also implemented an online tool for analysing occurrence of microsatellites with user-defined parameters. Furthermore, Primer3 was embedded to help users to design high-quality primers with customizable settings. To our knowledge, PSMD is the most extensive resource which is likely to be adopted by scientists engaged in biological, medical, environmental and agricultural research.
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Affiliation(s)
- Lianming Du
- Institute for Advanced Study, Chengdu University, Chengdu, China
| | - Qin Liu
- Key Laboratory of Bio-resources and Eco-environment, Ministry of Education, College of Life Science, Sichuan University, Chengdu, China.,College of Life Sciences and Food Engineering, Yibin University, Yibin, China
| | - Kelei Zhao
- Institute for Advanced Study, Chengdu University, Chengdu, China
| | - Jie Tang
- School of Pharmacy and Bioengineering, Chengdu University, Chengdu, China
| | - Xiuyue Zhang
- Key Laboratory of Bio-resources and Eco-environment, Ministry of Education, College of Life Science, Sichuan University, Chengdu, China
| | - Bisong Yue
- Key Laboratory of Bio-resources and Eco-environment, Ministry of Education, College of Life Science, Sichuan University, Chengdu, China
| | - Zhenxin Fan
- Key Laboratory of Bio-resources and Eco-environment, Ministry of Education, College of Life Science, Sichuan University, Chengdu, China
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9
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Abstract
The mitochondrial genome encodes proteins essential for the oxidative phosphorylation and, consequently, for proper mitochondrial function. Its localization and, possibly, structural organization contribute to higher DNA damage accumulation, when compared to the nuclear genome. In addition, the mitochondrial genome mutates at rates several times higher than the nuclear, although the causal relationship between these events are not clearly established. Maintaining mitochondrial DNA stability is critical for cellular function and organismal fitness, and several pathways contribute to that, including damage tolerance and bypass, degradation of damaged genomes and DNA repair. Despite initial evidence suggesting that mitochondria lack DNA repair activities, most DNA repair pathways have been at least partially characterized in mitochondria from several model organisms, including humans. In this chapter, we review what is currently known about how the main DNA repair pathways operate in mitochondria and contribute to mitochondrial DNA stability, with focus on the enzymology of mitochondrial DNA repair.
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Affiliation(s)
- Rebeca R Alencar
- Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil
| | - Caio M P F Batalha
- Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil
| | - Thiago S Freire
- Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil
| | - Nadja C de Souza-Pinto
- Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil.
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10
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Sharp NP, Sandell L, James CG, Otto SP. The genome-wide rate and spectrum of spontaneous mutations differ between haploid and diploid yeast. Proc Natl Acad Sci U S A 2018; 115:E5046-E5055. [PMID: 29760081 PMCID: PMC5984525 DOI: 10.1073/pnas.1801040115] [Citation(s) in RCA: 99] [Impact Index Per Article: 14.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022] Open
Abstract
By altering the dynamics of DNA replication and repair, alternative ploidy states may experience different rates and types of new mutations, leading to divergent evolutionary outcomes. We report a direct comparison of the genome-wide spectrum of spontaneous mutations arising in haploids and diploids following a mutation-accumulation experiment in the budding yeast Saccharomyces cerevisiae Characterizing the number, types, locations, and effects of thousands of mutations revealed that haploids were more prone to single-nucleotide mutations (SNMs) and mitochondrial mutations, while larger structural changes were more common in diploids. Mutations were more likely to be detrimental in diploids, even after accounting for the large impact of structural changes, contrary to the prediction that mutations would have weaker effects, due to masking, in diploids. Haploidy is expected to reduce the opportunity for conservative DNA repair involving homologous chromosomes, increasing the insertion-deletion rate, but we found little support for this idea. Instead, haploids were more susceptible to SNMs in late-replicating genomic regions, resulting in a ploidy difference in the spectrum of substitutions. In diploids, we detect mutation rate variation among chromosomes in association with centromere location, a finding that is supported by published polymorphism data. Diploids are not simply doubled haploids; instead, our results predict that the spectrum of spontaneous mutations will substantially shape the dynamics of genome evolution in haploid and diploid populations.
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Affiliation(s)
- Nathaniel P Sharp
- Department of Zoology, University of British Columbia, Vancouver, BC, Canada V6T 1Z4
| | - Linnea Sandell
- Department of Zoology, University of British Columbia, Vancouver, BC, Canada V6T 1Z4
| | - Christopher G James
- Department of Zoology, University of British Columbia, Vancouver, BC, Canada V6T 1Z4
| | - Sarah P Otto
- Department of Zoology, University of British Columbia, Vancouver, BC, Canada V6T 1Z4
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11
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Braga Goncalves I, Cornetti L, Couperus AS, van Damme CJG, Mobley KB. Phylogeography of the snake pipefish, Entelurus aequoreus (Family: Syngnathidae) in the northeastern Atlantic Ocean. Biol J Linn Soc Lond 2017. [DOI: 10.1093/biolinnean/blx112] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
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12
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Activation of Dun1 in response to nuclear DNA instability accounts for the increase in mitochondrial point mutations in Rad27/FEN1 deficient S. cerevisiae. PLoS One 2017; 12:e0180153. [PMID: 28678842 PMCID: PMC5497989 DOI: 10.1371/journal.pone.0180153] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2017] [Accepted: 06/09/2017] [Indexed: 11/25/2022] Open
Abstract
Rad27/FEN1 nuclease that plays important roles in the maintenance of DNA stability in the nucleus has recently been shown to reside in mitochondria. Accordingly, it has been established that Rad27 deficiency causes increased mutagenesis, but decreased microsatellite instability and homologous recombination in mitochondria. Our current analysis of mutations leading to erythromycin resistance indicates that only some of them arise in mitochondrial DNA and that the GC→AT transition is a hallmark of the mitochondrial mutagenesis in rad27 null background. We also show that the mitochondrial mutator phenotype resulting from Rad27 deficiency entirely depends on the DNA damage checkpoint kinase Dun1. DUN1 inactivation suppresses the mitochondrial mutator phenotype caused by Rad27 deficiency and this suppression is eliminated at least in part by subsequent deletion of SML1 encoding a repressor of ribonucleotide reductase. We conclude that Rad27 deficiency causes a mitochondrial mutator phenotype via activation of DNA damage checkpoint kinase Dun1 and that a Dun1-mediated increase of dNTP pools contributes to this phenomenon. These results point to the nuclear DNA instability as the source of mitochondrial mutagenesis. Consistently, we show that mitochondrial mutations occurring more frequently in yeast devoid of Rrm3, a DNA helicase involved in rDNA replication, are also dependent on Dun1. In addition, we have established that overproduction of Exo1, which suppresses DNA damage sensitivity and replication stress in nuclei of Rad27 deficient cells, but does not enter mitochondria, suppresses the mitochondrial mutagenesis. Exo1 overproduction restores also a great part of allelic recombination and microsatellite instability in mitochondria of Rad27 deficient cells. In contrast, the overproduction of Exo1 does not influence mitochondrial direct-repeat mediated deletions in rad27 null background, pointing to this homologous recombination pathway as the direct target of Rad27 activity in mitochondria.
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13
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14
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The influence of mitochondrial dynamics on mitochondrial genome stability. Curr Genet 2017; 64:199-214. [PMID: 28573336 DOI: 10.1007/s00294-017-0717-4] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2017] [Revised: 05/15/2017] [Accepted: 05/27/2017] [Indexed: 12/28/2022]
Abstract
Mitochondria are dynamic organelles that fuse and divide. These changes alter the number and distribution of mitochondrial structures throughout the cell in response to developmental and metabolic cues. We have demonstrated that mitochondrial fission is essential to the maintenance of mitochondrial DNA (mtDNA) under changing metabolic conditions in wild-type Saccharomyces cerevisiae. While increased loss of mtDNA integrity has been demonstrated for dnm1-∆ fission mutants after growth in a non-fermentable carbon source, we demonstrate that growth of yeast in different carbon sources affects the frequency of mtDNA loss, even when the carbon sources are fermentable. In addition, we demonstrate that the impact of fission on mtDNA maintenance during growth in different carbon sources is neither mediated by retrograde signaling nor mitophagy. Instead, we demonstrate that mitochondrial distribution and mtDNA maintenance phenotypes conferred by loss of Dnm1p are suppressed by the loss of Sod2p, the mitochondrial matrix superoxide dismutase.
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15
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Roles for the Rad27 Flap Endonuclease in Mitochondrial Mutagenesis and Double-Strand Break Repair in Saccharomyces cerevisiae. Genetics 2017; 206:843-857. [PMID: 28450457 DOI: 10.1534/genetics.116.195149] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2016] [Accepted: 04/18/2017] [Indexed: 01/07/2023] Open
Abstract
The structure-specific nuclease, Rad27p/FEN1, plays a crucial role in DNA repair and replication mechanisms in the nucleus. Genetic assays using the rad27-∆ mutant have shown altered rates of DNA recombination, microsatellite instability, and point mutation in mitochondria. In this study, we examined the role of Rad27p in mitochondrial mutagenesis and double-strand break (DSB) repair in Saccharomyces cerevisiae Our findings show that Rad27p is essential for efficient mitochondrial DSB repair by a pathway that generates deletions at a region flanked by direct repeat sequences. Mutant analysis suggests that both exonuclease and endonuclease activities of Rad27p are required for its role in mitochondrial DSB repair. In addition, we found that the nuclease activities of Rad27p are required for the prevention of mitochondrial DNA (mtDNA) point mutations, and in the generation of spontaneous mtDNA rearrangements. Overall, our findings underscore the importance of Rad27p in the maintenance of mtDNA, and demonstrate that it participates in multiple DNA repair pathways in mitochondria, unlinked to nuclear phenotypes.
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16
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Stein A, Kalifa L, Sia EA. Members of the RAD52 Epistasis Group Contribute to Mitochondrial Homologous Recombination and Double-Strand Break Repair in Saccharomyces cerevisiae. PLoS Genet 2015; 11:e1005664. [PMID: 26540255 PMCID: PMC4634946 DOI: 10.1371/journal.pgen.1005664] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2015] [Accepted: 10/22/2015] [Indexed: 11/19/2022] Open
Abstract
Mitochondria contain an independently maintained genome that encodes several proteins required for cellular respiration. Deletions in the mitochondrial genome have been identified that cause several maternally inherited diseases and are associated with certain cancers and neurological disorders. The majority of these deletions in human cells are flanked by short, repetitive sequences, suggesting that these deletions may result from recombination events. Our current understanding of the maintenance and repair of mtDNA is quite limited compared to our understanding of similar events in the nucleus. Many nuclear DNA repair proteins are now known to also localize to mitochondria, but their function and the mechanism of their action remain largely unknown. This study investigated the contribution of the nuclear double-strand break repair (DSBR) proteins Rad51p, Rad52p and Rad59p in mtDNA repair. We have determined that both Rad51p and Rad59p are localized to the matrix of the mitochondria and that Rad51p binds directly to mitochondrial DNA. In addition, a mitochondrially-targeted restriction endonuclease (mtLS-KpnI) was used to produce a unique double-strand break (DSB) in the mitochondrial genome, which allowed direct analysis of DSB repair in vivo in Saccharomyces cerevisiae. We find that loss of these three proteins significantly decreases the rate of spontaneous deletion events and the loss of Rad51p and Rad59p impairs the repair of induced mtDNA DSBs.
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Affiliation(s)
- Alexis Stein
- Department of Biology, University of Rochester, Rochester, New York, United States of America
| | - Lidza Kalifa
- Department of Biology, University of Rochester, Rochester, New York, United States of America
| | - Elaine A. Sia
- Department of Biology, University of Rochester, Rochester, New York, United States of America
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17
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Kooij PW, Aanen DK, Schiøtt M, Boomsma JJ. Evolutionarily advanced ant farmers rear polyploid fungal crops. J Evol Biol 2015; 28:1911-24. [PMID: 26265100 PMCID: PMC5014177 DOI: 10.1111/jeb.12718] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2014] [Accepted: 07/28/2015] [Indexed: 12/25/2022]
Abstract
Innovative evolutionary developments are often related to gene or genome duplications. The crop fungi of attine fungus-growing ants are suspected to have enhanced genetic variation reminiscent of polyploidy, but this has never been quantified with cytological data and genetic markers. We estimated the number of nuclei per fungal cell for 42 symbionts reared by 14 species of Panamanian fungus-growing ants. This showed that domesticated symbionts of higher attine ants are polykaryotic with 7-17 nuclei per cell, whereas nonspecialized crops of lower attines are dikaryotic similar to most free-living basidiomycete fungi. We then investigated how putative higher genetic diversity is distributed across polykaryotic mycelia, using microsatellite loci and evaluating models assuming that all nuclei are either heterogeneously haploid or homogeneously polyploid. Genetic variation in the polykaryotic symbionts of the basal higher attine genera Trachymyrmex and Sericomyrmex was only slightly enhanced, but the evolutionarily derived crop fungi of Atta and Acromyrmex leaf-cutting ants had much higher genetic variation. Our opposite ploidy models indicated that the symbionts of Trachymyrmex and Sericomyrmex are likely to be lowly and facultatively polyploid (just over two haplotypes on average), whereas Atta and Acromyrmex symbionts are highly and obligatorily polyploid (ca. 5-7 haplotypes on average). This stepwise transition appears analogous to ploidy variation in plants and fungi domesticated by humans and in fungi domesticated by termites and plants, where gene or genome duplications were typically associated with selection for higher productivity, but allopolyploid chimerism was incompatible with sexual reproduction.
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Affiliation(s)
- P W Kooij
- Centre for Social Evolution, Department of Biology, University of Copenhagen, Copenhagen, Denmark
| | - D K Aanen
- Laboratory of Genetics, Wageningen University, Wageningen, The Netherlands
| | - M Schiøtt
- Centre for Social Evolution, Department of Biology, University of Copenhagen, Copenhagen, Denmark
| | - J J Boomsma
- Centre for Social Evolution, Department of Biology, University of Copenhagen, Copenhagen, Denmark
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18
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Kaniak-Golik A, Skoneczna A. Mitochondria-nucleus network for genome stability. Free Radic Biol Med 2015; 82:73-104. [PMID: 25640729 DOI: 10.1016/j.freeradbiomed.2015.01.013] [Citation(s) in RCA: 62] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/18/2014] [Revised: 11/25/2014] [Accepted: 01/13/2015] [Indexed: 12/21/2022]
Abstract
The proper functioning of the cell depends on preserving the cellular genome. In yeast cells, a limited number of genes are located on mitochondrial DNA. Although the mechanisms underlying nuclear genome maintenance are well understood, much less is known about the mechanisms that ensure mitochondrial genome stability. Mitochondria influence the stability of the nuclear genome and vice versa. Little is known about the two-way communication and mutual influence of the nuclear and mitochondrial genomes. Although the mitochondrial genome replicates independent of the nuclear genome and is organized by a distinct set of mitochondrial nucleoid proteins, nearly all genome stability mechanisms responsible for maintaining the nuclear genome, such as mismatch repair, base excision repair, and double-strand break repair via homologous recombination or the nonhomologous end-joining pathway, also act to protect mitochondrial DNA. In addition to mitochondria-specific DNA polymerase γ, the polymerases α, η, ζ, and Rev1 have been found in this organelle. A nuclear genome instability phenotype results from a failure of various mitochondrial functions, such as an electron transport chain activity breakdown leading to a decrease in ATP production, a reduction in the mitochondrial membrane potential (ΔΨ), and a block in nucleotide and amino acid biosynthesis. The loss of ΔΨ inhibits the production of iron-sulfur prosthetic groups, which impairs the assembly of Fe-S proteins, including those that mediate DNA transactions; disturbs iron homeostasis; leads to oxidative stress; and perturbs wobble tRNA modification and ribosome assembly, thereby affecting translation and leading to proteotoxic stress. In this review, we present the current knowledge of the mechanisms that govern mitochondrial genome maintenance and demonstrate ways in which the impairment of mitochondrial function can affect nuclear genome stability.
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Affiliation(s)
- Aneta Kaniak-Golik
- Laboratory of Mutagenesis and DNA Repair, Institute of Biochemistry and Biophysics, Polish Academy of Science, 02-106 Warsaw, Poland
| | - Adrianna Skoneczna
- Laboratory of Mutagenesis and DNA Repair, Institute of Biochemistry and Biophysics, Polish Academy of Science, 02-106 Warsaw, Poland.
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Abstract
Human mitochondria harbor an essential, high copy number, 16,569 base pair, circular DNA genome that encodes 13 gene products required for electron transport and oxidative phosphorylation. Mutation of this genome can compromise cellular respiration, ultimately resulting in a variety of progressive metabolic diseases collectively known as 'mitochondrial diseases'. Mutagenesis of mtDNA and the persistence of mtDNA mutations in cells and tissues is a complex topic, involving the interplay of DNA replication, DNA damage and repair, purifying selection, organelle dynamics, mitophagy, and aging. We briefly review these general elements that affect maintenance of mtDNA, and we focus on nuclear genes encoding the mtDNA replication machinery that can perturb the genetic integrity of the mitochondrial genome.
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Affiliation(s)
- William C Copeland
- Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, Durham, NC 27709, USA.
| | - Matthew J Longley
- Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, Durham, NC 27709, USA
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20
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Muftuoglu M, Mori MP, de Souza-Pinto NC. Formation and repair of oxidative damage in the mitochondrial DNA. Mitochondrion 2014; 17:164-81. [PMID: 24704805 DOI: 10.1016/j.mito.2014.03.007] [Citation(s) in RCA: 66] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2013] [Revised: 03/18/2014] [Accepted: 03/18/2014] [Indexed: 12/13/2022]
Abstract
The mitochondrial DNA (mtDNA) encodes for only 13 polypeptides, components of 4 of the 5 oxidative phosphorylation complexes. But despite this apparently small numeric contribution, all 13 subunits are essential for the proper functioning of the oxidative phosphorylation circuit. Thus, accumulation of lesions, mutations and deletions/insertions in the mtDNA could have severe functional consequences, including mitochondrial diseases, aging and age-related diseases. The DNA is a chemically unstable molecule, which can be easily oxidized, alkylated, deaminated and suffer other types of chemical modifications, throughout evolution the organisms that survived were those who developed efficient DNA repair processes. In the last two decades, it has become clear that mitochondria have DNA repair pathways, which operate, at least for some types of lesions, as efficiently as the nuclear DNA repair pathways. The mtDNA is localized in a particularly oxidizing environment, making it prone to accumulate oxidatively generated DNA modifications (ODMs). In this article, we: i) review the major types of ODMs formed in mtDNA and the known repair pathways that remove them; ii) discuss the possible involvement of other repair pathways, just recently characterized in mitochondria, in the repair of these modifications; and iii) address the role of DNA repair in mitochondrial function and a possible cross-talk with other pathways that may potentially participate in mitochondrial genomic stability, such as mitochondrial dynamics and nuclear-mitochondrial signaling. Oxidative stress and ODMs have been increasingly implicated in disease and aging, and thus we discuss how variations in DNA repair efficiency may contribute to the etiology of such conditions or even modulate their clinical outcomes.
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Affiliation(s)
- Meltem Muftuoglu
- Department of Molecular Biology and Genetics, Acibadem University, Atasehir, 34752 Istanbul, Turkey
| | - Mateus P Mori
- Depto. de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, 05508-000 Brazil
| | - Nadja C de Souza-Pinto
- Depto. de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, 05508-000 Brazil.
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21
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Cross-species amplification and characterization of microsatellite loci in Pinus mugo Turra. Biologia (Bratisl) 2013. [DOI: 10.2478/s11756-013-0189-z] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
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22
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Characterization and evolutionary analysis of Brassica species-diverged sequences containing simple repeat units. Genes Genomics 2013. [DOI: 10.1007/s13258-013-0076-6] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
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23
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Cheung AWY, Brosnan JM, Phister T, Smart KA. Impact of dried, creamed and cake supply formats on the genetic variation and ethanol tolerance of three Saccharomyces cerevisiae distilling strains. JOURNAL OF THE INSTITUTE OF BREWING 2012. [DOI: 10.1002/jib.23] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/10/2022]
Affiliation(s)
- Annie W. Y. Cheung
- Bioenergy and Brewing Science, School of Biosciences; University of Nottingham; Sutton Bonington Campus; Loughborough; Leics; LE12 5RD; UK
| | - James M. Brosnan
- The Scotch Whisky Research Institute; The Robertson Trust Building, Research Avenue North, Riccarton; Edinburgh; EH14 4AP; UK
| | - Trevor Phister
- Bioenergy and Brewing Science, School of Biosciences; University of Nottingham; Sutton Bonington Campus; Loughborough; Leics; LE12 5RD; UK
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24
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Kalifa L, Quintana DF, Schiraldi LK, Phadnis N, Coles GL, Sia RA, Sia EA. Mitochondrial genome maintenance: roles for nuclear nonhomologous end-joining proteins in Saccharomyces cerevisiae. Genetics 2012; 190:951-64. [PMID: 22214610 PMCID: PMC3296257 DOI: 10.1534/genetics.111.138214] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2011] [Accepted: 12/31/2011] [Indexed: 12/27/2022] Open
Abstract
Mitochondrial DNA (mtDNA) deletions are associated with sporadic and inherited diseases and age-associated neurodegenerative disorders. Approximately 85% of mtDNA deletions identified in humans are flanked by short directly repeated sequences; however, mechanisms by which these deletions arise are unknown. A limitation in deciphering these mechanisms is the essential nature of the mitochondrial genome in most living cells. One exception is budding yeast, which are facultative anaerobes and one of the few organisms for which directed mtDNA manipulation is possible. Using this model system, we have developed a system to simultaneously monitor spontaneous direct-repeat-mediated deletions (DRMDs) in the nuclear and mitochondrial genomes. In addition, the mitochondrial DRMD reporter contains a unique KpnI restriction endonuclease recognition site that is not present in otherwise wild-type (WT) mtDNA. We have expressed KpnI fused to a mitochondrial localization signal to induce a specific mitochondrial double-strand break (mtDSB). Here we report that loss of the MRX (Mre11p, Rad50p, Xrs2p) and Ku70/80 (Ku70p, Ku80p) complexes significantly impacts the rate of spontaneous deletion events in mtDNA, and these proteins contribute to the repair of induced mtDSBs. Furthermore, our data support homologous recombination (HR) as the predominant pathway by which mtDNA deletions arise in yeast, and suggest that the MRX and Ku70/80 complexes are partially redundant in mitochondria.
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Affiliation(s)
- Lidza Kalifa
- Department of Biology, University of Rochester, Rochester, New York 14627
| | - Daniel F. Quintana
- Department of Biology, University of Rochester, Rochester, New York 14627
| | - Laura K. Schiraldi
- Department of Biology, University of Rochester, Rochester, New York 14627
- Department of Biology, The College at Brockport, State University of New York, Brockport, New York 14420
| | - Naina Phadnis
- Department of Biology, University of Rochester, Rochester, New York 14627
| | - Garry L. Coles
- Department of Biology, The College at Brockport, State University of New York, Brockport, New York 14420
| | - Rey A. Sia
- Department of Biology, The College at Brockport, State University of New York, Brockport, New York 14420
| | - Elaine A. Sia
- Department of Biology, University of Rochester, Rochester, New York 14627
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Kalifa L, Beutner G, Phadnis N, Sheu SS, Sia EA. Evidence for a role of FEN1 in maintaining mitochondrial DNA integrity. DNA Repair (Amst) 2009; 8:1242-9. [PMID: 19699691 DOI: 10.1016/j.dnarep.2009.07.008] [Citation(s) in RCA: 54] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2009] [Revised: 07/21/2009] [Accepted: 07/23/2009] [Indexed: 02/05/2023]
Abstract
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.
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Affiliation(s)
- Lidza Kalifa
- Department of Biology, University of Rochester, NY 14627, United States
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27
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Malc E, Dzierzbicki P, Kaniak A, Skoneczna A, Ciesla Z. Inactivation of the 20S proteasome maturase, Ump1p, leads to the instability of mtDNA in Saccharomyces cerevisiae. Mutat Res 2009; 669:95-103. [PMID: 19467248 DOI: 10.1016/j.mrfmmm.2009.05.008] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2009] [Revised: 04/30/2009] [Accepted: 05/14/2009] [Indexed: 01/29/2023]
Abstract
The proteasome plays fundamental roles in the removal of oxidized proteins and in normal degradation of short-lived proteins. Increasing evidence suggests that the proteasome may be an important factor in both oxidative stress response and cellular aging. Moreover, it was recently reported that proteasome inhibition leads to mitochondrial dysfunction. In this study, we have investigated whether proteasome impairment, caused by deletion of UMP1, a gene necessary for the 20S proteasome biogenesis, may influence the stability of the yeast mitochondrial genome. Here we show that an ump1Delta mutant displays enhanced mitochondrial point mutagenesis, measured by the frequency of oligomycin-resistant (Oli(r)) and erythromycin-resistant (Ery(r)) mutants, compared to that of the isogenic wild-type strain. Deletion of UMP1 significantly increases also the frequency of respiration-defective mutants having gross rearrangements of the mitochondrial genome. We show that this mitochondrial mutator phenotype of the ump1Delta strain is considerably reduced in the presence of a plasmid encoding Msh1p, the mitochondrial homologue of the bacterial mismatch protein MutS, which was shown previously to counteract oxidative lesion-induced instability of mtDNA. In search of the mechanism underlying the decreased stability of mtDNA in the ump1Delta deletion mutant, we have determined the level of reactive oxygen species (ROS) in the mutant cells and have found that they are exposed to endogenous oxidative stress. Furthermore, we show also that both cellular and intramitochondrial levels of Msh1p are significantly reduced in the mutant cells compared to the wild-type cells. We conclude, therefore, that both an increased ROS production and a markedly decreased level of Msh1p, a protein crucial for the repair of mtDNA, lead in S. cerevisiae cells with impaired proteasome activity to the increased instability of their mitochondrial genome.
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Affiliation(s)
- Ewa Malc
- Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland
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28
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Abstract
Mitochondrial DNA is thought to be especially prone to oxidative damage by reactive oxygen species generated through electron transport during cellular respiration. This damage is mitigated primarily by the base excision repair (BER) pathway, one of the few DNA repair pathways with confirmed activity on mitochondrial DNA. Through genetic epistasis analysis of the yeast Saccharomyces cerevisiae, we examined the genetic interaction between each of the BER proteins previously shown to localize to the mitochondria. In addition, we describe a series of genetic interactions between BER components and the MutS homolog MSH1, a respiration-essential gene. We show that, in addition to their variable effects on mitochondrial function, mutant msh1 alleles conferring partial function interact genetically at different points in mitochondrial BER. In addition to this separation of function, we also found that the role of Msh1p in BER is unlikely to be involved in the avoidance of large-scale deletions and rearrangements.
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Kaniak A, Dzierzbicki P, Rogowska AT, Malc E, Fikus M, Ciesla Z. Msh1p counteracts oxidative lesion-induced instability of mtDNA and stimulates mitochondrial recombination in Saccharomyces cerevisiae. DNA Repair (Amst) 2009; 8:318-29. [DOI: 10.1016/j.dnarep.2008.11.004] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2008] [Revised: 11/02/2008] [Accepted: 11/05/2008] [Indexed: 01/01/2023]
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30
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Creation of a chloroplast microsatellite reporter for detection of replication slippage in Chlamydomonas reinhardtii. EUKARYOTIC CELL 2008; 7:639-46. [PMID: 18263764 DOI: 10.1128/ec.00447-07] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
Microsatellites are composed of short tandem direct repeats; deletions or duplications of those repeats through the process of replication slippage result in microsatellite instability relative to other genomic loci. Variation in repeat number occurs so frequently that microsatellites can be used for genotyping and forensic analysis. However, an accurate assessment of the rates of change can be difficult because the presence of many repeats makes it difficult to determine whether changes have occurred through single or multiple events. The current study was undertaken to experimentally assess the rates of replication slippage that occur in vivo in the chloroplast DNA of Chlamydomonas reinhardtii. A reporter construct was created in which a stretch of AAAG repeats was inserted into a functional gene to allow changes to be observed when they occurred at the synthetic microsatellite. Restoration of the reading frame occurred through replication slippage in 15 of every million viable cells. Since only one-third of the potential insertion/deletion events would restore the reading frame, the frequency of change could be deduced to be 4.5 x 10(-5). Analysis of the slippage events showed that template slippage was the primary event, resulting in deletions rather than duplications. These findings contrasted with events observed in Escherichia coli during maintenance of the plasmid, where duplications were the rule.
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31
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Kalifa L, Sia EA. Analysis of Rev1p and Pol zeta in mitochondrial mutagenesis suggests an alternative pathway of damage tolerance. DNA Repair (Amst) 2007; 6:1732-9. [PMID: 17689152 PMCID: PMC2129123 DOI: 10.1016/j.dnarep.2007.06.005] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2007] [Revised: 06/08/2007] [Accepted: 06/14/2007] [Indexed: 10/23/2022]
Abstract
Ultraviolet light is a potent DNA damaging agent that induces bulky lesions in DNA which block the replicative polymerases. In order to ensure continued DNA replication and cell viability, specialized translesion polymerases bypass these lesions at the expense of introducing mutations in the nascent DNA strand. A recent study has shown that the N-terminal sequences of the nuclear translesion polymerases Rev1p and Pol zeta can direct GFP to the mitochondrial compartment of Saccharomyces cerevisiae. We have investigated the role of these polymerases in mitochondrial mutagenesis. Our analysis of mitochondrial DNA point mutations, microsatellite instability, and the spectra of mitochondrial mutations indicate that these translesion polymerases function in a less mutagenic pathway in the mitochondrial compartment than they do in the nucleus. Mitochondrial phenotypes resulting from the loss of Rev1p and Pol zeta suggest that although these polymerases are responsible for the majority of mitochondrial frameshift mutations, they do not greatly contribute to mitochondrial DNA point mutations. Analysis of spontaneous mitochondrial DNA point mutations suggests that Pol zeta may play a role in general mitochondrial DNA maintenance. In addition, we observe a 20-fold increase in UV-induced mitochondrial DNA point mutations in rev deficient strains. Our data provides evidence for an alternative damage tolerance pathway that is specific to the mitochondrial compartment.
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Affiliation(s)
- Lidza Kalifa
- Department of Biology, University of Rochester, Rochester, NY 14627, United States
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32
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Bonnefoy N, Remacle C, Fox TD. Genetic transformation of Saccharomyces cerevisiae and Chlamydomonas reinhardtii mitochondria. Methods Cell Biol 2007; 80:525-48. [PMID: 17445712 DOI: 10.1016/s0091-679x(06)80026-9] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Affiliation(s)
- Nathalie Bonnefoy
- Centre de Génétique Moléculaire, CNRS UPR2167, Avenue de la Terrasse, 91198 Gif-sur-Yvette cedex, France
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Pfeuty A, Dufresne C, Gueride M, Lecellier G. Mitochondrial upstream promoter sequences modulate in vivo the transcription of a gene in yeast mitochondria. Mitochondrion 2006; 6:289-98. [PMID: 17110175 DOI: 10.1016/j.mito.2006.10.003] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2006] [Revised: 09/28/2006] [Accepted: 10/10/2006] [Indexed: 10/24/2022]
Abstract
An in vivo study of the importance of the length and/or structures of sequences upstream of a mitochondrial promoter was undertaken in Saccharomyces cerevisiae. Short tandem mtDNA repeats were introduced upstream of the COX2 gene. Our data show that its expression is modulated by the sequence located over 200 bp upstream of the promoter. A deletion decreases the level of transcripts to about 50%. The initial level can be recovered by a fill-in AT-rich sequence or partially by the presence of a long repeat tract; on the contrary, a smaller number of copies tends to intensify the effect of the deletion. These results show that the length and base composition upstream of mitochondrial promoter are involved in vivo in the modulation of the gene expression.
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Affiliation(s)
- A Pfeuty
- Université de Versailles-Saint Quentin en Yvelines, Laboratoire de Génétique et Biologie Cellulaire, 45 Avenue des Etats-Unis, 78035 Versailles, Cedex, France
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34
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Phadnis N, Mehta R, Meednu N, Sia EA. Ntg1p, the base excision repair protein, generates mutagenic intermediates in yeast mitochondrial DNA. DNA Repair (Amst) 2006; 5:829-39. [PMID: 16730479 DOI: 10.1016/j.dnarep.2006.04.002] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2006] [Revised: 04/12/2006] [Accepted: 04/13/2006] [Indexed: 11/25/2022]
Abstract
Mitochondrial DNA is predicted to be highly prone to oxidative damage due to its proximity to free radicals generated by oxidative phosphorylation. Base excision repair (BER) is the primary repair pathway responsible for repairing oxidative damage in nuclear and mitochondrial genomes. In yeast mitochondria, three N-glycosylases have been identified so far, Ntg1p, Ogg1p and Ung1p. Ntg1p, a broad specificity N-glycosylase, takes part in catalyzing the first step of BER that involves the removal of the damaged base. In this study, we examined the role of Ntg1p in maintaining yeast mitochondrial genome integrity. Using genetic reporters and assays to assess mitochondrial mutations, we found that loss of Ntg1p suppresses mitochondrial point mutation rates, frameshifts and recombination rates. We also observed a suppression of respiration loss in the ntg1-Delta cells in response to ultraviolet light exposure implying an overlap between BER and UV-induced damage in the yeast mitochondrial compartment. Over-expression of the BER AP endonuclease, Apn1p, did not significantly affect the mitochondrial mutation rate in the presence of Ntg1p, whereas Apn1p over-expression in an ntg1-Delta background increased the frequency of mitochondrial mutations. In addition, loss of Apn1p also suppressed mitochondrial point mutations. Our work suggests that both Ntg1p and Apn1p generate mutagenic intermediates in the yeast mitochondrial genome.
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Affiliation(s)
- Naina Phadnis
- Department of Biology, University of Rochester, NY 14627-0211, USA
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35
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Graziewicz MA, Longley MJ, Copeland WC. DNA polymerase gamma in mitochondrial DNA replication and repair. Chem Rev 2006; 106:383-405. [PMID: 16464011 DOI: 10.1021/cr040463d] [Citation(s) in RCA: 211] [Impact Index Per Article: 11.1] [Reference Citation Analysis] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Affiliation(s)
- Maria A Graziewicz
- Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA
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36
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Mookerjee SA, Sia EA. 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 2006; 595:91-106. [PMID: 16337661 DOI: 10.1016/j.mrfmmm.2005.10.006] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2005] [Revised: 09/22/2005] [Accepted: 10/20/2005] [Indexed: 05/05/2023]
Abstract
The mechanisms that govern mutation avoidance in the mitochondrial genome, though believed to be numerous, are poorly understood. The identification of individual genes has implicated mismatch repair and several recombination pathways in maintaining the fidelity and structural stability of mitochondrial DNA. However, the majority of genes in these pathways have not been identified and the interactions between different pathways have not been extensively studied. Additionally, the multicopy presence of the mitochondrial genome affects the occurrence and persistence of mutant phenotypes, making mitochondrial DNA transmission and sorting important factors affecting mutation accumulation. We present new evidence that the putative recombination genes CCE1, DIN7, and MHR1 have overlapping function with the mismatch repair homolog MSH1 in point mutation avoidance and suppression of aberrant recombination events. In addition, we demonstrate a novel role for Msh1p in mtDNA transmission, a role not predicted by studies of its nuclear homologs.
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Affiliation(s)
- Shona A Mookerjee
- Department of Biology, University of Rochester, Rochester, NY 14627-0211, USA
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37
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Oliveira EJ, Pádua JG, Zucchi MI, Vencovsky R, Vieira MLC. Origin, evolution and genome distribution of microsatellites. Genet Mol Biol 2006. [DOI: 10.1590/s1415-47572006000200018] [Citation(s) in RCA: 204] [Impact Index Per Article: 10.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022] Open
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Phadnis N, Sia RA, Sia EA. Analysis of repeat-mediated deletions in the mitochondrial genome of Saccharomyces cerevisiae. Genetics 2005; 171:1549-59. [PMID: 16157666 PMCID: PMC1456083 DOI: 10.1534/genetics.105.047092] [Citation(s) in RCA: 37] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2005] [Accepted: 08/26/2005] [Indexed: 11/18/2022] Open
Abstract
Mitochondrial DNA deletions and point mutations accumulate in an age-dependent manner in mammals. The mitochondrial genome in aging humans often displays a 4977-bp deletion flanked by short direct repeats. Additionally, direct repeats flank two-thirds of the reported mitochondrial DNA deletions. The mechanism by which these deletions arise is unknown, but direct-repeat-mediated deletions involving polymerase slippage, homologous recombination, and nonhomologous end joining have been proposed. We have developed a genetic reporter to measure the rate at which direct-repeat-mediated deletions arise in the mitochondrial genome of Saccharomyces cerevisiae. Here we analyze the effect of repeat size and heterology between repeats on the rate of deletions. We find that the dependence on homology for repeat-mediated deletions is linear down to 33 bp. Heterology between repeats does not affect the deletion rate substantially. Analysis of recombination products suggests that the deletions are produced by at least two different pathways, one that generates only deletions and one that appears to generate both deletions and reciprocal products of recombination. We discuss how this reporter may be used to identify the proteins in yeast that have an impact on the generation of direct-repeat-mediated deletions.
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Affiliation(s)
- Naina Phadnis
- Department of Biological Sciences, University of Rochester, Rochester, NY 14627, USA
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Vongsamphanh R, Wagner JR, Ramotar D. Saccharomyces cerevisiae Ogg1 prevents poly(GT) tract instability in the mitochondrial genome. DNA Repair (Amst) 2005; 5:235-42. [PMID: 16293446 DOI: 10.1016/j.dnarep.2005.10.003] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2005] [Revised: 10/03/2005] [Accepted: 10/06/2005] [Indexed: 01/10/2023]
Abstract
Reactive oxygen species can attack the mitochondrial genome to produce a vast array of oxidative DNA lesions including 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo). We assess the role of the Saccharomyces cerevisiae 8-oxo-dGuo DNA glycosylase, Ogg1, in the maintenance of a poly(GT) tract reporter system present in the mitochondrial genome. Deletion in the poly(GT) tract causes the reporter system to produce arginine-independent (Arg+) colonies. We show that the mitochondrial form of Ogg1 is functionally active at processing 8-oxo-dGuo lesions and that Ogg1-deficient cells exhibit nearly six-fold elevated rate of Arg+ mutants under normal growth condition, as compared to the parent. Overexpression of Ogg1 completely suppressed the high rate of Arg+ mutations to levels lower than the parental, suggesting that Ogg1 function could be limited in the mitochondria. Further analysis revealed that the Arg+ mutations can be prevented if the cells are grown under anaerobic conditions. These findings provide in vivo evidence that oxidative stress induces the formation of lesions, most likely 8-oxo-dGuo, which must be repaired by Ogg1, otherwise the lesions can trigger poly(GT) tract instability in the mitochondrial genome. We also demonstrate that overproduction of the major apurinic/apyrimidinic (AP) endonuclease Apn1, a nuclear and mitochondrial enzyme with multiple DNA repair activities, substantially elevated the rate of Arg+ mutants, but which was counteracted by Ogg1 overexpression. We suggest that Ogg1 might bind to AP sites and protect this lesion from the spurious action of Apn1 overproduction. Thus, cleavage of AP site located within or in the vicinity of the poly(GT) tract could destabilize this repeat.
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Affiliation(s)
- Ratsavarinh Vongsamphanh
- University of Montreal, Maisonneuve-Rosemont Hospital, Guy-Bernier Research Centre, 5415 de l'Assomption, Montreal, Que., Canada H1T 2M4
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40
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Larsen NB, Rasmussen M, Rasmussen LJ. Nuclear and mitochondrial DNA repair: similar pathways? Mitochondrion 2005; 5:89-108. [PMID: 16050976 DOI: 10.1016/j.mito.2005.02.002] [Citation(s) in RCA: 212] [Impact Index Per Article: 10.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2004] [Revised: 01/31/2005] [Accepted: 02/03/2005] [Indexed: 02/08/2023]
Abstract
Mitochondrial DNA (mtDNA) alterations are implicated in a broad range of human diseases and alterations of the mitochondrial genome are assumed to be a result of its high susceptibility to oxidative damage and its limited DNA repair compared to nuclear DNA (nDNA). Characterization of DNA repair mechanisms has generally focused on these processes in nDNA but increasing interest and research effort have contributed to our knowledge of the mechanisms underlying DNA repair in mitochondria. In this review, we make comparisons between nDNA and mtDNA repair pathways and propose a model for how these pathways interact in mitochondria.
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Affiliation(s)
- Nicolai Balle Larsen
- Department of Life Sciences and Chemistry, Roskilde University, Universitetsvej 1, 4000 Roskilde, Denmark
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41
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Mayer F, Kerth G. Microsatellite evolution in the mitochondrial genome of Bechstein's bat (Myotis bechsteinii). J Mol Evol 2005; 61:408-16. [PMID: 16082564 DOI: 10.1007/s00239-005-0040-4] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2005] [Accepted: 05/05/2005] [Indexed: 11/28/2022]
Abstract
Being highly polymorphic, microsatellites are widely used genetic markers. They are abundant throughout the nuclear genomes of eukaryotes but rare in the mitochondrial genomes (mtDNA) of animals. We describe a short but highly polymorphic AT microsatellite in the mtDNA control region of Bechstein's bat and discuss the role of mutation, genetic drift, and selection in maintaining its variability. As heteroplasmy and hence mutation rate were positively correlated with repeat number, a simple mutation model cannot explain the observed frequency distribution of AT copy numbers. Because of the unimodal distribution of repeat numbers found in heteroplasmic individuals, single step mutations are likely to be the predominant mechanism of copy number alternations. Above a certain copy number (seven repeats), deletions of single dinucleotide repeats seem to be more common than additions, which results in a decrease in frequency of long alleles. Heteroplasmy was inherited from mothers to their offspring and no evidence of paternal inheritance of mitochondria was found. Genetic differences accumulated with more distant ancestry, which suggests that microsatellites can be useful genetic markers in population genetics.
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Affiliation(s)
- Frieder Mayer
- Institute of Zoology II, University of Erlangen-Nürnberg, Staudtstrasse 5, Erlangen, D-91058, Germany.
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42
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Mookerjee SA, Lyon HD, Sia EA. Analysis of the functional domains of the mismatch repair homologue Msh1p and its role in mitochondrial genome maintenance. Curr Genet 2004; 47:84-99. [PMID: 15611870 DOI: 10.1007/s00294-004-0537-1] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2004] [Revised: 09/14/2004] [Accepted: 09/20/2004] [Indexed: 01/16/2023]
Abstract
Mitochondrial DNA (mtDNA) repair occurs in all eukaryotic organisms and is essential for the maintenance of mitochondrial function. Evidence from both humans and yeast suggests that mismatch repair is one of the pathways that functions in overall mtDNA stability. In the mitochondria of the yeast Saccharomyces cerevisiae, the presence of a homologue to the bacterial MutS mismatch repair protein, MSH1, has long been known to be essential for mitochondrial function. The mechanisms for which it is essential are unclear, however. Here, we analyze the effects of two point mutations, msh1-F105A and msh1-G776D, both predicted to be defective in mismatch repair; and we show that they are both able to maintain partial mitochondrial function. Moreover, there are significant differences in the severity of mitochondrial disruption between the two mutants that suggest multiple roles for Msh1p in addition to mismatch repair. Our overall findings suggest that these additional predicted functions of Msh1p, including recombination surveillance and heteroduplex rejection, may be primarily responsible for its essential role in mtDNA stability.
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Affiliation(s)
- Shona A Mookerjee
- Department of Biology, University of Rochester, RC Box 270211, Rochester, NY 14627-0211, USA
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43
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Dzierzbicki P, Koprowski P, Fikus MU, Malc E, Ciesla Z. Repair of oxidative damage in mitochondrial DNA of Saccharomyces cerevisiae: involvement of the MSH1-dependent pathway. DNA Repair (Amst) 2004; 3:403-11. [PMID: 15010316 DOI: 10.1016/j.dnarep.2003.12.005] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2003] [Revised: 12/18/2003] [Accepted: 12/22/2003] [Indexed: 10/26/2022]
Abstract
Mitochondrial DNA (mtDNA) is located close to the respiratory chain, a major source of reactive oxygen species (ROS). This proximity makes mtDNA more vulnerable than nuclear DNA to damage by ROS. Therefore, the efficient repair of oxidative lesions in mtDNA is essential for maintaining the stability of the mitochondrial genome. A series of genetic and biochemical studies has indicated that eukaryotic cells, including the model organism Saccharomyces cerevisiae, use several alternative strategies to prevent mutagenesis induced by endogenous oxidative damage to nuclear DNA. However, apart from base excision repair (BER), no other pathways involved in the repair of oxidative damage in mtDNA have been identified. In this study, we have examined mitochondrial mutagenesis in S. cerevisiae cells which lack the activity of the Ogg1 glycosylase, an enzyme playing a crucial role in the removal of 8-oxoG, the most abundant oxidative lesion of DNA. We show that the overall frequency of the mitochondrial oligomycin-resistant (Olir) mutants is increased in the ogg1 strain by about one order of magnitude compared to that of the wild-type strain. Noteworthy, in the mitochondrial oli1 gene, G:C to T:A transversions are generated approximately 50-fold more frequently in the ogg1 mutant relative to the wild-type strain. We also demonstrate that the increased frequency of Olir mutants in the ogg1 strain is markedly reduced by the presence of plasmids encoding Msh1p, a homologue of the bacterial mismatch protein MutS, which specifically functions in mitochondria. This suppression of the mitochondrial mutator phenotype of the ogg1 strain seems to be specific, since overexpression of the mutant allele msh1-R813W failed to exert this effect. Finally, we also show that the increased frequency of Olir mutants arising in an msh1/MSH1 heterozygote grown in glucose-containing medium is further enhanced if the cells are cultivated in glycerol-containing medium, i.e. under conditions when the respiratory chain is fully active. Taken together, these results strongly suggest that MSH1-dependent repair represents a significant back-up to mtBER in the repair of oxidative damage in mtDNA.
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Affiliation(s)
- Piotr Dzierzbicki
- Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5A, 02-106 Warsaw, Poland
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Koprowski P, Fikus MU, Dzierzbicki P, Mieczkowski P, Lazowska J, Ciesla Z. Enhanced expression of the DNA damage-inducible gene DIN7 results in increased mutagenesis of mitochondrial DNA in Saccharomyces cerevisiae. Mol Genet Genomics 2003; 269:632-9. [PMID: 12827502 DOI: 10.1007/s00438-003-0873-8] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2003] [Accepted: 05/30/2003] [Indexed: 11/26/2022]
Abstract
We reported previously that the product of DIN7, a DNA damage-inducible gene of Saccharomyces cerevisiae, belongs to the XPG family of proteins, which are involved in DNA repair and replication. This family includes the S. cerevisiae protein Rad2p and its human homolog XPGC, Rad27p and its mammalian homolog FEN-1, and Exonuclease I (Exo I). Interestingly, Din7p is the only member of the XPG family which specifically functions in mitochondria. We reported previously that overexpression of DIN7 results in a mitochondrial mutator phenotype. In the present study we wished to test the hypothesis that this phenotype is dependent on the nuclease activity of Din7p. For this purpose, we constructed two alleles, din7-D78A and din7-D173A, which encode proteins in which highly conserved aspartates important for the nuclease activity of the XPG proteins have been replaced by alanines. Here, we report that overexpression of the mutant alleles, in contrast to DIN7, fails to increase the frequency of mitochondrial petite mutants or erythromycin-resistant (Er) mutants. Also, overproduction of din7-D78Ap does not result in destabilization of poly GT tracts in mitochondrial DNA (mtDNA), the phenotype observed in cells that overexpress Din7p. We also show that petite mutants induced by enhanced synthesis of wild-type Din7p exhibit gross rearrangements of mtDNA, and that this correlates with enhanced recombination within the mitochondrial cyt b gene. These results suggest that the stability of the mitochondrial genome of S. cerevisiae is modulated by the level of the nuclease Din7p.
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Affiliation(s)
- P Koprowski
- Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 5A Pawinskiego St., 02-106 Warsaw, Poland
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Strand MK, Stuart GR, Longley MJ, Graziewicz MA, Dominick OC, Copeland WC. POS5 gene of Saccharomyces cerevisiae encodes a mitochondrial NADH kinase required for stability of mitochondrial DNA. EUKARYOTIC CELL 2003; 2:809-20. [PMID: 12912900 PMCID: PMC178377 DOI: 10.1128/ec.2.4.809-820.2003] [Citation(s) in RCA: 90] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
In a search for nuclear genes that affect mutagenesis of mitochondrial DNA in Saccharomyces cerevisiae, an ATP-NAD (NADH) kinase, encoded by POS5, that functions exclusively in mitochondria was identified. The POS5 gene product was overproduced in Escherichia coli and purified without a mitochondrial targeting sequence. A direct biochemical assay demonstrated that the POS5 gene product utilizes ATP to phosphorylate both NADH and NAD(+), with a twofold preference for NADH. Disruption of POS5 increased minus-one frameshift mutations in mitochondrial DNA 50-fold, as measured by the arg8(m) reversion assay, with no increase in nuclear mutations. Also, a dramatic increase in petite colony formation and slow growth on glycerol or limited glucose were observed. POS5 was previously described as a gene required for resistance to hydrogen peroxide. Consistent with a role in the mitochondrial response to oxidative stress, a pos5 deletion exhibited a 28-fold increase in oxidative damage to mitochondrial proteins and hypersensitivity to exogenous copper. Furthermore, disruption of POS5 induced mitochondrial biogenesis as a response to mitochondrial dysfunction. Thus, the POS5 NADH kinase is required for mitochondrial DNA stability with a critical role in detoxification of reactive oxygen species. These results predict a role for NADH kinase in human mitochondrial diseases.
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Affiliation(s)
- Micheline K Strand
- Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA
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46
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Weetman D, Hauser L, Carvalho GR. Reconstruction of microsatellite mutation history reveals a strong and consistent deletion bias in invasive clonal snails, Potamopyrgus antipodarum. Genetics 2002; 162:813-22. [PMID: 12399391 PMCID: PMC1462296 DOI: 10.1093/genetics/162.2.813] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Direct observations of mutations and comparative analyses suggest that nuclear microsatellites show a tendency to expand, with reports of deletion biases limited to very long alleles or a few loci in multilocus studies. Here we investigate microsatellite evolution in clonal snails, Potamopyrgus antipodarum, since their introduction to Britain in the 19th century, using an analysis based on minimum spanning networks of multilocus microsatellite genotypes. British populations consist of a small number of highly distinct genotype groups with very few outlying genotypes, suggesting clonal lineages containing minor variation generated by mutation. Network patterns suggest that a single introduced genotype was the ancestor of all extant variation and also provide support for wholly apomictic reproduction within the most common clonal lineage (group A). Microsatellites within group A showed a strong tendency to delete repeats, with an overall bias exceeding 88%, irrespective of the exact method used to infer mutations. This highly unusual pattern of deletion bias is consistent across populations and loci and is unrelated to allele size. We suggest that for persistence of microsatellites in this clone, some change in the mutation mechanism must have occurred in relatively recent evolutionary time. Possible causes of such a change in mechanism are discussed.
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Affiliation(s)
- David Weetman
- Department of Biological Sciences, University of Hull, United Kingdom.
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47
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Abstract
We have used microsatellite sequences to evaluate the influence of the mismatch repair system on mutation bias in D. melanogaster. While mismatch-proficient cells have the highest mutation rate at (GT)(n) repeats, (AT)(n) repeats were the least stable ones in spel1(-/-) flies lacking functional mismatch repair. Furthermore, the mutation spectrum of long microsatellite alleles in spel1(-/-) was slightly upward biased, resulting in a gain of repeats, whereas wild-type flies have a strong downward bias. Interestingly, this mismatch repair-mediated downward mutation bias is reflected in the genome composition of D. melanogaster. When compared to other species, D. melanogaster has significantly shorter microsatellites. Our results suggest that the mismatch repair system may have an important role in shaping genome composition.
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Affiliation(s)
- Bettina Harr
- Institut für Tierzucht und Genetik, Veterinärmedizinische Universität Wien, Veterinärplatz 1, Austria
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Craven RJ, Greenwell PW, Dominska M, Petes TD. Regulation of genome stability by TEL1 and MEC1, yeast homologs of the mammalian ATM and ATR genes. Genetics 2002; 161:493-507. [PMID: 12072449 PMCID: PMC1462148 DOI: 10.1093/genetics/161.2.493] [Citation(s) in RCA: 85] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/14/2022] Open
Abstract
In eukaryotes, a family of related protein kinases (the ATM family) is involved in regulating cellular responses to DNA damage and telomere length. In the yeast Saccharomyces cerevisiae, two members of this family, TEL1 and MEC1, have functionally redundant roles in both DNA damage repair and telomere length regulation. Strains with mutations in both genes are very sensitive to DNA damaging agents, have very short telomeres, and undergo cellular senescence. We find that strains with the double mutant genotype also have approximately 80-fold increased rates of mitotic recombination and chromosome loss. In addition, the tel1 mec1 strains have high rates of telomeric fusions, resulting in translocations, dicentrics, and circular chromosomes. Similar chromosome rearrangements have been detected in mammalian cells with mutations in ATM (related to TEL1) and ATR (related to MEC1) and in mammalian cells that approach cell crisis.
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Affiliation(s)
- Rolf J Craven
- Department of Biology and Curriculum in Genetics and Molecular Biology, University of North Carolina, Chapel Hill, North Carolina 27599-3280, USA
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49
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Abstract
Unpaired and mispaired bases in DNA can arise by replication errors, spontaneous or induced base modifications, and during recombination. The major pathway for correction of mismatches arising during replication is the MutHLS pathway of Escherichia coli and related pathways in other organisms. MutS initiates repair by binding to the mismatch, and activates together with MutL the MutH endonuclease, which incises at hemimethylated dam sites and thereby mediates strand discrimination. Multiple MutS and MutL homologues exist in eukaryotes, which play different roles in the mismatch repair (MMR) pathway or in recombination. No MutH homologues have been identified in eukaryotes, suggesting that strand discrimination is different to E. coli. Repair can be initiated by the heterodimers MSH2-MSH6 (MutSalpha) and MSH2-MSH3 (MutSbeta). Interestingly, MSH3 (and thus MutSbeta) is missing in some genomes, as for example in Drosophila, or is present as in Schizosaccharomyces pombe but appears to play no role in MMR. MLH1-PMS1 (MutLalpha) is the major MutL homologous heterodimer. Again some, but not all, eukaryotes have additional MutL homologues, which all form a heterodimer with MLH1 and which play a minor role in MMR. Additional factors with a possible function in eukaryotic MMR are PCNA, EXO1, and the DNA polymerases delta and epsilon. MMR-independent pathways or factors that can process some types of mismatches in DNA are nucleotide-excision repair (NER), some base excision repair (BER) glycosylases, and the flap endonuclease FEN-1. A pathway has been identified in Saccharomyces cerevisiae and human that corrects loops with about 16 to several hundreds of unpaired nucleotides. Such large loops cannot be processed by MMR.
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Affiliation(s)
- Thomas M Marti
- Institute of Cell Biology, University of Bern, Bern, Switzerland
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50
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Longley MJ, Nguyen D, Kunkel TA, Copeland WC. The fidelity of human DNA polymerase gamma with and without exonucleolytic proofreading and the p55 accessory subunit. J Biol Chem 2001; 276:38555-62. [PMID: 11504725 DOI: 10.1074/jbc.m105230200] [Citation(s) in RCA: 178] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Mutations in human mitochondrial DNA influence aging, induce severe neuromuscular pathologies, cause maternally inherited metabolic diseases, and suppress apoptosis. Since the genetic stability of mitochondrial DNA depends on the accuracy of DNA polymerase gamma (pol gamma), we investigated the fidelity of DNA synthesis by human pol gamma. Comparison of the wild-type 140-kDa catalytic subunit to its exonuclease-deficient derivative indicates pol gamma has high base substitution fidelity that results from high nucleotide selectivity and exonucleolytic proofreading. pol gamma is also relatively accurate for single-base additions and deletions in non-iterated and short repetitive sequences. However, when copying homopolymeric sequences longer than four nucleotides, pol gamma has low frameshift fidelity and also generates base substitutions inferred to result from a primer dislocation mechanism. The ability of pol gamma both to make and to proofread dislocation intermediates is the first such evidence for a family A polymerase. Including the p55 accessory subunit, which confers processivity to the pol gamma catalytic subunit, decreases frameshift and base substitution fidelity. Kinetic analyses indicate that p55 promotes extension of mismatched termini to lower the fidelity. These data suggest that homopolymeric runs in mitochondrial DNA may be particularly prone to frameshift mutation in vivo due to replication errors by pol gamma.
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Affiliation(s)
- M J Longley
- Laboratory of Molecular Genetics and the Laboratory of Structural Biology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA
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