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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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Richard GF. The Startling Role of Mismatch Repair in Trinucleotide Repeat Expansions. Cells 2021; 10:cells10051019. [PMID: 33925919 PMCID: PMC8145212 DOI: 10.3390/cells10051019] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2021] [Revised: 04/20/2021] [Accepted: 04/21/2021] [Indexed: 12/26/2022] Open
Abstract
Trinucleotide repeats are a peculiar class of microsatellites whose expansions are responsible for approximately 30 human neurological or developmental disorders. The molecular mechanisms responsible for these expansions in humans are not totally understood, but experiments in model systems such as yeast, transgenic mice, and human cells have brought evidence that the mismatch repair machinery is involved in generating these expansions. The present review summarizes, in the first part, the role of mismatch repair in detecting and fixing the DNA strand slippage occurring during microsatellite replication. In the second part, key molecular differences between normal microsatellites and those that show a bias toward expansions are extensively presented. The effect of mismatch repair mutants on microsatellite expansions is detailed in model systems, and in vitro experiments on mismatched DNA substrates are described. Finally, a model presenting the possible roles of the mismatch repair machinery in microsatellite expansions is proposed.
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Affiliation(s)
- Guy-Franck Richard
- Institut Pasteur, CNRS UMR3525, 25 rue du Docteur Roux, 75015 Paris, France
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Abstract
Trinucleotide repeats are a peculiar class of microsatellites involved in many neurological as well as developmental disorders. Their propensity to generate very large expansions over time is supposedly due to their capacity to form specific secondary structures, such as imperfect hairpins, triple helices, or G-quadruplexes. These unusual structures were proposed to trigger expansions in vivo. Here, I review known technical issues linked to these structures, such as slippage during polymerase chain reaction and aberrant migration of long trinucleotide repeats during agarose gel electrophoresis. Our current understanding of interactions between trinucleotide repeat secondary structures and the mismatch-repair machinery is also quickly reviewed, and critical questions relevant to these interactions are addressed.
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Affiliation(s)
- Guy-Franck Richard
- Department Genomes & Genetics, Institut Pasteur, CNRS UMR3525, Paris, France.
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Bonjoch L, Mur P, Arnau-Collell C, Vargas-Parra G, Shamloo B, Franch-Expósito S, Pineda M, Capellà G, Erman B, Castellví-Bel S. Approaches to functionally validate candidate genetic variants involved in colorectal cancer predisposition. Mol Aspects Med 2019; 69:27-40. [PMID: 30935834 DOI: 10.1016/j.mam.2019.03.004] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2019] [Revised: 03/26/2019] [Accepted: 03/26/2019] [Indexed: 02/07/2023]
Abstract
Most next generation sequencing (NGS) studies identified candidate genetic variants predisposing to colorectal cancer (CRC) but do not tackle its functional interpretation to unequivocally recognize a new hereditary CRC gene. Besides, germline variants in already established hereditary CRC-predisposing genes or somatic variants share the same need when trying to categorize those with relevant significance. Functional genomics approaches have an important role in identifying the causal links between genetic architecture and phenotypes, in order to decipher cellular function in health and disease. Therefore, functional interpretation of identified genetic variants by NGS platforms is now essential. Available approaches nowadays include bioinformatics, cell and molecular biology and animal models. Recent advances, such as the CRISPR-Cas9, ZFN and TALEN systems, have been already used as a powerful tool with this objective. However, the use of cell lines is of limited value due to the CRC heterogeneity and its close interaction with microenvironment. Access to tridimensional cultures or organoids and xenograft models that mimic the in vivo tissue architecture could revolutionize functional analysis. This review will focus on the application of state-of-the-art functional studies to better tackle new genes involved in germline predisposition to this neoplasm.
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Affiliation(s)
- Laia Bonjoch
- Gastroenterology Department, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), University of Barcelona, Barcelona, Spain
| | - Pilar Mur
- Hereditary Cancer Program, Catalan Institute of Oncology, Institut d'Investigació Biomèdica de Bellvitge (IDIBELL), ONCOBELL Program, L'Hospitalet de Llobregat, Barcelona, Spain; Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Spain
| | - Coral Arnau-Collell
- Gastroenterology Department, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), University of Barcelona, Barcelona, Spain
| | - Gardenia Vargas-Parra
- Hereditary Cancer Program, Catalan Institute of Oncology, Institut d'Investigació Biomèdica de Bellvitge (IDIBELL), ONCOBELL Program, L'Hospitalet de Llobregat, Barcelona, Spain; Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Spain
| | - Bahar Shamloo
- Molecular Biology, Genetics, and Bioengineering Department, Legacy Research Institute, Portland, OR, USA
| | - Sebastià Franch-Expósito
- Gastroenterology Department, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), University of Barcelona, Barcelona, Spain
| | - Marta Pineda
- Hereditary Cancer Program, Catalan Institute of Oncology, Institut d'Investigació Biomèdica de Bellvitge (IDIBELL), ONCOBELL Program, L'Hospitalet de Llobregat, Barcelona, Spain; Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Spain
| | - Gabriel Capellà
- Hereditary Cancer Program, Catalan Institute of Oncology, Institut d'Investigació Biomèdica de Bellvitge (IDIBELL), ONCOBELL Program, L'Hospitalet de Llobregat, Barcelona, Spain; Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Spain
| | - Batu Erman
- Molecular Biology, Genetics and Bioengineering Program, Faculty of Engineering and Natural Sciences, Sabanci University, Istanbul, Turkey
| | - Sergi Castellví-Bel
- Gastroenterology Department, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), University of Barcelona, Barcelona, Spain.
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Abstract
The fission yeast Schizosaccharomyces pombe is an excellent model organism to study DNA metabolism, in which the DNA replication and repair mechanisms are evolutionarily conserved. In this introduction we describe a range of methods commonly used to study aspects of DNA metabolism in fission yeast, focusing on approaches used for the analysis of genome stability, DNA replication, and DNA repair. We describe the use of a minichromosome, Ch16, for monitoring different aspects of genome stability. We introduce two-dimensional gel electrophoresis and immunofluorescent visualization of combed DNA molecules for the analysis of DNA replication. Further, we introduce a pulsed field gel electrophoresis (PFGE) assay to physically monitor chromosome integrity, which can be used in conjunction with a DNA double-strand break (DSB) repair assay to genetically quantitate different DSB repair and misrepair outcomes, including gross chromosomal rearrangements, in fission yeast.
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Affiliation(s)
- Francisco Antequera
- Instituto de Biología Funcional y Genómica, CSIC/Universidad de Salamanca, Salamanca 37007, Spain
| | - Timothy C Humphrey
- CRUK-MRC Oxford Institute for Radiation Oncology, University of Oxford, OX3 7DQ, United Kingdom
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A mutated dph3 gene causes sensitivity of Schizosaccharomyces pombe cells to cytotoxic agents. Curr Genet 2017; 63:1081-1091. [PMID: 28555368 PMCID: PMC5668335 DOI: 10.1007/s00294-017-0711-x] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2017] [Revised: 05/11/2017] [Accepted: 05/23/2017] [Indexed: 12/11/2022]
Abstract
Dph3 is involved in diphthamide modification of the eukaryotic translation elongation factor eEF2 and in Elongator-mediated modifications of tRNAs, where a 5-methoxycarbonyl-methyl moiety is added to wobble uridines. Lack of such modifications affects protein synthesis due to inaccurate translation of mRNAs at ribosomes. We have discovered that integration of markers at the msh3 locus of Schizosaccharomyces pombe impaired the function of the nearby located dph3 gene. Such integrations rendered cells sensitive to the cytotoxic drugs hydroxyurea and methyl methanesulfonate. We constructed dph3 and msh3 strains with mutated ATG start codons (ATGmut), which allowed investigating drug sensitivity without potential interference by marker insertions. The dph3-ATGmut and a dph3::loxP-ura4-loxM gene disruption strain, but not msh3-ATGmut, turned out to be sensitive to hydroxyurea and methyl methanesulfonate, likewise the strains with cassettes integrated at the msh3 locus. The fungicide sordarin, which inhibits diphthamide modified eEF2 of Saccharomyces cerevisiae, barely affected survival of wild type and msh3Δ S. pombe cells, while the dph3Δ mutant was sensitive. The msh3-ATG mutation, but not dph3Δ or the dph3-ATG mutation caused a defect in mating-type switching, indicating that the ura4 marker at the dph3 locus did not interfere with Msh3 function. We conclude that Dph3 is required for cellular resistance to the fungicide sordarin and to the cytotoxic drugs hydroxyurea and methyl methanesulfonate. This is likely mediated by efficient translation of proteins in response to DNA damage and replication stress.
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Schizosaccharomyces pombe MutSα and MutLα Maintain Stability of Tetra-Nucleotide Repeats and Msh3 of Hepta-Nucleotide Repeats. G3-GENES GENOMES GENETICS 2017; 7:1463-1473. [PMID: 28341698 PMCID: PMC5427490 DOI: 10.1534/g3.117.040816] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 11/18/2022]
Abstract
Defective mismatch repair (MMR) in humans is associated with colon cancer and instability of microsatellites, that is, DNA sequences with one or several nucleotides repeated. Key factors of eukaryotic MMR are the heterodimers MutSα (Msh2-Msh6), which recognizes base-base mismatches and unpaired nucleotides in DNA, and MutLα (Mlh1-Pms1), which facilitates downstream steps. In addition, MutSβ (Msh2-Msh3) recognizes DNA loops of various sizes, although our previous data and the data presented here suggest that Msh3 of Schizosaccharomyces pombe does not play a role in MMR. To test microsatellite stability in S. pombe and hence DNA loop repair, we have inserted tetra-, penta-, and hepta-nucleotide repeats in the ade6 gene and determined their Ade+ reversion rates and spectra in wild type and various mutants. Our data indicate that loops with four unpaired nucleotides in the nascent and the template strand are the upper limit of MutSα- and MutLα-mediated MMR in S. pombe Stability of hepta-nucleotide repeats requires Msh3 and Exo1 in MMR-independent processes as well as the DNA repair proteins Rad50, Rad51, and Rad2FEN1 Most strikingly, mutation rates in the double mutants msh3 exo1 and msh3 rad51 were decreased when compared to respective single mutants, indicating that Msh3 prevents error prone processes carried out by Exo1 and Rad51. We conclude that Msh3 has no obvious function in MMR in S. pombe, but contributes to DNA repeat stability in MMR-independent processes.
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Esteban-Jurado C, Giménez-Zaragoza D, Muñoz J, Franch-Expósito S, Álvarez-Barona M, Ocaña T, Cuatrecasas M, Carballal S, López-Cerón M, Marti-Solano M, Díaz-Gay M, van Wezel T, Castells A, Bujanda L, Balmaña J, Gonzalo V, Llort G, Ruiz-Ponte C, Cubiella J, Balaguer F, Aligué R, Castellví-Bel S. POLE and POLD1 screening in 155 patients with multiple polyps and early-onset colorectal cancer. Oncotarget 2017; 8:26732-26743. [PMID: 28423643 PMCID: PMC5432293 DOI: 10.18632/oncotarget.15810] [Citation(s) in RCA: 32] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2017] [Accepted: 02/18/2017] [Indexed: 12/31/2022] Open
Abstract
Germline mutations in POLE and POLD1 have been shown to cause predisposition to colorectal multiple polyposis and a wide range of neoplasms, early-onset colorectal cancer being the most prevalent. In order to find additional mutations affecting the proofreading activity of these polymerases, we sequenced its exonuclease domain in 155 patients with multiple polyps or an early-onset colorectal cancer phenotype without alterations in the known hereditary colorectal cancer genes. Interestingly, none of the previously reported mutations in POLE and POLD1 were found. On the other hand, among the genetic variants detected, only two of them stood out as putative pathogenic in the POLE gene, c.1359 + 46del71 and c.1420G > A (p.Val474Ile). The first variant, detected in two families, was not proven to alter correct RNA splicing. Contrarily, c.1420G > A (p.Val474Ile) was detected in one early-onset colorectal cancer patient and located right next to the exonuclease domain. The pathogenicity of this change was suggested by its rarity and bioinformatics predictions, and it was further indicated by functional assays in Schizosaccharomyces pombe. This is the first study to functionally analyze a POLE genetic variant outside the exonuclease domain and widens the spectrum of genetic changes in this DNA polymerase that could lead to colorectal cancer predisposition.
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Affiliation(s)
- Clara Esteban-Jurado
- Gastroenterology Department, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), University of Barcelona, Barcelona, Catalonia, Spain
| | - David Giménez-Zaragoza
- Biomedical Sciences Department, School of Medicine, University de Barcelona, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Catalonia, Spain
| | - Jenifer Muñoz
- Gastroenterology Department, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), University of Barcelona, Barcelona, Catalonia, Spain
| | - Sebastià Franch-Expósito
- Gastroenterology Department, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), University of Barcelona, Barcelona, Catalonia, Spain
| | - Miriam Álvarez-Barona
- Galician Public Foundation of Genomic Medicine (FPGMX), Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Genomics Medicine Group, Hospital Clínico, Santiago de Compostela, University of Santiago de Compostela, Galicia, Spain
| | - Teresa Ocaña
- Gastroenterology Department, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), University of Barcelona, Barcelona, Catalonia, Spain
| | - Miriam Cuatrecasas
- Department of Pathology, Hospital Clinic, Biobanc Clinic-IDIBAPS, Barcelona, Catalonia, Spain
| | - Sabela Carballal
- Gastroenterology Department, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), University of Barcelona, Barcelona, Catalonia, Spain
| | - María López-Cerón
- Gastroenterology Department, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), University of Barcelona, Barcelona, Catalonia, Spain
| | - Maria Marti-Solano
- Department of Pharmaceutical Chemistry, Philipps-University Marburg, Marburg, Germany
| | - Marcos Díaz-Gay
- Gastroenterology Department, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), University of Barcelona, Barcelona, Catalonia, Spain
| | - Tom van Wezel
- Leiden University Medical Center (LUMC), Leiden, Netherlands
| | - Antoni Castells
- Gastroenterology Department, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), University of Barcelona, Barcelona, Catalonia, Spain
| | - Luis Bujanda
- Gastroenterology Department, Hospital Donostia–Instituto Biodonostia, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Basque Country University (UPV/EHU), San Sebastián, Spain
| | - Judith Balmaña
- High Risk and Cancer Prevention Unit, Medical Oncology Department, University Hospital Vall d'Hebron and Vall d'Hebron Institute of Oncology, Barcelona, Spain
| | - Victoria Gonzalo
- Gastroenterology Department, Hospital Universitari Mútua de Terrassa, Terrassa, Barcelona, Spain
| | - Gemma Llort
- Clinical Oncology Department, Corporacio Parc Tauli, Sabadell, Barcelona, Spain
| | - Clara Ruiz-Ponte
- Galician Public Foundation of Genomic Medicine (FPGMX), Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Genomics Medicine Group, Hospital Clínico, Santiago de Compostela, University of Santiago de Compostela, Galicia, Spain
| | - Joaquín Cubiella
- Gastroenterology Department, Complexo Hospitalario Universitario de Ourense, Instituto de Investigación Biomédica Ourense, Pontevedra y Vigo, Ourense, Spain
| | - Francesc Balaguer
- Gastroenterology Department, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), University of Barcelona, Barcelona, Catalonia, Spain
| | - Rosa Aligué
- Biomedical Sciences Department, School of Medicine, University de Barcelona, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Catalonia, Spain
| | - Sergi Castellví-Bel
- Gastroenterology Department, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), University of Barcelona, Barcelona, Catalonia, Spain
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Genome-Wide Estimates of Mutation Rates and Spectrum in Schizosaccharomyces pombe Indicate CpG Sites are Highly Mutagenic Despite the Absence of DNA Methylation. G3-GENES GENOMES GENETICS 2015; 6:149-60. [PMID: 26564949 PMCID: PMC4704713 DOI: 10.1534/g3.115.022129] [Citation(s) in RCA: 40] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
We accumulated mutations for 1952 generations in 79 initially identical, haploid lines of the fission yeast Schizosaccharomyces pombe, and then performed whole-genome sequencing to determine the mutation rates and spectrum. We captured 696 spontaneous mutations across the 79 mutation accumulation (MA) lines. We compared the mutation spectrum and rate to a recently published equivalent experiment on the same species, and to another model ascomycetous yeast, the budding yeast Saccharomyces cerevisiae. While the two species are approximately 600 million years diverged from each other, they share similar life histories, genome size and genomic G/C content. We found that Sc. pombe and S. cerevisiae have similar mutation rates, but Sc. pombe exhibits a stronger insertion bias. Intriguingly, we observed an increased mutation rate at cytosine nucleotides, specifically CpG nucleotides, which is also seen in S. cerevisiae. However, the absence of methylation in Sc. pombe and the pattern of mutation at these sites, primarily C → A as opposed to C → T, strongly suggest that the increased mutation rate is not caused by deamination of methylated cytosines. This result implies that the high mutability of CpG dinucleotides in other species may be caused in part by a methylation-independent mechanism. Many of our findings mirror those seen in the recent study, despite the use of different passaging conditions, indicating that MA is a reliable method for estimating mutation rates and spectra.
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Protacio RU, Storey AJ, Davidson MK, Wahls WP. Nonsense codon suppression in fission yeast due to mutations of tRNA(Ser.11) and translation release factor Sup35 (eRF3). Curr Genet 2015; 61:165-73. [PMID: 25519804 PMCID: PMC4393767 DOI: 10.1007/s00294-014-0465-7] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2014] [Revised: 12/01/2014] [Accepted: 12/03/2014] [Indexed: 02/07/2023]
Abstract
In the fission yeast Schizosaccharomyces pombe, sup9 mutations can suppress the termination of translation at nonsense (stop) codons. We localized sup9 physically to the spctrnaser.11 locus and confirmed that one allele (sup9-UGA) alters the anticodon of a serine tRNA. We also found that another purported allele is not allelic. Instead, strains with that suppressor (renamed sup35-F592S) have a single base pair substitution (T1775C) that introduces an amino acid substitution in the Sup35 protein (Sup35-F592S). Reduced functionality of Sup35 (eRF3), the ubiquitous guanine nucleotide-responsive translation release factor of eukaryotes, increases read-through of stop codons. Tetrad dissection revealed that suppression is tightly linked to (inseparable from) the sup35-F592S mutation and that there are no additional extragenic modifiers. The Mendelian inheritance indicates that the Sup35-F592S protein does not adopt an infectious amyloid state ([PSI (+)] prion) to affect suppression, consistent with recent evidence that fission yeast Sup35 does not form prions. We also report that sup9-UGA and sup35-F592S exhibit different strengths of suppression for opal stop codons of ade6-M26 and ade6-M375. We discuss possible mechanisms for the variation in suppressibility exhibited by the two alleles.
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Affiliation(s)
- Reine U. Protacio
- Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, 4301 West Markham Street (slot 516), Little Rock, AR 72205-7199, USA
| | - Aaron J. Storey
- Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, 4301 West Markham Street (slot 516), Little Rock, AR 72205-7199, USA
| | - Mari K. Davidson
- Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, 4301 West Markham Street (slot 516), Little Rock, AR 72205-7199, USA
| | - Wayne P. Wahls
- Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, 4301 West Markham Street (slot 516), Little Rock, AR 72205-7199, USA
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11
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Tosti E, Katakowski JA, Schaetzlein S, Kim HS, Ryan CJ, Shales M, Roguev A, Krogan NJ, Palliser D, Keogh MC, Edelmann W. Evolutionarily conserved genetic interactions with budding and fission yeast MutS identify orthologous relationships in mismatch repair-deficient cancer cells. Genome Med 2014; 6:68. [PMID: 25302077 PMCID: PMC4189729 DOI: 10.1186/s13073-014-0068-4] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2013] [Accepted: 08/28/2014] [Indexed: 12/13/2022] Open
Abstract
Background The evolutionarily conserved DNA mismatch repair (MMR) system corrects base-substitution and insertion-deletion mutations generated during erroneous replication. The mutation or inactivation of many MMR factors strongly predisposes to cancer, where the resulting tumors often display resistance to standard chemotherapeutics. A new direction to develop targeted therapies is the harnessing of synthetic genetic interactions, where the simultaneous loss of two otherwise non-essential factors leads to reduced cell fitness or death. High-throughput screening in human cells to directly identify such interactors for disease-relevant genes is now widespread, but often requires extensive case-by-case optimization. Here we asked if conserved genetic interactors (CGIs) with MMR genes from two evolutionary distant yeast species (Saccharomyces cerevisiae and Schizosaccharomyzes pombe) can predict orthologous genetic relationships in higher eukaryotes. Methods High-throughput screening was used to identify genetic interaction profiles for the MutSα and MutSβ heterodimer subunits (msh2Δ, msh3Δ, msh6Δ) of fission yeast. Selected negative interactors with MutSβ (msh2Δ/msh3Δ) were directly analyzed in budding yeast, and the CGI with SUMO-protease Ulp2 further examined after RNA interference/drug treatment in MSH2-deficient and -proficient human cells. Results This study identified distinct genetic profiles for MutSα and MutSβ, and supports a role for the latter in recombinatorial DNA repair. Approximately 28% of orthologous genetic interactions with msh2Δ/msh3Δ are conserved in both yeasts, a degree consistent with global trends across these species. Further, the CGI between budding/fission yeast msh2 and SUMO-protease Ulp2 is maintained in human cells (MSH2/SENP6), and enhanced by Olaparib, a PARP inhibitor that induces the accumulation of single-strand DNA breaks. This identifies SENP6 as a promising new target for the treatment of MMR-deficient cancers. Conclusion Our findings demonstrate the utility of employing evolutionary distance in tractable lower eukaryotes to predict orthologous genetic relationships in higher eukaryotes. Moreover, we provide novel insights into the genome maintenance functions of a critical DNA repair complex and propose a promising targeted treatment for MMR deficient tumors. Electronic supplementary material The online version of this article (doi:10.1186/s13073-014-0068-4) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Elena Tosti
- Department of Cell Biology, Albert Einstein College of Medicine, New York, USA
| | - Joseph A Katakowski
- Department of Microbiology & Immunology, Albert Einstein College of Medicine, New York, USA
| | - Sonja Schaetzlein
- Department of Cell Biology, Albert Einstein College of Medicine, New York, USA
| | - Hyun-Soo Kim
- Department of Cell Biology, Albert Einstein College of Medicine, New York, USA
| | - Colm J Ryan
- Department of Cellular & Molecular Pharmacology, UCSF, San Francisco, USA ; California Institute for Quantitative Biosciences, San Francisco, USA ; School of Medicine and Medical Science, University College Dublin, Dublin, Ireland
| | - Michael Shales
- Department of Cellular & Molecular Pharmacology, UCSF, San Francisco, USA
| | - Assen Roguev
- Department of Cellular & Molecular Pharmacology, UCSF, San Francisco, USA
| | - Nevan J Krogan
- Department of Cellular & Molecular Pharmacology, UCSF, San Francisco, USA ; California Institute for Quantitative Biosciences, San Francisco, USA ; J. David Gladstone Institutes, San Francisco, USA
| | - Deborah Palliser
- Department of Microbiology & Immunology, Albert Einstein College of Medicine, New York, USA
| | | | - Winfried Edelmann
- Department of Cell Biology, Albert Einstein College of Medicine, New York, USA
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Richard GF, Kerrest A, Dujon B. Comparative genomics and molecular dynamics of DNA repeats in eukaryotes. Microbiol Mol Biol Rev 2008; 72:686-727. [PMID: 19052325 PMCID: PMC2593564 DOI: 10.1128/mmbr.00011-08] [Citation(s) in RCA: 339] [Impact Index Per Article: 19.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Repeated elements can be widely abundant in eukaryotic genomes, composing more than 50% of the human genome, for example. It is possible to classify repeated sequences into two large families, "tandem repeats" and "dispersed repeats." Each of these two families can be itself divided into subfamilies. Dispersed repeats contain transposons, tRNA genes, and gene paralogues, whereas tandem repeats contain gene tandems, ribosomal DNA repeat arrays, and satellite DNA, itself subdivided into satellites, minisatellites, and microsatellites. Remarkably, the molecular mechanisms that create and propagate dispersed and tandem repeats are specific to each class and usually do not overlap. In the present review, we have chosen in the first section to describe the nature and distribution of dispersed and tandem repeats in eukaryotic genomes in the light of complete (or nearly complete) available genome sequences. In the second part, we focus on the molecular mechanisms responsible for the fast evolution of two specific classes of tandem repeats: minisatellites and microsatellites. Given that a growing number of human neurological disorders involve the expansion of a particular class of microsatellites, called trinucleotide repeats, a large part of the recent experimental work on microsatellites has focused on these particular repeats, and thus we also review the current knowledge in this area. Finally, we propose a unified definition for mini- and microsatellites that takes into account their biological properties and try to point out new directions that should be explored in a near future on our road to understanding the genetics of repeated sequences.
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Affiliation(s)
- Guy-Franck Richard
- Institut Pasteur, Unité de Génétique Moléculaire des Levures, CNRS, URA2171, Université Pierre et Marie Curie, UFR927, 25 rue du Dr. Roux, F-75015, Paris, France.
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13
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Sharma S, Brosh RM. Unique and important consequences of RECQ1 deficiency in mammalian cells. Cell Cycle 2008; 7:989-1000. [PMID: 18414032 DOI: 10.4161/cc.7.8.5707] [Citation(s) in RCA: 45] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
Abstract
Five members of the RecQ subfamily of DEx-H-containing DNA helicases have been identified in both human and mouse, and mutations in BLM, WRN, and RECQ4 are associated with human diseases of premature aging, cancer, and chromosomal instability. Although a genetic disease has not been linked to RECQ1 mutations, RECQ1 helicase is the most highly expressed of the human RecQ helicases, suggesting an important role in cellular DNA metabolism. Recent advances have elucidated a unique role of RECQ1 to suppress genomic instability. Embryonic fibroblasts from RECQ1-deficient mice displayed aneuploidy, chromosomal instability, and increased load of DNA damage.(1) Acute depletion of human RECQ1 renders cells sensitive to DNA damage and results in spontaneous gamma-H2AX foci and elevated sister chromatid exchanges, indicating aberrant repair of DNA breaks.(2) Consistent with a role in DNA repair, RECQ1 relocalizes to irradiation-induced nuclear foci and associates with chromatin.(2) RECQ1 catalytic activities(3) and interactions with DNA repair proteins(2,4,5) are likely to be important for its molecular functions in genome homeostasis. Collectively, these studies provide the first evidence for an important role of RECQ1 to confer chromosomal stability that is unique from that of other RecQ helicases and suggest its potential involvement in tumorigenesis.
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Affiliation(s)
- Sudha Sharma
- Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, Department of Health and Human Services, Baltimore, Maryland, USA
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14
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Jaendling A, Ramayah S, Pryce DW, McFarlane RJ. Functional characterisation of the Schizosaccharomyces pombe homologue of the leukaemia-associated translocation breakpoint binding protein translin and its binding partner, TRAX. BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 2007; 1783:203-13. [PMID: 18062930 DOI: 10.1016/j.bbamcr.2007.10.014] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/09/2007] [Revised: 09/10/2007] [Accepted: 10/25/2007] [Indexed: 11/25/2022]
Abstract
Translin is a conserved protein which associates with the breakpoint junctions of chromosomal translocations linked with the development of some human cancers. It binds to both DNA and RNA and has been implicated in mRNA metabolism and regulation of genome stability. It has a binding partner, translin-associated protein X (TRAX), levels of which are regulated by the translin protein in higher eukaryotes. In this study we find that this regulatory function is conserved in the lower eukaryotes, suggesting that translin and TRAX have important functions which provide a selective advantage to both unicellular and multi-cellular eukaryotes, indicating that this function may not be tissue-specific in nature. However, to date, the biological importance of translin and TRAX remains unclear. Here we systematically investigate proposals that suggest translin and TRAX play roles in controlling mitotic cell proliferation, DNA damage responses, genome stability, meiotic/mitotic recombination and stability of GT-rich repeat sequences. We find no evidence for translin and/or TRAX primary function in these pathways, indicating that the conserved biochemical function of translin is not implicated in primary pathways for regulating genome stability and/or segregation.
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Affiliation(s)
- Alessa Jaendling
- North West Cancer Research Fund Institute, University of Wales Bangor, Bangor, Gwynedd, LL57 2UW, United Kingdom
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15
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Yamada-Inagawa T, Klar AJS, Dalgaard JZ. Schizosaccharomyces pombe switches mating type by the synthesis-dependent strand-annealing mechanism. Genetics 2007; 177:255-65. [PMID: 17660548 PMCID: PMC2013724 DOI: 10.1534/genetics.107.076315] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022] Open
Abstract
Schizosaccharomyces pombe cells can switch between two mating types, plus (P) and minus (M). The change in cell type occurs due to a replication-coupled recombination event that transfers genetic information from one of the silent-donor loci, mat2P or mat3M, into the expressed mating-type determining mat1 locus. The mat1 locus can as a consequence contain DNA encoding either P or M information. A molecular mechanism, known as synthesis-dependent strand annealing, has been proposed for the underlying recombination event. A key feature of this model is that only one DNA strand of the donor locus provides the information that is copied into the mat1. Here we test the model by constructing strains that switch using two different mutant P cassettes introduced at the donor loci, mat2 and mat3. We show that in such strains wild-type P-cassette DNA is efficiently generated at mat1 through heteroduplex DNA formation and repair. The present data provide an in vivo genetic test of the proposed molecular recombination mechanism.
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16
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Holmberg C, Fleck O, Hansen HA, Liu C, Slaaby R, Carr AM, Nielsen O. Ddb1 controls genome stability and meiosis in fission yeast. Genes Dev 2005; 19:853-62. [PMID: 15805471 PMCID: PMC1074322 DOI: 10.1101/gad.329905] [Citation(s) in RCA: 80] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
The human UV-damaged DNA-binding protein Ddb1 associates with cullin 4 ubiquitin ligases implicated in nucleotide excision repair (NER). These complexes also contain the signalosome (CSN), but NER-relevant ubiquitination targets have not yet been identified. We report that fission yeast Ddb1, Cullin 4 (Pcu4), and CSN subunits Csn1 and Csn2 are required for degradation of the ribonucleotide reductase (RNR) inhibitor protein Spd1. Ddb1-deficient cells have >20-fold increased spontaneous mutation rate. This is partly dependent on the error-prone translesion DNA polymerases. Spd1 deletion substantially reduced the mutation rate, suggesting that insufficient RNR activity accounts for approximately 50% of observed mutations. Epistasis analysis indicated that Ddb1 contributed to mutation avoidance and tolerance to DNA damage in a pathway distinct from NER. Finally, we show that Ddb1/Csn1/Cullin 4-mediated Spd1 degradation becomes essential when cells differentiate into meiosis. These results suggest that Ddb1, along with Cullin 4 and the signalosome, constitute a major pathway controlling genome stability, repair, and differentiation via RNR regulation.
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Affiliation(s)
- Christian Holmberg
- Department of Genetics, Institute of Molecular Biology, University of Copenhagen, DK-1353 Copenhagen K, Denmark
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17
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Doherty KM, Sharma S, Uzdilla LA, Wilson TM, Cui S, Vindigni A, Brosh RM. RECQ1 helicase interacts with human mismatch repair factors that regulate genetic recombination. J Biol Chem 2005; 280:28085-94. [PMID: 15886194 DOI: 10.1074/jbc.m500265200] [Citation(s) in RCA: 73] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/30/2023] Open
Abstract
Understanding the molecular and cellular functions of RecQ helicases has attracted considerable interest since several human diseases characterized by premature aging and/or cancer have been genetically linked to mutations in genes of the RecQ family. Although a human disease has not yet been genetically linked to a mutation in RECQ1, the prominent roles of RecQ helicases in the maintenance of genome stability suggest that RECQ1 helicase is likely to be important in vivo. To acquire a better understanding of RECQ1 cellular and molecular functions, we have investigated its protein interactions. Using a co-immunoprecipitation approach, we have identified several DNA repair factors that are associated with RECQ1 in vivo. Direct physical interaction of these repair factors with RECQ1 was confirmed with purified recombinant proteins. Importantly, RECQ1 stimulates the incision activity of human exonuclease 1 and the mismatch repair recognition complex MSH2/6 stimulates RECQ1 helicase activity. These protein interactions suggest a role of RECQ1 in a pathway involving mismatch repair factors. Regulation of genetic recombination, a proposed role for RecQ helicases, is supported by the identified RECQ1 protein interactions and is discussed.
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Affiliation(s)
- Kevin M Doherty
- Laboratory of Molecular Gerontology, NIA, National Institutes of Health, Baltimore, Maryland 21224, USA
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18
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Tran PT, Erdeniz N, Symington LS, Liskay RM. EXO1-A multi-tasking eukaryotic nuclease. DNA Repair (Amst) 2004; 3:1549-59. [PMID: 15474417 DOI: 10.1016/j.dnarep.2004.05.015] [Citation(s) in RCA: 167] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2004] [Accepted: 05/26/2004] [Indexed: 12/14/2022]
Abstract
Exo1 was first isolated as a 5' --> 3' exonuclease activity induced during meiosis in fission yeast and since that time has been implicated in a multitude of eukaryotic DNA metabolic pathways that include DNA repair, recombination, replication, and telomere integrity. Involvement in multiple pathways affecting genomic stability makes EXO1 a logical target for mutation during oncogenesis. Here, we review studies in several experimental systems that shed light on the role of Exo1 in these DNA transaction pathways, particularly those that may relate to oncogenesis.
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Affiliation(s)
- Phuoc T Tran
- Graduate Medical Education, St. Mary's Medical Center, San Francisco, CA 94117, USA
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19
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Estes S, Phillips PC, Denver DR, Thomas WK, Lynch M. Mutation accumulation in populations of varying size: the distribution of mutational effects for fitness correlates in Caenorhabditis elegans. Genetics 2004; 166:1269-79. [PMID: 15082546 PMCID: PMC1470770 DOI: 10.1534/genetics.166.3.1269] [Citation(s) in RCA: 82] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/20/2023] Open
Abstract
The consequences of mutation for population-genetic and evolutionary processes depend on the rate and, especially, the frequency distribution of mutational effects on fitness. We sought to approximate the form of the distribution of mutational effects by conducting divergence experiments in which lines of a DNA repair-deficient strain of Caenorhabditis elegans, msh-2, were maintained at a range of population sizes. Assays of these lines conducted in parallel with the ancestral control suggest that the mutational variance is dominated by contributions from highly detrimental mutations. This was evidenced by the ability of all but the smallest population-size treatments to maintain relatively high levels of mean fitness even under the 100-fold increase in mutational pressure caused by knocking out the msh-2 gene. However, we show that the mean fitness decline experienced by larger populations is actually greater than expected on the basis of our estimates of mutational parameters, which could be consistent with the existence of a common class of mutations with small individual effects. Further, comparison of the total mutation rate estimated from direct sequencing of DNA to that detected from phenotypic analyses implies the existence of a large class of evolutionarily relevant mutations with no measurable effect on laboratory fitness.
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Affiliation(s)
- Suzanne Estes
- Center for Ecology and Evolutionary Biology, University of Oregon, Eugene, Oregon 97403, USA.
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20
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Abstract
DNA mismatch repair (MMR) guards the integrity of the genome in virtually all cells. It contributes about 1000-fold to the overall fidelity of replication and targets mispaired bases that arise through replication errors, during homologous recombination, and as a result of DNA damage. Cells deficient in MMR have a mutator phenotype in which the rate of spontaneous mutation is greatly elevated, and they frequently exhibit microsatellite instability at mono- and dinucleotide repeats. The importance of MMR in mutation avoidance is highlighted by the finding that defects in MMR predispose individuals to hereditary nonpolyposis colorectal cancer. In addition to its role in postreplication repair, the MMR machinery serves to police homologous recombination events and acts as a barrier to genetic exchange between species.
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Affiliation(s)
- Mark J Schofield
- Genetics and Biochemistry Branch, National Institute of Diabetes, and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
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21
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Tran PT, Erdeniz N, Dudley S, Liskay RM. Characterization of nuclease-dependent functions of Exo1p in Saccharomyces cerevisiae. DNA Repair (Amst) 2002; 1:895-912. [PMID: 12531018 DOI: 10.1016/s1568-7864(02)00114-3] [Citation(s) in RCA: 106] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
Exo1p is a member of the Rad2p family of structure-specific nucleases that contain conserved N and I nuclease domains. Exo1p has been implicated in numerous DNA metabolic processes, such as recombination, double-strand break repair and DNA mismatch repair (MMR). In this report, we describe in vitro and in vivo characterization of full-length wild-type and mutant forms of Exo1p. Herein, we demonstrate that full-length yeast Exo1p possesses an intrinsic 5'-3' exonuclease activity as reported previously, but also possesses a flap-endonuclease activity. Our study indicates that Exo1p shares similar, but not identical structure-function relationships to other characterized members of the Rad2p family in the N and I nuclease domains. The two exo1p mutants we examined, showed deficiencies for both double-stranded DNA (dsDNA) 5'-3' exonuclease and flap-endonuclease activities. Examining the genetic interaction of these two exo1 mutations with rad27Delta suggest that the Exo1p flap-endonuclease activity and not the dsDNA 5'-3' exonuclease is redundant to Rad27p for viability. In addition, our in vivo results also indicate that many exo1Delta phenotypes are dependent on the complete catalytic activities of Exo1p. Finally, our findings plus those of other investigators suggest that Exo1p functions both in a catalytic and a structural capacity during DNA MMR.
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Affiliation(s)
- Phuoc T Tran
- Department of Molecular and Medical Genetics, Oregon Health & Science University, Portland, OR 97201, USA
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22
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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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23
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Current awareness on yeast. Yeast 2001; 18:1269-76. [PMID: 11561294 DOI: 10.1002/yea.689] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022] Open
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24
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Hohl M, Christensen O, Kunz C, Naegeli H, Fleck O. Binding and repair of mismatched DNA mediated by Rhp14, the fission yeast homologue of human XPA. J Biol Chem 2001; 276:30766-72. [PMID: 11408483 DOI: 10.1074/jbc.m104039200] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Rhp14 of Schizosaccharomyces pombe is homologous to human XPA and Saccharomyces cerevisiae Rad14, which act in nucleotide excision repair of DNA damages induced by ultraviolet light and chemical agents. Cells with disrupted rhp14 were highly sensitive to ultraviolet light, and epistasis analysis with swi10 (nucleotide excision repair) and rad2 (Uve1-dependent ultraviolet light damage repair pathway) revealed that Rhp14 is an important component of nucleotide excision repair for ultraviolet light-induced damages. Moreover, defective rhp14 caused instability of a GT repeat, similar to swi10 and synergistically with msh2 and exo1. Recombinant Rhp14 with an N-terminal hexahistidine tag was purified from Escherichia coli. Complementation studies with a rhp14 mutant demonstrated that the tagged Rhp14 is functional in repair of ultraviolet radiation-induced damages and in mitotic mutation avoidance. In bandshift assays, Rhp14 showed a preference to substrates with mismatched and unpaired nucleotides. Similarly, XPA bound more efficiently to C/C, A/C, and T/C mismatches than to homoduplex DNA. Our data show that mismatches and loops in DNA are substrates of nucleotide excision repair. Rhp14 is likely part of the recognition complex but alone is not sufficient for the high discrimination of nucleotide excision repair for modified DNA.
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Affiliation(s)
- M Hohl
- Institute of Cell Biology, University of Bern, Baltzerstrasse 4, CH-3012 Bern, Switzerland
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Tornier C, Bessone S, Varlet I, Rudolph C, Darmon M, Fleck O. Requirement for Msh6, but not for Swi4 (Msh3), in Msh2-dependent repair of base-base mismatches and mononucleotide loops in Schizosaccharomyces pombe. Genetics 2001; 158:65-75. [PMID: 11333218 PMCID: PMC1461642 DOI: 10.1093/genetics/158.1.65] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/14/2022] Open
Abstract
The msh6 mismatch repair gene of Schizosaccharomyces pombe was cloned, sequenced, and inactivated. Strains bearing all combinations of inactivated msh6, msh2, and swi4 (the S. pombe MSH3 ortholog) alleles were tested for their defects in mitotic and meiotic mismatch repair. Mitotic mutation rates were similarly increased in msh6 and msh2 mutants, both for reversion of a base-base substitution as well as of an insertion of one nucleotide in a mononucleotide run. Tetrad analysis and intragenic two-factor crosses revealed that meiotic mismatch repair was affected in msh6 to the same extent as in msh2 background. In contrast, loss of Swi4 likely did not cause a defect in mismatch repair, but rather resulted in reduced recombination frequency. Consistently, a mutated swi4 caused a two- to threefold reduction of recombinants in intergenic crosses, while msh2 and msh6 mutants were not significantly different from wild type. In summary, our study showed that Msh6 plays the same important role as Msh2 in the major mismatch repair pathway of S. pombe, while Swi4 rather functions in recombination.
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Affiliation(s)
- C Tornier
- Laboratory of Medical Biochemistry, University of Bordeaux 2, F-33076 Bordeaux Cedex, France
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