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Results for “Recombinational DNA Repair”

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At least 145 records · Page 8Linked to original sources

PubMed1993

Resolution of Holliday intermediates in recombination and DNA repair: indirect suppression of ruvA, ruvB, and ruvC mutations.

The ruvA, ruvB, and ruvC genes of Escherichia coli provide activities that catalyze branch migration and resolution of Holliday junction intermediates in recombination. Mutation of any one of these genes interferes with recombination and reduces the ability of the cell to repair damage to DNA. A suppressor of ruv mutations was identified on the basis of its ability to restore resistance to mitomycin and UV light and to allow normal levels of recombination in a recBC sbcBC strain carrying a Tn10 insertion in ruvA. The mutation responsible was located at 12.5 min on the genetic map and defines a new locus which has been designated rus. The rus suppressor works just as well in recBC sbcA and rec+ sbc+ backgrounds and is not allele specific. Mutations in ruvB and ruvC are suppressed to an intermediate level, except when ruvA is also inactive, in which case suppression is complete. In all cases, suppression depends on RecG protein, a DNA-dependent ATPase that catalyzes branch migration of Holliday junctions. The rus mutation activates an additional factor that probably works with RecG to process Holliday junction intermediates independently of the RuvAB and RuvC proteins. The possibility that this additional factor is a junction-specific resolvase is discussed.

Bacterial Proteins↗

PubMed1978

Cell-cycle variation in the induction of lethality and mitotic recombination after treatment with UV and nitrous acid in the yeast, Saccharomyces cerevisiae.

Exponentially growing yeast cultures separated into discrete periods of the cell cycle by zonal rotor centrifugation show cyclic variation in both UV and nitrous acid induced cell lethality, mitotic gene conversion and mitotic crossing-over. Maximum cell survival after UV treatment was observed in the S and G2 phases of the cell cycle at a time when UV induction of both types of mitotic recombination was at a minimum. In contrast, cell inactivation by the chemical mutagen nitrous acid showed a single discrete period of sensitivity which occurred in S phase cells which are undergoing DNA synthesis. Mitotic gene conversion and mitotic crossing-over were induced by nitrous acid in cells at all stages of the cell cycle with a peak of induction of both events occurring at the time of maximum cell lethality. The lack of correlation observed between maximum cell and the maximum induction of mitotic intragenic recombination suggest that other DNA-repair mechanisms besides DNA-recombination repair are involved in the recovery of inactivated yeast cells during the cell cycle.

Cell Cycle↗

PubMed2005

Multiple roles of vertebrate REV genes in DNA repair and recombination.

In yeast, Rev1, Rev3, and Rev7 are involved in translesion synthesis over various kinds of DNA damage and spontaneous and UV-induced mutagenesis. Here, we disrupted Rev1, Rev3, and Rev7 in the chicken B-lymphocyte line DT40. REV1-/- REV3-/- REV7-/- cells showed spontaneous cell death, chromosomal instability/fragility, and hypersensitivity to various genotoxic treatments as observed in each of the single mutants. Surprisingly, the triple-knockout cells showed a suppressed level of sister chromatid exchanges (SCEs), which may reflect postreplication repair events mediated by homologous recombination, while each single mutant showed an elevated SCE level. Furthermore, REV1-/- cells as well as triple mutants showed a decreased level of immunoglobulin gene conversion, suggesting participation of Rev1 in a recombination-based pathway. The present study gives us a new insight into cooperative function of three Rev molecules and the Polzeta (Rev3-Rev7)-independent role of Rev1 in vertebrate cells.

Animals↗

PubMed2004

Transformation and DNA repair: linkage by DNA recombination.

The stability of microbial genomes is constantly challenged by horizontal gene transfer, recombination and DNA damage. Mechanisms for rapid genome variation, adaptation and maintenance are a necessity to ensure microbial fitness and survival in changing environments. Indeed, genome sequences reveal that most, if not all, bacterial species have numerous gene functions for DNA repair and recombination. These important topics were addressed at the Second Genome Maintenance Meeting (GMM2).

DNA Damage↗

PubMed1988

Present and future applications of new biotechnology to problems of radiation damage and repair.

Recombinant DNA technology is having a profound impact on the way radiation responses are being studied today, and the impact should be even greater in the future. Five basic tools of the new technology are described in this article: restriction endonucleases, hybridization, DNA cloning, DNA sequencing, and specialized uses of cloning vectors. Examples of the present uses of these tools for the elucidation and modification of radiation responses are given, along with some likely future directions.

Animals↗

PubMed2004

Analysis of recombinational repair of DNA double-strand breaks in mammalian cells with I-SceI nuclease.

Eukaryotes repair DNA double-strand breaks (DSBs) by homologous recombination (HR) or by nonhomologous end-joining (NHEJ). DSBs are a natural consequence of DNA metabolism, occurring, for example, during DNA replication and meiosis. DSBs are also induced by chemicals and radiation. I-SceI endonuclease recognizes an 18-bp sequence with little degeneracy; therefore I-SceI is highly specific, and its recognition sequence is predicted to occur by chance less than once in even the largest known genomes. As such, I-SceI can be used to introduce a DSB into a defined (engineered) site in a mammalian chromosome, and this facilitates detailed studies of DSB repair. DSBs induced in repeated regions can be repaired by several different HR processes, including gene conversion with or without associated crossovers, or single-strand annealing. The specific types of HR events that can be scored depend on the configuration of the repeated regions and whether selection for recombinants is imposed. Nonselective assays detect both HR and NHEJ events. This chapter focuses on the systems for delivering I-SceI nuclease to mammalian cells and the strategies for detecting various outcomes of DSB repair.

Amino Acid Sequence↗

PubMed2001

C. elegans mre-11 is required for meiotic recombination and DNA repair but is dispensable for the meiotic G(2) DNA damage checkpoint.

We investigated the roles of Caenorhabditis elegans MRE-11 in multiple cellular processes required to maintain genome integrity. Although yeast Mre11 is known to promote genome stability through several diverse pathways, inviability of vertebrate cells that lack Mre11 has hindered elucidation of the in vivo roles of this conserved protein in metazoan biology. Worms homozygous for an mre-11 null mutation are viable, allowing us to demonstrate in vivo requirements for MRE-11 in meiotic recombination and DNA repair. In mre-11 mutants, meiotic crossovers are not detected, and oocyte chromosomes lack chiasmata but appear otherwise intact. gamma-irradiation of mre-11 mutant germ cells during meiotic prophase eliminates progeny survivorship and induces chromosome fragmentation and other cytologically visible abnormalities, indicating a defect in repair of radiation-induced chromosome damage. Whereas mre-11 mutant germ cells are repair-deficient, they retain function of the meiotic G(2) DNA damage checkpoint that triggers germ cell apoptosis in response to ionizing radiation. Although mre-11/mre-11 animals derived from heterozygous parents are viable and produce many embryos, there is a marked drop both in the number and survivorship of embryos produced by succeeding generations. This progressive loss of fecundity and viability indicates that MRE-11 performs a function essential for maintaining reproductive capacity in the species.

Amino Acid Sequence↗

PubMed1990

Induced rates of mitotic crossing over and possible mitotic gene conversion per wing anlage cell in Drosophila melanogaster by X rays and fission neutrons.

As a model for chromosome aberrations, radiation-induced mitotic recombination of mwh and flr genes in Drosophila melanogaster strain (mwh +/+ flr) was quantitatively studied. Fission neutrons were five to six times more effective than X rays per unit dose in producing either crossover-mwh/flr twins and mwh singles-or flr singles, indicating that common processes are involved in the production of crossover and flr singles. The X-ray-induced rate/wing anlage cell/Gy for flr singles was 1 X 10(-5), whereas that of crossover was 2 x 10(-4); the former and the latter rate are of the same order of magnitude as those of gene conversion and crossover in yeast, respectively. Thus, we conclude that proximal-marker "flr" singles induced in the transheterozygote are gene convertants. Using the model based on yeast that recombination events result from repair of double-strand breaks or gaps, we propose that mitotic recombination in the fly is a secondary result of recombinational DNA repair. Evidence for recombinational misrepair in the fly is given. The relative ratio of radiation-induced mitotic crossover to spontaneous meiotic crossover is one order of magnitude higher in the fly than in yeast and humans.

Animals↗

PubMed2005

Good timing in the cell cycle for precise DNA repair by BRCA1.

It is now clear that large DNA-binding proteins have evolved in mammals to orchestrate the relatively ancient process of DNA recombinational repair. These proteins are recruited to accurately repair DNA double strand breaks (DSBs)--the frequent, potentially lethal and mutagenic lesions in the genomes of all organisms. An essential mammalian regulator of DSB repair is BRCA1. Heterozygous BRCA1 mutations predispose individuals to breast, ovarian and other secondary cancers. BRCA1-defective cells exhibit reduced DSB repair, sensitivity to a wide range of DNA damaging agents, genomic instability and defects in the S-phase checkpoint, transcription and chromatin remodelling. DSBs can be repaired by RAD51/RPA-dependent homologous recombination (HR) or DNA-PK-dependent non-homologous end-joining (NHEJ). Both of these pathways can be imprecise and mutagenic. BRCA1 plays a central role in promoting accurate repair by both HR and NHEJ. Consistent with recent evidence, we have assembled a novel cell-cycle-dependent model in which DNA-PK inhibits RPA in S-phase of the cell cycle, while BRCA1 inhibits the exonuclease processivity of the MRE11/RAD50/NBS1 (MRN) complex and facilitates the removal of RPA in S and G2 phase. This model provides an explanation for how BRCA1 promotes accurate DSB repair during various phases of the cell cycle and also accounts for the dual effects that BRCA1 and MRN activity have upon DNA repair and S-phase arrest.

Acid Anhydride Hydrolases↗

PubMed2003

Identification and characterization of BCL-3-binding protein: implications for transcription and DNA repair or recombination.

A putative oncogene bcl-3 was originally identified and cloned at the breakpoint in the recurring chromosome translocation t(14;19) found in some cases of B cell chronic lymphocytic leukemia. Studies of bcl-3-deficient mice demonstrated a critical role for bcl-3 in the development of a normal immune response and the formation of germinal centers in secondary lymphoid organs. However, the molecular mechanism that underlies B cell leukemogenesis and the knockout mouse phenotype remains unclear. Here we have identified and characterized BCL-3-binding protein (B3BP) as a protein interacting specifically with the bcl-3 gene product (BCL-3) by a yeast two-hybrid screen. We found that B3BP associates with not only BCL-3 but also p300/CBP histone acetyltransferases. The N-terminal region of B3BP that contains the ATP-binding site is important for the interaction with BCL-3 and p300/CBP. Homology searches indicate that the ATP-binding region of B3BP, which contains a typical Walker-type ATP-binding P-loop, most resembles that of 2',3'-cyclic nucleotide 3'-phosphodiesterase of mammals and polynucleotide kinase of T4 bacteriophage. In fact B3BP shows intrinsic ATP binding and hydrolyzing activity. Furthermore, we demonstrated that B3BP is a 5'-polynucleotide kinase. We also found a small MutS-related domain, which is thought to be involved in the DNA repair or recombination reaction, in the C-terminal region of B3BP, and it shows nicking endonuclease activity. These observations might help to gain new insights into the function of BCL-3 and p300/CBP, especially the coupling of transcription with repair or recombination.

Adenosine Triphosphate↗

PubMed1994

Cloning of human and mouse genes homologous to RAD52, a yeast gene involved in DNA repair and recombination.

The RAD52 gene of Saccharomyces cerevisiae is required for recombinational repair of double-strand breaks. Using degenerate oligonucleotides based on conserved amino acid sequences of RAD52 and rad22, its counterpart from Schizosaccharomyces pombe, RAD52 homologs from man and mouse were cloned by the polymerase chain reaction. DNA sequence analysis revealed an open reading frame of 418 amino acids for the human RAD52 homolog and of 420 amino acid residues for the mouse counterpart. The identity between the two proteins is 69% and the overall similarity 80%. The homology of the mammalian proteins with their counterparts from yeast is primarily concentrated in the N-terminal region. Low amounts of RAD52 RNA were observed in adult mouse tissues. A relatively high level of gene expression was observed in testis and thymus, suggesting that the mammalian RAD52 protein, like its homolog from yeast, plays a role in recombination. The mouse RAD52 gene is located near the tip of chromosome 6 in region G3. The human equivalent maps to region p13.3 of chromosome 12. Until now, this human chromosome has not been implicated in any of the rodent mutants with a defect in the repair of double-strand breaks.

Amino Acid Sequence↗

PubMed2000

Contribution of base excision repair, nucleotide excision repair, and DNA recombination to alkylation resistance of the fission yeast Schizosaccharomyces pombe.

DNA damage is unavoidable, and organisms across the evolutionary spectrum possess DNA repair pathways that are critical for cell viability and genomic stability. To understand the role of base excision repair (BER) in protecting eukaryotic cells against alkylating agents, we generated Schizosaccharomyces pombe strains mutant for the mag1 3-methyladenine DNA glycosylase gene. We report that S. pombe mag1 mutants have only a slightly increased sensitivity to methylation damage, suggesting that Mag1-initiated BER plays a surprisingly minor role in alkylation resistance in this organism. We go on to show that other DNA repair pathways play a larger role than BER in alkylation resistance. Mutations in genes involved in nucleotide excision repair (rad13) and recombinational repair (rhp51) are much more alkylation sensitive than mag1 mutants. In addition, S. pombe mutant for the flap endonuclease rad2 gene, whose precise function in DNA repair is unclear, were also more alkylation sensitive than mag1 mutants. Further, mag1 and rad13 interact synergistically for alkylation resistance, and mag1 and rhp51 display a surprisingly complex genetic interaction. A model for the role of BER in the generation of alkylation-induced DNA strand breaks in S. pombe is discussed.

Adenine↗

PubMed2002

Differential expression and requirements for Schizosaccharomyces pombe RAD52 homologs in DNA repair and recombination.

In fission yeast two RAD52 homologs have been identified, rad22A(+) and rad22B(+). Two-hybrid experiments and GST pull-down assays revealed physical interaction between Rad22A and Rad22B, which is dependent on the N-terminal regions. Interaction with Rhp51 is dependent on the C-terminal parts of either protein. Both Rad22A and Rad22B also interact with RPA. The expression of rad22B(+) in mitotically dividing cells is very low in comparison with rad22A(+) but is strongly enhanced after induction of meiosis, in contrast to rad22A(+). Rad22B mutant cells are not hypersensitive to DNA-damaging agents (X-rays, UV and cisplatin) and display normal levels of recombination. In these respects the Schizosaccharomyces pombe rad22B mutant resembles the weak phenotype of vertebrate cells deficient for RAD52. Mutation of rad22A(+) leads to severe sensitivity to DNA-damaging agents and to defects in recombination. In a rad22Arad22B double mutant a further increase in sensitivity to DNA-damaging agents and additional mitotic recombination defects were observed. The data presented here indicate that Rad22A and Rad22B have overlapping roles in repair and recombination, although specialized functions for each protein cannot be excluded.

DNA Helicases↗

PubMed1995

A nuclear mutation defective in mitochondrial recombination in yeast.

Homologous recombination (crossing over and gene conversion) is generally essential for heritage and DNA repair, and occasionally causes DNA aberrations, in nuclei of eukaryotes. However, little is known about the roles of homologous recombination in the inheritance and stability of mitochondrial DNA which is continuously damaged by reactive oxygen species, by-products of respiration. Here, we report the first example of a nuclear recessive mutation which suggests an essential role for homologous recombination in the stable inheritance of mitochondrial DNA. For the detection of this class of mutants, we devised a novel procedure, 'mitochondrial crossing in haploid', which has enabled us to examine many mutant clones. Using this procedure, we examined mutants of Saccharomyces cerevisiae that showed an elevated UV induction of respiration-deficient mutations. We obtained a mutant that was defective in both the omega-intron homing and Endo.SceI-induced homologous gene conversion. We found that the mutant cells are temperature sensitive in the maintenance of mitochondrial DNA. A tetrad analysis indicated that elevated UV induction of respiration-deficient mutations, recombination deficiency and temperature sensitivity are all caused by a single nuclear mutation (mhr1) on chromosome XII. The pleiotropic characteristics of the mutant suggest an essential role for the MHR1 gene in DNA repair, recombination and the maintenance of DNA in mitochondria.

Base Sequence↗

PubMed2003

In vitro and in vivo studies of MutS, MutL and MutH mutants: correlation of mismatch repair and DNA recombination.

We have used the recently determined crystal structures of Escherichia coli (E. coli) MutS, MutL and MutH to guide construction of 47 amino-acid substitutions in these proteins and analyzed their behavior in mismatch repair and recombination in vitro and in vivo. We find that the active site of the MutH endonuclease is composed of regions from two separate structural domains and that the C-terminal 5 residues of MutH influence both DNA binding and cleavage. We also find that the non-specific DNA-binding activity of MutL is required for mismatch repair and probably functions after strand cleavage by MutH. Alteration of residues in either the mismatch recognition domain, the ATPase active site, or the domain interfaces linking the two activities can diminish the differential binding of MutS to homoduplex versus heteroduplex and results in the loss of mismatch-specific MutH activation. Finally, every mutation that abolishes mismatch repair is deficient in blocking homeologous recombination, suggesting that mismatch repair and prevention of homeologous recombination use the same MutS-MutL complexes for signaling in E. coli.

Adenosine Triphosphatases↗

PubMed1995

Interactions among mutations affecting spontaneous mutation, mitotic recombination, and DNA repair in yeast.

The mutant alleles mms9-1, mms13-1, or mms21-1 of Saccharomyces cerevisiae confer pleiotropic effects, including sensitivity to the alkylating agent methyl methanesulfonate, elevations in spontaneous mutation and mitotic recombination, defects in meiosis, and cross-sensitivity to radiation. We constructed double-mutant strains containing an mms mutation and a defect in either excision repair, mutagenic repair, or recombinational repair and measured the levels of spontaneous mutation and mitotic recombination. Double mutants lacking excision repair show elevations in spontaneous mutation but with predominantly unchanged levels of mitotic recombination. RAD52 function was required for the expression of the hyper-recombination phenotype of the mms9-1, mms13-1, and mms21-1 alleles; double mutants displayed the very low recombination levels characteristic of rad52 mutants. Phenotypes of double mutants containing one of the mms alleles and either of the hyper-recombination/mutator rad6-1 or rad3-102 alleles suggest that the mutagenic lesions in mms strains may not be identical to the recombinogenic lesions.

Adenosine Triphosphatases↗

PubMed1984

An analysis of DNA repair and recombination functions of bacteriophage T4 by means of suppressors: the role of das.

Previous studies have indicated that the bacteriophage T4 das mutations partially suppressed the DNA replication defects in gene 46 and 47 mutations. Here it is shown that the das mutation also suppresses the DNA repair defects but not the DNA replication defects of the uvsX and uvsY mutations. In contrast, the das mutation suppressed both the DNA replication and repair defects of gene 46 and 47 mutations. These characteristics of das as well as those of the other suppressors, including uvsW(dar) and two new suppressors sur and uvsU have been used for the analysis of the DNA repair pathway. Based on the functions of these suppressors, a sequence in which the gene products in this pathway might act is suggested.

DNA Repair↗

PubMed2004

Proteomics of herpes simplex virus replication compartments: association of cellular DNA replication, repair, recombination, and chromatin remodeling proteins with ICP8.

In this study, we have used immunoprecipitation and mass spectrometry to identify over 50 cellular and viral proteins that are associated with the herpes simplex virus 1 (HSV-1) ICP8 single-stranded DNA-binding protein. Many of the coprecipitating cellular proteins are known members of large cellular complexes involved in (i) DNA replication or damage repair, including RPA and MSH6; (ii) nonhomologous and homologous recombination, including the catalytic subunit of the DNA-dependent protein kinase, Ku86, and Rad50; and (iii) chromatin remodeling, including BRG1, BRM, hSNF2H, BAF155, mSin3a, and histone deacetylase 2. It appears that DNA mediates the association of certain proteins with ICP8, while more direct protein-protein interactions mediate the association with other proteins. A number of these proteins accumulate in viral replication compartments in the infected cell nucleus, indicating that these proteins may have a role in viral replication. WRN, which functions in cellular recombination pathways via its helicase and exonuclease activities, is not absolutely required for viral replication, as viral yields are only very slightly, if at all, decreased in WRN-deficient human primary fibroblasts compared to control cells. In Ku70-deficient murine embryonic fibroblasts, viral yields are increased by almost 50-fold, suggesting that the cellular nonhomologous end-joining pathway inhibits HSV replication. We hypothesize that some of the proteins coprecipitating with ICP8 are involved in HSV replication and may give new insight into viral replication mechanisms.

Animals↗