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Molecular Cell Oct 2021Currently favored models for meiotic recombination posit that both noncrossover and crossover recombination are initiated by DNA double-strand breaks but form by...
Currently favored models for meiotic recombination posit that both noncrossover and crossover recombination are initiated by DNA double-strand breaks but form by different mechanisms: noncrossovers by synthesis-dependent strand annealing and crossovers by formation and resolution of double Holliday junctions centered around the break. This dual mechanism hypothesis predicts different hybrid DNA patterns in noncrossover and crossover recombinants. We show that these predictions are not upheld, by mapping with unprecedented resolution parental strand contributions to recombinants at a model locus. Instead, break repair in both noncrossovers and crossovers involves synthesis-dependent strand annealing, often with multiple rounds of strand invasion. Crossover-specific double Holliday junction formation occurs via processes involving branch migration as an integral feature, one that can be separated from repair of the break itself. These findings reveal meiotic recombination to be a highly dynamic process and prompt a new view of the relationship between crossover and noncrossover recombination.
Topics: Crossing Over, Genetic; DNA Breaks, Double-Stranded; DNA, Cruciform; DNA, Fungal; Meiosis; Recombinational DNA Repair; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sister Chromatid Exchange; Templates, Genetic
PubMed: 34453891
DOI: 10.1016/j.molcel.2021.08.003 -
RNA Biology 2016The transfer of genetic information from RNA to DNA is considered an extraordinary process in molecular biology. Despite the fact that cells transcribe abundant amount...
The transfer of genetic information from RNA to DNA is considered an extraordinary process in molecular biology. Despite the fact that cells transcribe abundant amount of RNA with a wide range of functions, it has been difficult to uncover whether RNA can serve as a template for DNA repair and recombination. An increasing number of experimental evidences suggest a direct role of RNA in DNA modification. Recently, we demonstrated that endogenous transcript RNA can serve as a template to repair a DNA double-strand break (DSB), the most harmful DNA lesion, not only indirectly via formation of a DNA copy (cDNA) intermediate, but also directly in a homology driven mechanism in budding yeast. These results point out that the transfer of genetic information from RNA to DNA is more general than previously thought. We found that transcript RNA is more efficient in repairing a DSB in its own DNA (in cis) than in a homologous but ectopic locus (in trans). Here, we summarize current knowledge about the process of RNA-driven DNA repair and recombination, and provide further data in support of our model of DSB repair by transcript RNA in cis. We show that a DSB is precisely repaired predominately by transcript RNA and not by residual cDNA in conditions in which formation of cDNA by reverse transcription is inhibited. Additionally, we demonstrate that defects in ribonuclease (RNase) H stimulate precise DSB repair by homologous RNA or cDNA sequence, and not by homologous DNA sequence carried on a plasmid. These results highlight an antagonistic role of RNase H in RNA-DNA recombination. Ultimately, we discuss several questions that should be addressed to better understand mechanisms and implications of RNA-templated DNA repair and recombination.
Topics: DNA; DNA Breaks, Double-Stranded; DNA Repair; RNA; Recombination, Genetic; Reverse Transcription; Ribonuclease H
PubMed: 26637053
DOI: 10.1080/15476286.2015.1116676 -
BMC Microbiology Feb 2017Natural transformation enables acquisition of adaptive traits and drives genome evolution in prokaryotes. Yet, the selective forces responsible for the evolution and...
BACKGROUND
Natural transformation enables acquisition of adaptive traits and drives genome evolution in prokaryotes. Yet, the selective forces responsible for the evolution and maintenance of natural transformation remain elusive since taken-up DNA has also been hypothesized to provide benefits such as nutrients or templates for DNA repair to individual cells.
RESULTS
We investigated the immediate effects of DNA uptake and recombination on the naturally competent bacterium Acinetobacter baylyi in both benign and genotoxic conditions. In head-to-head competition experiments between DNA uptake-proficient and -deficient strains, we observed a fitness benefit of DNA uptake independent of UV stress. This benefit was found with both homologous and heterologous DNA and was independent of recombination. Recombination with taken-up DNA reduced survival of transformed cells with increasing levels of UV-stress through interference with nucleotide excision repair, suggesting that DNA strand breaks occur during recombination attempts with taken-up DNA. Consistent with this, we show that absence of RecBCD and RecFOR recombinational DNA repair pathways strongly decrease natural transformation.
CONCLUSIONS
Our data show a physiological benefit of DNA uptake unrelated to recombination. In contrast, recombination during transformation is a strand break inducing process that represents a previously unrecognized cost of natural transformation.
Topics: Acinetobacter; Bacterial Proteins; Biological Evolution; Cost-Benefit Analysis; DNA Damage; DNA Repair; DNA, Bacterial; Exodeoxyribonuclease V; Gene Deletion; Gene Transfer, Horizontal; Genes, Bacterial; Membrane Proteins; Mutation; Phenotype; Recombination, Genetic; Stress, Physiological; Survival; Transformation, Bacterial; Ultraviolet Rays
PubMed: 28202049
DOI: 10.1186/s12866-017-0953-2 -
Mutation Research Dec 2006Vanillin (VAN) and cinnamaldehyde (CIN) are dietary antimutagens that effectively inhibit both induced and spontaneous mutations. We have shown previously that VAN and...
Vanillin (VAN) and cinnamaldehyde (CIN) are dietary antimutagens that effectively inhibit both induced and spontaneous mutations. We have shown previously that VAN and CIN reduced the spontaneous mutant frequency in Salmonella TA104 (hisG428, rfa, DeltauvrB, pKM101) by approximately 50% and that both compounds significantly reduced mutations at GC sites but not at AT sites. Previous studies have suggested that VAN and CIN may reduce mutations in bacterial model systems by modulating DNA repair pathways, particularly by enhancing recombinational repair. To further explore the basis for inhibition of spontaneous mutation by VAN and CIN, we have determined the effects of these compounds on survival and mutant frequency in five Escherichia coli strains derived from the wild-type strain NR9102 with different DNA repair backgrounds. At nontoxic doses, both VAN and CIN significantly reduced mutant frequency in the wild-type strain NR9102, in the nucleotide excision repair-deficient strain NR11634 (uvrB), and in the recombination-proficient but SOS-deficient strain NR11475 (recA430). In contrast, in the recombination-deficient and SOS-deficient strain NR11317 (recA56), both VAN and CIN not only failed to inhibit the spontaneous mutant frequency but actually increased the mutant frequency. In the mismatch repair-defective strain NR9319 (mutL), only CIN was antimutagenic. Our results show that the antimutagenicity of VAN and CIN against spontaneous mutation required the RecA recombination function but was independent of the SOS and nucleotide excision repair pathways. Thus, we propose the counterintuitive notion that these antimutagens actually produce a type of DNA damage that elicits recombinational repair (but not mismatch, SOS, or nucleotide excision repair), which then repairs not only the damage induced by VAN and CIN but also other DNA damage-resulting in an antimutagenic effect on spontaneous mutation.
Topics: Acrolein; Antimutagenic Agents; Benzaldehydes; DNA Repair; Dose-Response Relationship, Drug; Escherichia coli; Mutagenesis; Recombination, Genetic; SOS Response, Genetics
PubMed: 16999979
DOI: 10.1016/j.mrfmmm.2006.08.006 -
Expert Review of Proteomics Aug 2017Defects in tissue repair or wound healing pose a clinical, economic and social problem worldwide. Despite decades of studies, there have been few effective therapeutic... (Review)
Review
Defects in tissue repair or wound healing pose a clinical, economic and social problem worldwide. Despite decades of studies, there have been few effective therapeutic treatments. Areas covered: We discuss the possible reasons for why growth factor therapy did not succeed. We point out the lack of human disorder-relevant animal models as another blockade for therapeutic development. We summarize the recent discovery of secreted heat shock protein-90 (Hsp90) as a novel wound healing agent. Expert commentary: Wound healing is a highly complex and multistep process that requires participations of many cell types, extracellular matrices and soluble molecules to work together in a spatial and temporal fashion within the wound microenvironment. The time that wounds remain open directly correlates with the clinical mortality associated with wounds. This time urgency makes the healing process impossible to regenerate back to the unwounded stage, rather forces it to take many shortcuts in order to protect life. Therefore, for therapeutic purpose, it is crucial to identify so-called 'driver genes' for the life-saving phase of wound closure. Keratinocyte-secreted Hsp90α was discovered in 2007 and has shown the promise by overcoming several key hurdles that have blocked the effectiveness of growth factors during wound healing.
Topics: Animals; Disease Models, Animal; Exosomes; HSP90 Heat-Shock Proteins; Humans; Intercellular Signaling Peptides and Proteins; Recombinant Proteins; Wound Healing
PubMed: 28715921
DOI: 10.1080/14789450.2017.1355244 -
Genes To Cells : Devoted To Molecular &... Feb 1998Recombinational DNA repair is both the most complex and least understood of DNA repair pathways. In bacterial cells grown under normal laboratory conditions (without a... (Review)
Review
Recombinational DNA repair is both the most complex and least understood of DNA repair pathways. In bacterial cells grown under normal laboratory conditions (without a DNA damaging treatment other than an aerobic environment), a substantial number (10-50%) of the replication forks originating at oriC encounter a DNA lesion or strand break. When this occurs, repair is mediated by an elaborate set of recombinational DNA repair pathways which encompass most of the enzymes involved in DNA metabolism. Four steps are discussed: (i) The replication fork stalls and/or collapses. (ii) Recombination enzymes are recruited to the location of the lesion, and function with nearly perfect efficiency and fidelity. (iii) Additional enzymatic systems, including the phiX174-type primosome (or repair primosome), then function in the origin-independent reassembly of the replication fork. (iv) Frequent recombination associated with recombinational DNA repair leads to the formation of dimeric chromosomes, which are monomerized by the XerCD site-specific recombination system.
Topics: Bacteria; Bacterial Proteins; DNA; DNA Damage; DNA Repair; DNA Replication; Recombination, Genetic
PubMed: 9605402
DOI: 10.1046/j.1365-2443.1998.00175.x -
PLoS Genetics Sep 2009Resection of DNA double-strand break (DSB) ends is generally considered a critical determinant in pathways of DSB repair and genome stability. Unlike for enzymatically...
Resection of DNA double-strand break (DSB) ends is generally considered a critical determinant in pathways of DSB repair and genome stability. Unlike for enzymatically induced site-specific DSBs, little is known about processing of random "dirty-ended" DSBs created by DNA damaging agents such as ionizing radiation. Here we present a novel system for monitoring early events in the repair of random DSBs, based on our finding that single-strand tails generated by resection at the ends of large molecules in budding yeast decreases mobility during pulsed field gel electrophoresis (PFGE). We utilized this "PFGE-shift" to follow the fate of both ends of linear molecules generated by a single random DSB in circular chromosomes. Within 10 min after gamma-irradiation of G2/M arrested WT cells, there is a near-synchronous PFGE-shift of the linearized circular molecules, corresponding to resection of a few hundred bases. Resection at the radiation-induced DSBs continues so that by the time of significant repair of DSBs at 1 hr there is about 1-2 kb resection per DSB end. The PFGE-shift is comparable in WT and recombination-defective rad52 and rad51 strains but somewhat delayed in exo1 mutants. However, in rad50 and mre11 null mutants the initiation and generation of resected ends at radiation-induced DSB ends is greatly reduced in G2/M. Thus, the Rad50/Mre11/Xrs2 complex is responsible for rapid processing of most damaged ends into substrates that subsequently undergo recombinational repair. A similar requirement was found for RAD50 in asynchronously growing cells. Among the few molecules exhibiting shift in the rad50 mutant, the residual resection is consistent with resection at only one of the DSB ends. Surprisingly, within 1 hr after irradiation, double-length linear molecules are detected in the WT and rad50, but not in rad52, strains that are likely due to crossovers that are largely resection- and RAD50-independent.
Topics: Chromosomes, Fungal; DNA Breaks, Double-Stranded; DNA Repair; DNA, Fungal; DNA-Binding Proteins; Dimerization; Dose-Response Relationship, Radiation; Electrophoresis, Gel, Pulsed-Field; Exodeoxyribonucleases; G2 Phase; Gamma Rays; Mitosis; Mutation; Rad51 Recombinase; Rad52 DNA Repair and Recombination Protein; Recombination, Genetic; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 19763170
DOI: 10.1371/journal.pgen.1000656 -
BioEssays : News and Reviews in... Mar 2009Eukaryotic genomes harbor a large number of homologous repeat sequences that are capable of recombining. Their potential to disrupt genome stability highlights the need... (Review)
Review
Eukaryotic genomes harbor a large number of homologous repeat sequences that are capable of recombining. Their potential to disrupt genome stability highlights the need to understand how homologous recombination processes are coordinated. The Saccharomyces cerevisiae Rad1-Rad10 endonuclease performs an essential role in recombination between repeated sequences, by processing 3' single-stranded intermediates formed during single-strand annealing and gene conversion events. Several recent studies have focused on factors involved in Rad1-Rad10-dependent removal of 3' nonhomologous tails during homologous recombination, including Msh2-Msh3, Slx4, and the newly identified Saw1 protein. Together, this new work provides a model for how Rad1-Rad10-dependent end processing is coordinated: Msh2-Msh3 stabilizes and prepares double-strand/single-strand junctions for Rad1-Rad10 cleavage, Saw1 recruits Rad1-Rad10 to 3' tails, and Slx4 mediates crosstalk between the DNA damage checkpoint machinery and Rad1-Rad10.
Topics: Animals; DNA; DNA Repair; Exonucleases; Humans; Models, Genetic; Recombination, Genetic
PubMed: 19260026
DOI: 10.1002/bies.200800195 -
Molecular Microbiology Mar 2004Recent findings suggest that DNA nicks stimulate homologous recombination by being converted into double-strand breaks, which are mended by RecA-catalysed...
Recent findings suggest that DNA nicks stimulate homologous recombination by being converted into double-strand breaks, which are mended by RecA-catalysed recombinational repair and are lethal if not repaired. Hyper-rec mutants, in which DNA nicks become detectable, are synthetic-lethal with recA inactivation, substantiating the idea. Escherichia coli dut mutants are the only known hyper-recs in which presumed nicks in DNA do not cause inviability with recA, suggesting that nicks stimulate homologous recombination directly. Here, we show that dut recA mutants are synthetic-lethal; specifically, dut mutants depend on the RecBC-RuvABC recombinational repair pathway that mends double-strand DNA breaks. Although induced for SOS, dut mutants are not rescued by full SOS induction if RecA is not available, suggesting that recombinational rather than regulatory functions of RecA are needed for their viability. We also detected chromosomal fragmentation in dut rec mutants, indicating double-strand DNA breaks. Both the synthetic lethality and chromosomal fragmentation of dut rec mutants are suppressed by preventing uracil excision via inactivation of uracil DNA-glycosylase or by preventing dUTP production via inactivation of dCTP deaminase. We suggest that nicks become substrates for recombinational repair after being converted into double-strand DNA breaks.
Topics: Amino Acid Sequence; Chromosomes, Bacterial; DNA Damage; DNA Fragmentation; DNA Repair; DNA, Bacterial; Escherichia coli; Escherichia coli Proteins; Models, Molecular; Molecular Sequence Data; Mutation; Protein Structure, Tertiary; Pyrophosphatases; Rec A Recombinases; Recombination, Genetic; SOS Response, Genetics; Sequence Alignment; Uracil
PubMed: 14982624
DOI: 10.1111/j.1365-2958.2003.03924.x -
The Journal of Biological Chemistry Apr 2004The XPF/ERCC1 heterodimer is a DNA structure-specific endonuclease that participates in nucleotide excision repair and homology-dependent recombination reactions,...
The XPF/ERCC1 heterodimer is a DNA structure-specific endonuclease that participates in nucleotide excision repair and homology-dependent recombination reactions, including DNA single strand annealing and gene targeting. Here we show that XPF/ERCC1 is stably associated with hRad52, a recombinational repair protein, in human cell-free extracts and that these factors interact directly via the N-terminal domain of hRad52 and the XPF protein. Complex formation between hRad52 and XPF/ERCC1 concomitantly stimulates the DNA structure-specific endonuclease activity of XPF/ERCC1 and attenuates the DNA strand annealing activity of hRad52. Our results reveal a novel role for hRad52 as a subunit of a DNA structure-specific endonuclease and are congruent with evidence implicating both hRad52 and XPF/ERCC1 in a number of homologous recombination reactions. We propose that the ternary complex of hRad52 and XPF/ERCC1 is the active species that processes recombination intermediates generated during the repair of DNA double strand breaks and in homology-dependent gene targeting events.
Topics: DNA; DNA Repair; DNA-Binding Proteins; Endonucleases; Enzyme Activation; HeLa Cells; Humans; Protein Structure, Tertiary; Proteins; Rad52 DNA Repair and Recombination Protein; Recombination, Genetic
PubMed: 14734547
DOI: 10.1074/jbc.M313779200