Gene conversion adjacent to regions of double-strand break repair

The repair of double-strand breaks and gaps can be studied in vegetative yeast cells by transforming the DNA with restriction enzyme-cut plasmids. Postulated models for this repair process require the formation of heteroduplex DNA on either side of the region of break or gap repair. We describe the use of restriction site mutations in the his3 gene to detect conversion events flanking but outside of a region of a double-strand break repair. The frequency with which a mutation was converted declined with increasing distance between the mutation and the edge of the gap repair region. The data are consistent with heteroduplex DNA tracts of at least several hundred base pairs adjacent to regions of double-strand break repair.

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A Test of the Double-Strand Break Repair Model for Meiotic Recombination in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/144.1.27 ◽

1996 ◽

Vol 144 (1) ◽

pp. 27-41 ◽

Cited By ~ 7

Author(s):

Larry A Gilbertson ◽

Franklin W Stahl

Keyword(s):

Meiotic Recombination ◽

Strand Break ◽

Double Strand Break ◽

Double Strand Break Repair ◽

Holliday Junction ◽

Single Event ◽

Heteroduplex Dna ◽

Holliday Junction Resolvase ◽

Break Repair ◽

Two Sides

Abstract We tested predictions of the double-strand break repair (DSBR) model for meiotic recombination by examining the segregation patterns of small palindromic insertions, which frequently escape mismatch repair when in heteroduplex DNA. The palindromes flanked a well characterized DSB site at the ARC4 locus. The “canonical” DSBR model, in which only 5′ ends are degraded and resolution of the four-stranded intermediate is by Holliday junction resolvase, predicts that hDNA will frequently occur on both participating chromatids in a single event. Tetrads reflecting this configuration of hDNA were rare. In addition, a class of tetrads not predicted by the canonical DSBR model was identified. This class represented events that produced hDNA in a “trans” configuration, on opposite strands of the same duplex on the two sides of the DSB site. Whereas most classes of convertant tetrads had typical frequencies of associated crossovers, tetrads with trans hDNA were parental for flanking markers. Modified versions of the DSBR model, including one that uses a topoisomerase to resolve the canonical DSBR intermediate, are supported by these data.

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Regulation of Error-Prone DNA Double-Strand Break Repair and Its Impact on Genome Evolution

Cells ◽

10.3390/cells9071657 ◽

2020 ◽

Vol 9 (7) ◽

pp. 1657 ◽

Cited By ~ 2

Author(s):

Terrence Hanscom ◽

Mitch McVey

Keyword(s):

Strand Break ◽

Double Strand Break ◽

Dna Lesions ◽

Double Strand Break Repair ◽

End Joining ◽

Genetic Changes ◽

Strand Breaks ◽

Dna Double Strand Break ◽

Alternative End Joining ◽

Break Repair

Double-strand breaks are one of the most deleterious DNA lesions. Their repair via error-prone mechanisms can promote mutagenesis, loss of genetic information, and deregulation of the genome. These detrimental outcomes are significant drivers of human diseases, including many cancers. Mutagenic double-strand break repair also facilitates heritable genetic changes that drive organismal adaptation and evolution. In this review, we discuss the mechanisms of various error-prone DNA double-strand break repair processes and the cellular conditions that regulate them, with a focus on alternative end joining. We provide examples that illustrate how mutagenic double-strand break repair drives genome diversity and evolution. Finally, we discuss how error-prone break repair can be crucial to the induction and progression of diseases such as cancer.

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DNA Strand Breaks Signal the Induction of DNA Double-Strand Break Repair in Saccharomyces cerevisiae

Radiation Research ◽

10.1667/rr3460.1 ◽

2005 ◽

Vol 164 (6) ◽

pp. 781-790 ◽

Cited By ~ 6

Author(s):

Rakesh Kumar Singh ◽

Malini Krishna

Keyword(s):

Saccharomyces Cerevisiae ◽

Strand Break ◽

Dna Strand Breaks ◽

Double Strand Break ◽

Double Strand Break Repair ◽

Strand Breaks ◽

Dna Double Strand Break ◽

Break Repair ◽

Dna Strand

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Polymerase Mu Is a DNA-Directed DNA/RNA Polymerase

Molecular and Cellular Biology ◽

10.1128/mcb.23.7.2309-2315.2003 ◽

2003 ◽

Vol 23 (7) ◽

pp. 2309-2315 ◽

Cited By ~ 121

Author(s):

Stephanie A. Nick McElhinny ◽

Dale A. Ramsden

Keyword(s):

High Specificity ◽

Strand Break ◽

Double Strand Break ◽

Nonhomologous End Joining ◽

Double Strand Break Repair ◽

End Joining ◽

Strand Breaks ◽

Break Repair ◽

The Impact

ABSTRACT DNA polymerases are defined as such because they use deoxynucleotides instead of ribonucleotides with high specificity. We show here that polymerase mu (pol μ), implicated in the nonhomologous end-joining pathway for repair of DNA double-strand breaks, incorporates both ribonucleotides and deoxynucleotides in a template-directed manner. pol μ has an approximately 1,000-fold-reduced ability to discriminate against ribonucleotides compared to that of the related pol β, although pol μ's substrate specificity is similar to that of pol β in most other respects. Moreover, pol μ more frequently incorporates ribonucleotides when presented with nucleotide concentrations that approximate cellular pools. We therefore addressed the impact of ribonucleotide incorporation on the activities of factors required for double-strand break repair by nonhomologous end joining. We determined that the ligase required for this pathway readily joined strand breaks with terminal ribonucleotides. Most significantly, pol μ frequently introduced ribonucleotides into the repair junctions of an in vitro nonhomologous end-joining reaction, an activity that would be expected to have important consequences in the context of cellular double-strand break repair.

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Repair of DNA Double-Strand Breaks following UV Damage in Three Sulfolobussolfataricus Strains

Journal of Bacteriology ◽

10.1128/jb.00667-10 ◽

2010 ◽

Vol 192 (19) ◽

pp. 4954-4962 ◽

Cited By ~ 24

Author(s):

Michael L. Rolfsmeier ◽

Marian F. Laughery ◽

Cynthia A. Haseltine

Keyword(s):

Sulfolobus Solfataricus ◽

Strand Break ◽

Double Strand Break ◽

Double Strand Breaks ◽

Double Strand Break Repair ◽

Hyperthermophilic Archaea ◽

Chromosomal Damage ◽

Strand Breaks ◽

Break Repair ◽

Uv C

ABSTRACT DNA damage repair mechanisms have been most thoroughly explored in the eubacterial and eukaryotic branches of life. The methods by which members of the archaeal branch repair DNA are significantly less well understood but have been gaining increasing attention. In particular, the approaches employed by hyperthermophilic archaea have been a general source of interest, since these organisms thrive under conditions that likely lead to constant chromosomal damage. In this work we have characterized the responses of three Sulfolobus solfataricus strains to UV-C irradiation, which often results in double-strand break formation. We examined S. solfataricus strain P2 obtained from two different sources and S. solfataricus strain 98/2, a popular strain for site-directed mutation by homologous recombination. Cellular recovery, as determined by survival curves and the ability to return to growth after irradiation, was found to be strain specific and differed depending on the dose applied. Chromosomal damage was directly visualized using pulsed-field gel electrophoresis and demonstrated repair rate variations among the strains following UV-C irradiation-induced double-strand breaks. Several genes involved in double-strand break repair were found to be significantly upregulated after UV-C irradiation. Transcript abundance levels and temporal expression patterns for double-strand break repair genes were also distinct for each strain, indicating that these Sulfolobus solfataricus strains have differential responses to UV-C-induced DNA double-strand break damage.

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Intermolecular recombination between DNAs introduced into mouse L cells is mediated by a nonconservative pathway that leads to crossover products.

Molecular and Cellular Biology ◽

10.1128/mcb.10.1.103 ◽

1990 ◽

Vol 10 (1) ◽

pp. 103-112 ◽

Cited By ~ 48

Author(s):

F L Lin ◽

K Sperle ◽

N Sternberg

Keyword(s):

Mammalian Cells ◽

Dna Recombination ◽

Strand Break ◽

Double Strand Break ◽

Yeast Cells ◽

Double Strand Break Repair ◽

L Cells ◽

Single Strand Annealing ◽

Break Repair ◽

Strand Annealing

We describe experiments designed to measure the efficiency of intermolecular recombination between mutant herpesvirus thymidine kinase (tk) genes introduced into mouse L cells. Recombinants were scored as stable transformants containing a functional tk gene. The two recombination substrates used were ptkB8, a pBR322-based plasmid containing a mutant tk gene, with a BamHI linker in an SphI restriction site that is centrally located within the gene, and mp10tk delta 3' delta 5', an mp10 vector with a tk gene deleted at both the 3' and 5' ends. The only homology shared by the two DNAs is 885 base pairs within the tk gene. To determine whether the double-strand break repair model that has been used to explain recombination in yeast cells (J. W. Szostak, T. L. Orr-Weaver, R. J. Rothstein, and F. W. Stahl, Cell 33:25-35, 1983) can account for recombination during the introduction of these DNAs into mammalian cells, we transformed cells with BamHI-linearized ptkB8 and supercoiled mp10tk delta 3' delta 5' replicative-form DNA. These two DNAs should recombine efficiently according to that model and should generate gene conversion products. In this reaction, the supercoiled DNA acts as the donor of information to repair the cleaved tk gene. Our results indicated that the efficiency of this reaction was very low (less than 10 transformants were obtained per 0.1 microgram of each DNA used in the reaction per 10(6) cells). In contrast, if BamHI-cleaved ptkB8 DNA was cotransformed into cells along with a circular DNA molecule containing a tk gene deleted only at its 3' end or only at its 5' end (mp10tk delta 3' or mp10tk delta 5'), then the efficiency of recombination could be more than 4 orders of magnitude higher than it was with circular mp10tk delta 3' delta 5' DNA. Recombination frequencies were highest when the tk delta 3' or tk delta 5' DNA used was cleaved at the tk deletion junction. Southern analyses of DNA from TK+ transformants generated with BamHI-cleaved ptkB8 and BamHI-cleaved mp10tk delta 3' DNAs indicated that recombination was almost always associated with the reassortment of markers flanking the reconstructed tk DNA. Together, these results are more consistent with the nonconservative single-strand annealing model for recombination that we proposed several years ago (F.-L. Lin, K. Sperle, and N. Sternberg, Mol. Cell. Biol. 4:1020-1034, 1984) than they are with the double-strand break repair model.

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YGR042W/MTE1 Functions in Double-Strand Break Repair with MPH1

10.1101/032581 ◽

2015 ◽

Author(s):

Askar Yimit ◽

TaeHyung Kim ◽

Ranjith Anand ◽

Sarah Meister ◽

Jiongwen Ou ◽

...

Keyword(s):

Dna Damage ◽

Strand Break ◽

Double Strand Break ◽

Dna Helicase ◽

Double Strand Breaks ◽

Double Strand Break Repair ◽

Dependent Manner ◽

Dna Breaks ◽

Strand Breaks ◽

Break Repair

Double-strand DNA breaks occur upon exposure of cells to agents such as ionizing radiation and ultraviolet light or indirectly through replication fork collapse at DNA damage sites. If left unrepaired double-strand breaks can cause genome instability and cell death. In response to DNA damage, proteins involved in double-strand break repair by homologous recombination re-localize into discrete nuclear foci. We identified 29 proteins that co-localize with the recombination repair protein Rad52 in response to DNA damage. Of particular interest, Ygr042w/Mte1, a protein of unknown function, showed robust colocalization with Rad52. Mte1 foci fail to form when the DNA helicase Mph1 is absent. Mte1 and Mph1 form a complex, and are recruited to double-strand breaks in vivo in a mutually dependent manner. Mte1 is important for resolution of Rad52 foci during double-strand break repair, and for suppressing break-induced replication. Together our data indicate that Mte1 functions with Mph1 in double-strand break repair.

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In Vitro Repair of Gaps in Bacteriophage T7 DNA

Journal of Bacteriology ◽

10.1128/jb.180.23.6193-6202.1998 ◽

1998 ◽

Vol 180 (23) ◽

pp. 6193-6202 ◽

Cited By ~ 3

Author(s):

Ying-Ta Lai ◽

Warren Masker

Keyword(s):

Strand Break ◽

Double Strand Break ◽

Double Strand Breaks ◽

Double Strand Break Repair ◽

Bacteriophage T7 ◽

Dna Molecules ◽

Strand Breaks ◽

Highly Efficient ◽

Break Repair

ABSTRACT An in vitro system based upon extracts of Escherichia coli infected with bacteriophage T7 was used to study the mechanism of double-strand break repair. Double-strand breaks were placed in T7 genomes by cutting with a restriction endonuclease which recognizes a unique site in the T7 genome. These molecules were allowed to repair under conditions where the double-strand break could be healed by (i) direct joining of the two partial genomes resulting from the break, (ii) annealing of complementary versions of 17-bp sequences repeated on either side of the break, or (iii) recombination with intact T7 DNA molecules. The data show that while direct joining and single-strand annealing contributed to repair of double-strand breaks, these mechanisms made only minor contributions. The efficiency of repair was greatly enhanced when DNA molecules that bridge the region of the double-strand break (referred to as donor DNA) were provided in the reaction mixtures. Moreover, in the presence of the donor DNA most of the repaired molecules acquired genetic markers from the donor DNA, implying that recombination between the DNA molecules was instrumental in repairing the break. Double-strand break repair in this system is highly efficient, with more than 50% of the broken molecules being repaired within 30 min under some experimental conditions. Gaps of 1,600 nucleotides were repaired nearly as well as simple double-strand breaks. Perfect homology between the DNA sequence near the break site and the donor DNA resulted in minor (twofold) improvement in the efficiency of repair. However, double-strand break repair was still highly efficient when there were inhomogeneities between the ends created by the double-strand break and the T7 genome or between the ends of the donor DNA molecules and the genome. The distance between the double-strand break and the ends of the donor DNA molecule was critical to the repair efficiency. The data argue that ends of DNA molecules formed by double-strand breaks are typically digested by between 150 and 500 nucleotides to form a gap that is subsequently repaired by recombination with other DNA molecules present in the same reaction mixture or infected cell.

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Intermolecular recombination between DNAs introduced into mouse L cells is mediated by a nonconservative pathway that leads to crossover products

Molecular and Cellular Biology ◽

10.1128/mcb.10.1.103-112.1990 ◽

1990 ◽

Vol 10 (1) ◽

pp. 103-112

Author(s):

F L Lin ◽

K Sperle ◽

N Sternberg

Keyword(s):

Mammalian Cells ◽

Dna Recombination ◽

Strand Break ◽

Double Strand Break ◽

Yeast Cells ◽

Double Strand Break Repair ◽

L Cells ◽

Single Strand Annealing ◽

Break Repair ◽

Strand Annealing

We describe experiments designed to measure the efficiency of intermolecular recombination between mutant herpesvirus thymidine kinase (tk) genes introduced into mouse L cells. Recombinants were scored as stable transformants containing a functional tk gene. The two recombination substrates used were ptkB8, a pBR322-based plasmid containing a mutant tk gene, with a BamHI linker in an SphI restriction site that is centrally located within the gene, and mp10tk delta 3' delta 5', an mp10 vector with a tk gene deleted at both the 3' and 5' ends. The only homology shared by the two DNAs is 885 base pairs within the tk gene. To determine whether the double-strand break repair model that has been used to explain recombination in yeast cells (J. W. Szostak, T. L. Orr-Weaver, R. J. Rothstein, and F. W. Stahl, Cell 33:25-35, 1983) can account for recombination during the introduction of these DNAs into mammalian cells, we transformed cells with BamHI-linearized ptkB8 and supercoiled mp10tk delta 3' delta 5' replicative-form DNA. These two DNAs should recombine efficiently according to that model and should generate gene conversion products. In this reaction, the supercoiled DNA acts as the donor of information to repair the cleaved tk gene. Our results indicated that the efficiency of this reaction was very low (less than 10 transformants were obtained per 0.1 microgram of each DNA used in the reaction per 10(6) cells). In contrast, if BamHI-cleaved ptkB8 DNA was cotransformed into cells along with a circular DNA molecule containing a tk gene deleted only at its 3' end or only at its 5' end (mp10tk delta 3' or mp10tk delta 5'), then the efficiency of recombination could be more than 4 orders of magnitude higher than it was with circular mp10tk delta 3' delta 5' DNA. Recombination frequencies were highest when the tk delta 3' or tk delta 5' DNA used was cleaved at the tk deletion junction. Southern analyses of DNA from TK+ transformants generated with BamHI-cleaved ptkB8 and BamHI-cleaved mp10tk delta 3' DNAs indicated that recombination was almost always associated with the reassortment of markers flanking the reconstructed tk DNA. Together, these results are more consistent with the nonconservative single-strand annealing model for recombination that we proposed several years ago (F.-L. Lin, K. Sperle, and N. Sternberg, Mol. Cell. Biol. 4:1020-1034, 1984) than they are with the double-strand break repair model.

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