scholarly journals Multiple Heterologies Increase Mitotic Double-Strand Break-Induced Allelic Gene Conversion Tract Lengths in Yeast

Genetics ◽  
1999 ◽  
Vol 153 (2) ◽  
pp. 665-679 ◽  
Author(s):  
Jac A Nickoloff ◽  
Douglas B Sweetser ◽  
Jennifer A Clikeman ◽  
Guru Jot Khalsa ◽  
Sarah L Wheeler
Keyword(s):  
Gene Conversion ◽  
Mismatch Repair ◽  
Frameshift Mutation ◽  
Strand Break ◽  
Double Strand Break ◽  
Chromosome Loss ◽  
Allelic Gene ◽  
Break Induced Replication ◽  
Gene Conversion Tract ◽  
Conversion Tract

Abstract Spontaneous and double-strand break (DSB)-induced allelic recombination in yeast was investigated in crosses between ura3 heteroalleles inactivated by an HO site and a +1 frameshift mutation, with flanking markers defining a 3.4-kbp interval. In some crosses, nine additional phenotypically silent RFLP mutations were present at ∼100-bp intervals. Increasing heterology from 0.2 to 1% in this interval reduced spontaneous, but not DSB-induced, recombination. For DSB-induced events, 75% were continuous tract gene conversions without a crossover in this interval; discontinuous tracts and conversions associated with a crossover each comprised ∼7% of events, and 10% also converted markers in unbroken alleles. Loss of heterozygosity was seen for all markers centromere distal to the HO site in 50% of products; such loss could reflect gene conversion, break-induced replication, chromosome loss, or G2 crossovers. Using telomere-marked strains we determined that nearly all allelic DSB repair occurs by gene conversion. We further show that most allelic conversion results from mismatch repair of heteroduplex DNA. Interestingly, markers shared between the sparsely and densely marked interval converted at higher rates in the densely marked interval. Thus, the extra markers increased gene conversion tract lengths, which may reflect mismatch repair-induced recombination, or a shift from restoration- to conversion-type repair.

scholarly journals Rad51-Independent Interchromosomal Double-Strand Break Repair by Gene Conversion Requires Rad52 but Not Rad55, Rad57, or Dmc1

2007 ◽  
Vol 28 (3) ◽  
pp. 897-906 ◽  
Author(s):  
Thomas J. Pohl ◽  
Jac A. Nickoloff
Keyword(s):  
Gene Conversion ◽  
Strand Break ◽  
Double Strand Break ◽  
Single Strand ◽  
Homology Search ◽  
Wild Type ◽  
Dsb Repair ◽  
Single Strand Annealing ◽  
In The Wild ◽  
Break Induced Replication

ABSTRACT Homologous recombination (HR) is critical for DNA double-strand break (DSB) repair and genome stabilization. In yeast, HR is catalyzed by the Rad51 strand transferase and its “mediators,” including the Rad52 single-strand DNA-annealing protein, two Rad51 paralogs (Rad55 and Rad57), and Rad54. A Rad51 homolog, Dmc1, is important for meiotic HR. In wild-type cells, most DSB repair results in gene conversion, a conservative HR outcome. Because Rad51 plays a central role in the homology search and strand invasion steps, DSBs either are not repaired or are repaired by nonconservative single-strand annealing or break-induced replication mechanisms in rad51Δ mutants. Although DSB repair by gene conversion in the absence of Rad51 has been reported for ectopic HR events (e.g., inverted repeats or between plasmids), Rad51 has been thought to be essential for DSB repair by conservative interchromosomal (allelic) gene conversion. Here, we demonstrate that DSBs stimulate gene conversion between homologous chromosomes (allelic conversion) by >30-fold in a rad51Δ mutant. We show that Rad51-independent allelic conversion and break-induced replication occur independently of Rad55, Rad57, and Dmc1 but require Rad52. Unlike DSB-induced events, spontaneous allelic conversion was detected in both rad51Δ and rad52Δ mutants, but not in a rad51Δ rad52Δ double mutant. The frequencies of crossovers associated with DSB-induced gene conversion were similar in the wild type and the rad51Δ mutant, but discontinuous conversion tracts were fivefold more frequent and tract lengths were more widely distributed in the rad51Δ mutant, indicating that heteroduplex DNA has an altered structure, or is processed differently, in the absence of Rad51.

scholarly journals Sgs1 Regulates Gene Conversion Tract Lengths and Crossovers Independently of Its Helicase Activity

2006 ◽  
Vol 26 (11) ◽  
pp. 4086-4094 ◽  
Author(s):  
Yi-Chen Lo ◽  
Kimberly S. Paffett ◽  
Or Amit ◽  
Jennifer A. Clikeman ◽  
Rosa Sterk ◽  
...  
Keyword(s):  
Gene Conversion ◽  
Genome Stability ◽  
Synthetic Lethality ◽  
Chromosome Loss ◽  
Helicase Activity ◽  
Tract Length ◽  
Independent Functions ◽  
Gene Conversion Tract ◽  
Conversion Tract

ABSTRACT RecQ helicases maintain genome stability and suppress tumors in higher eukaryotes through roles in replication and DNA repair. The yeast RecQ homolog Sgs1 interacts with Top3 topoisomerase and Rmi1. In vitro, Sgs1 binds to and branch migrates Holliday junctions (HJs) and the human RecQ homolog BLM, with Top3α, resolves synthetic double HJs in a noncrossover sense. Sgs1 suppresses crossovers during the homologous recombination (HR) repair of DNA double-strand breaks (DSBs). Crossovers are associated with long gene conversion tracts, suggesting a model in which Sgs1 helicase catalyzes reverse branch migration and convergence of double HJs for noncrossover resolution by Top3. Consistent with this model, we show that allelic crossovers and gene conversion tract lengths are increased in sgs1Δ. However, crossover and tract length suppression was independent of Sgs1 helicase activity, which argues against helicase-dependent HJ convergence. HJs may converge passively by a “random walk,” and Sgs1 may play a structural role in stimulating Top3-dependent resolution. In addition to the new helicase-independent functions for Sgs1 in crossover and tract length control, we define three new helicase-dependent functions, including the suppression of chromosome loss, chromosome missegregation, and synthetic lethality in srs2Δ. We propose that Sgs1 has helicase-dependent functions in replication and helicase-independent functions in DSB repair by HR.

Evidence for Independent Mismatch Repair Processing on Opposite Sides of a Double-Strand Break in Saccharomyces cerevisiae

Genetics ◽  
1998 ◽  
Vol 148 (1) ◽  
pp. 59-70
Author(s):  
Yi-shin Weng ◽  
Jac A Nickoloff
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Conversion ◽  
Mismatch Repair ◽  
Strand Break ◽  
Double Strand Break ◽  
Stem Loop ◽  
Mitotic Gene Conversion ◽  
Low Levels ◽  
Mat Locus ◽  
Insertion Mutations

Abstract Double-strand break (DSB) induced gene conversion in Saccharomyces cerevisiae during meiosis and MAT switching is mediated primarily by mismatch repair of heteroduplex DNA (hDNA). We used nontandem ura3 duplications containing palindromic frameshift insertion mutations near an HO nuclease recognition site to test whether mismatch repair also mediates DSB-induced mitotic gene conversion at a non-MAT locus. Palindromic insertions included in hDNA are expected to produce a stem-loop mismatch, escape repair, and segregate to produce a sectored (Ura+/−) colony. If conversion occurs by gap repair, the insertion should be removed on both strands, and converted colonies will not be sectored. For both a 14-bp palindrome, and a 37-bp near-palindrome, ~75% of recombinant colonies were sectored, indicating that most DSB-induced mitotic gene conversion involves mismatch repair of hDNA. We also investigated mismatch repair of well-repaired markers flanking an unrepaired palindrome. As seen in previous studies, these additional markers increased loop repair (likely reflecting corepair). Among sectored products, few had additional segregating markers, indicating that the lack of repair at one marker is not associated with inefficient repair at nearby markers. Clear evidence was obtained for low levels of short tract mismatch repair. As seen with full gene conversions, donor alleles in sectored products were not altered. Markers on the same side of the DSB as the palindrome were involved in hDNA less often among sectored products than nonsectored products, but markers on the opposite side of the DSB showed similar hDNA involvement among both product classes. These results can be explained in terms of corepair, and they suggest that mismatch repair on opposite sides of a DSB involves distinct repair tracts.

scholarly journals Genetic Requirements for RAD51- andRAD54-Independent Break-Induced Replication Repair of a Chromosomal Double-Strand Break

2001 ◽  
Vol 21 (6) ◽  
pp. 2048-2056 ◽  
Author(s):  
Laurence Signon ◽  
Anna Malkova ◽  
Maria L. Naylor ◽  
Hannah Klein ◽  
James E. Haber
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Homologous Recombination ◽  
Gene Conversion ◽  
Strand Break ◽  
Double Strand Break ◽  
Telomere Maintenance ◽  
Diploid Cells ◽  
Break Induced Replication ◽  
In Cells

ABSTRACT Broken chromosomes can be repaired by several homologous recombination mechanisms, including gene conversion and break-induced replication (BIR). In Saccharomyces cerevisiae, an HO endonuclease-induced double-strand break (DSB) is normally repaired by gene conversion. Previously, we have shown that in the absence ofRAD52, repair is nearly absent and diploid cells lose the broken chromosome; however, in cells lacking RAD51, gene conversion is absent but cells can repair the DSB by BIR. We now report that gene conversion is also abolished when RAD54, RAD55, and RAD57 are deleted but BIR occurs, as withrad51Δ cells. DSB-induced gene conversion is not significantly affected when RAD50, RAD59, TID1(RDH54), SRS2, or SGS1 is deleted. Various double mutations largely eliminate both gene conversion and BIR, including rad51Δ rad50Δ, rad51Δ rad59Δ, andrad54Δ tid1Δ. These results demonstrate that there is aRAD51- and RAD54-independent BIR pathway that requires RAD59, TID1, RAD50, and presumablyMRE11 and XRS2. The similar genetic requirements for BIR and telomere maintenance in the absence of telomerase also suggest that these two processes proceed by similar mechanisms.

scholarly journals Mre11 and Ku regulation of double-strand break repair by gene conversion and break-induced replication

DNA Repair ◽  
2007 ◽  
Vol 6 (6) ◽  
pp. 797-808 ◽  
Author(s):  
Sanchita Krishna ◽  
Brant M. Wagener ◽  
Hui Ping Liu ◽  
Yi-Chen Lo ◽  
Rosa Sterk ◽  
...  
Keyword(s):  
Gene Conversion ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Break Repair ◽  
Break Repair ◽  
Break Induced Replication

scholarly journals The Conversion Gradient at HIS4 of Saccharomyces cerevisiae. II. A Role for Mismatch Repair Directed by Biased Resolution of the Recombinational Intermediate

Genetics ◽  
1999 ◽  
Vol 153 (2) ◽  
pp. 573-583 ◽  
Author(s):  
Henriette M Foss ◽  
Kenneth J Hillers ◽  
Franklin W Stahl
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Synthesis ◽  
Mismatch Repair ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Break Repair ◽  
Holliday Junction ◽  
Crossing Over ◽  
Gradient Type ◽  
Salient Features

AbstractSalient features of recombination at ARG4 of Saccharomyces provoke a variation of the double-strand-break repair (DSBR) model that has the following features: (1) Holliday junction cutting is biased in favor of strands upon which DNA synthesis occurred during formation of the joint molecule (this bias ensures that cutting both junctions of the joint-molecule intermediate arising during DSBR usually leads to crossing over); (2) cutting only one junction gives noncrossovers; and (3) repair of mismatches that are semirefractory to mismatch repair and/or far from the DSB site is directed primarily by junction resolution. The bias in junction resolution favors restoration of 4:4 segregation when such mismatches and the directing junction are on the same side of the DSB site. Studies at HIS4 confirmed the predicted influence of the bias in junction resolution on the conversion gradient, type of mismatch repair, and frequency of aberrant 5:3 segregation, as well as the predicted relationship between mismatch repair and crossing over.

scholarly journals Use of a Small Palindrome Genetic Marker to Investigate Mechanisms of Double-Strand-Break Repair in Mammalian Cells

Genetics ◽  
2000 ◽  
Vol 154 (3) ◽  
pp. 1281-1289 ◽  
Author(s):  
Julang Li ◽  
Mark D Baker
Keyword(s):  
Mismatch Repair ◽  
Mammalian Cells ◽  
Indirect Evidence ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Break Repair ◽  
Chromosomal Markers ◽  
Vector Borne ◽  
Break Repair ◽  
The One

Abstract We examined mechanisms of mammalian homologous recombination using a gene targeting assay in which the vector-borne region of homology to the chromosome bore small palindrome insertions that frequently escape mismatch repair when encompassed within heteroduplex DNA (hDNA). Our assay permitted the product(s) of each independent recombination event to be recovered for molecular analysis. The results revealed the following: (i) vector-borne double-strand break (DSB) processing usually did not yield a large double-strand gap (DSG); (ii) in 43% of the recombinants, the results were consistent with crossover at or near the DSB; and (iii) in the remaining recombinants, hDNA was an intermediate. The sectored (mixed) genotypes observed in 38% of the recombinants provided direct evidence for involvement of hDNA, while indirect evidence was obtained from the patterns of mismatch repair (MMR). Individual hDNA tracts were either long or short and asymmetric or symmetric on the one side of the DSB examined. Clonal analysis of the sectored recombinants revealed how vector-borne and chromosomal markers were linked in each strand of individual hDNA intermediates. As expected, vector-borne and chromosomal markers usually resided on opposite strands. However, in one recombinant, they were linked on the same strand. The results are discussed with particular reference to the double-strand-break repair (DSBR) model of recombination.

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