Overexpression of Rad51 inhibits double-strand break-induced homologous recombination but does not affect gene conversion tract lengths

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.

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Spontaneous and double-strand break-induced recombination, and gene conversion tract lengths, are differentially affected by overexpression of wild-type or ATPase-defective yeast Rad54

Nucleic Acids Research ◽

10.1093/nar/gkf413 ◽

2002 ◽

Vol 30 (13) ◽

pp. 2727-2735 ◽

Cited By ~ 22

Author(s):

P. M. Kim

Keyword(s):

Gene Conversion ◽

Strand Break ◽

Double Strand Break ◽

Wild Type ◽

Gene Conversion Tract ◽

Conversion Tract

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

Molecular and Cellular Biology ◽

10.1128/mcb.21.6.2048-2056.2001 ◽

2001 ◽

Vol 21 (6) ◽

pp. 2048-2056 ◽

Cited By ~ 104

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.

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The Role of Blm Helicase in Homologous Recombination, Gene Conversion Tract Length, and Recombination Between Diverged Sequences inDrosophilamelanogaster

Genetics ◽

10.1534/genetics.117.300285 ◽

2017 ◽

Vol 207 (3) ◽

pp. 923-933 ◽

Cited By ~ 6

Author(s):

Henry A. Ertl ◽

Daniel P. Russo ◽

Noori Srivastava ◽

Joseph T. Brooks ◽

Thu N. Dao ◽

...

Keyword(s):

Homologous Recombination ◽

Gene Conversion ◽

Tract Length ◽

Blm Helicase ◽

Gene Conversion Tract ◽

Conversion Tract ◽

Recombination Gene

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Mechanisms for gene conversion and homologous recombination: The double-strand break repair model and the successive half crossing-over model

Advances in Biophysics ◽

10.1016/0065-227x(92)90023-k ◽

1992 ◽

Vol 28 ◽

pp. 81-133 ◽

Cited By ~ 32

Author(s):

I Kobayashi

Keyword(s):

Homologous Recombination ◽

Gene Conversion ◽

Strand Break ◽

Double Strand Break ◽

Double Strand Break Repair ◽

Crossing Over ◽

Break Repair

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Mitotic gene conversion tracts associated with repair of a defined double-strand break in Saccharomyces cerevisiae

10.1101/132167 ◽

2017 ◽

Author(s):

Yee Fang Hum ◽

Sue Jinks-Robertson

Keyword(s):

Saccharomyces Cerevisiae ◽

Homologous Recombination ◽

Gene Conversion ◽

Strand Break ◽

Double Strand Break ◽

The Other ◽

Homologous Chromosomes ◽

Molecular Features ◽

Single Side ◽

Mitotic Gene Conversion

AbstractMitotic recombination between homologous chromosomes can lead to loss-of-heterozygosity (LOH), which is an important contributor to human disease. In the current study, a defined double-strand break (DSB) on chromosome IV was used to initiate LOH in a yeast strain with sequence-diverged chromosomes. Associated gene conversion tracts, which reflect the repair of mismatches formed when diverged chromosomes exchange single strands, were mapped using microarrays. LOH events reflected two broken chromosomes, one of which was repaired as a crossover and the other as a noncrossover. Gene conversion tracts associated with individual crossover and noncrossover events were similar in size and position, with half of the tracts unexpectedly mapping to only a single side of the initiating break. Although the molecular features of DSB-initiated events generally agree with those predicted by current models of homologous recombination, there were unexpected complexities in associated gene conversion tracts.

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DNA Polymerase δ Is Preferentially Recruited during Homologous Recombination To Promote Heteroduplex DNA Extension

Molecular and Cellular Biology ◽

10.1128/mcb.01651-07 ◽

2007 ◽

Vol 28 (4) ◽

pp. 1373-1382 ◽

Cited By ~ 73

Author(s):

Laurent Maloisel ◽

Francis Fabre ◽

Serge Gangloff

Keyword(s):

Homologous Recombination ◽

Gene Conversion ◽

Dna Polymerase ◽

Dna Polymerases ◽

Strand Break ◽

Gene Encoding ◽

Repair Synthesis ◽

Dna Polymerase Δ ◽

Strand Annealing ◽

Gene Conversion Tract

ABSTRACT DNA polymerases play a central role during homologous recombination (HR), but the identity of the enzyme(s) implicated remains elusive. The pol3-ct allele of the gene encoding the catalytic subunit of DNA polymerase δ (Polδ) has highlighted a role for this polymerase in meiotic HR. We now address the ubiquitous role of Polδ during HR in somatic cells. We find that pol3-ct affects gene conversion tract length during mitotic recombination whether the event is initiated by single-strand gaps following UV irradiation or by site-specific double-strand breaks. We show that the pol3-ct effects on gene conversion are completely independent of mismatch repair, indicating that shorter gene conversion tracts in pol3-ct correspond to shorter extensions of primed DNA synthesis. Interestingly, we find that shorter repair tracts do not favor synthesis-dependent strand annealing at the expense of double-strand-break repair. Finally, we show that the DNA polymerases that have been previously suspected to mediate HR repair synthesis (Polε and Polη) do not affect gene conversion during induced HR, including in the pol3-ct background. Our results argue strongly for the preferential recruitment of Polδ during HR.

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The 9-1-1 DNA Clamp Is Required for Immunoglobulin Gene Conversion

Molecular and Cellular Biology ◽

10.1128/mcb.00156-08 ◽

2008 ◽

Vol 28 (19) ◽

pp. 6113-6122 ◽

Cited By ~ 20

Author(s):

Alihossein Saberi ◽

Makoto Nakahara ◽

Julian E. Sale ◽

Koji Kikuchi ◽

Hiroshi Arakawa ◽

...

Keyword(s):

Homologous Recombination ◽

Gene Conversion ◽

Translesion Synthesis ◽

Strand Break ◽

Double Strand Break ◽

Immunoglobulin Gene ◽

Abasic Sites ◽

Immunoglobulin Locus ◽

Activation Induced Deaminase ◽

Dt40 Cells

ABSTRACT Chicken DT40 cells deficient in the 9-1-1 checkpoint clamp exhibit hypersensitivity to a variety of DNA-damaging agents. Although recent work suggests that, in addition to its role in checkpoint activation, this complex may play a role in homologous recombination and translesion synthesis, the cause of this hypersensitivity has not been studied thoroughly. The immunoglobulin locus of DT40 cells allows monitoring of homologous recombination and translesion synthesis initiated by activation-induced deaminase (AID)-dependent abasic sites. We show that both the RAD9 −/− and RAD17 −/− mutants exhibit substantially reduced immunoglobulin gene conversion. However, the level of nontemplated immunoglobulin point mutation increased in these mutants, a finding that is reminiscent of the phenotype resulting from the loss of RAD51 paralogs or Brca2. This suggests that the 9-1-1 complex does not play a central role in translesion synthesis in this context. Despite reduced immunoglobulin gene conversion, the RAD9 −/− and RAD17 −/− cells do not exhibit a prominent defect in double-strand break-induced gene conversion or a sensitivity to camptothecin. This suggests that the roles of Rad9 and Rad17 may be confined to a subset of homologous recombination reactions initiated by replication-stalling lesions rather than those associated with double-strand break repair.

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