Analysis of Chromosome/Allele Loss in Genetically Unstable Yeast by Quantitative Real-Time PCR

Lo, Yi‐Chen; Kurtz, Richard; Nickoloff, Jac A.

doi:10.2144/05385bm01

Cited by 5 publications

(4 citation statements)

References 14 publications

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“…The HIS3:telV marker on broken chromosomes was assayed by quantitative real-time PCR to determine chromosome loss among Ura Ϫ His Ϫ products, as described previously (21). His Ϫ products can result from crossovers in G 2 cells, and these have an associated product with two copies of HIS3:telV (His ϩϩ ), identified by PCR.…”

Section: Methodsmentioning

confidence: 99%

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

Pohl

Nickoloff

2008

Molecular and Cellular Biology

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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 breakinduced replication mechanisms in rad51⌬ 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 rad51⌬ 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 rad51⌬ and rad52⌬ mutants, but not in a rad51⌬ rad52⌬ double mutant. The frequencies of crossovers associated with DSB-induced gene conversion were similar in the wild type and the rad51⌬ mutant, but discontinuous conversion tracts were fivefold more frequent and tract lengths were more widely distributed in the rad51⌬ mutant, indicating that heteroduplex DNA has an altered structure, or is processed differently, in the absence of Rad51.

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Section: Methodsmentioning

confidence: 99%

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

Pohl

Nickoloff

2008

Molecular and Cellular Biology

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“…HR frequencies were calculated as the number of recombinants per YPD colony; typically, 1,000 to 1,500 colonies were scored in each of four determinations per strain. Broken chromosomes have a telomereproximal HIS3 gene (HIS3:telV), and chromosome loss was scored directly among Ura Ϫ His Ϫ products as described previously (33). Crossovers can produce His Ϫ and an associated product with two copies of HIS3:telV (His ϩϩ ), which was identified by PCR among at least 40 His ϩ products per strain.…”

Section: Methodsmentioning

confidence: 99%

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

Paffett

Amit

et al. 2006

Molecular and Cellular Biology

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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 Top3␣, 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 sgs1⌬. 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 srs2⌬. We propose that Sgs1 has helicasedependent functions in replication and helicase-independent functions in DSB repair by HR.The repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) is critical for maintaining genome stability and cancer suppression. DSBs are produced by ionizing radiation, genotoxic chemicals, and nucleases and when replication forks encounter DNA damage. Broken ends are converted to Rad51 nucleoprotein filaments that search for and invade homologous duplex DNA, producing a Holliday junction (HJ) intermediate. Branch migration of HJs extends or eliminates heteroduplex DNA (hDNA), and mismatches in hDNA are repaired, resulting in a region of localized loss of heterozygosity termed a gene conversion tract.Crossovers accompany some gene conversions, posing risks of deletions, inversions, translocations, and large-scale loss of heterozygosity (31,45,54). The mechanisms that suppress tract lengths and crossovers are important to elucidate because they determine the extent and frequency of the loss of heterozygosity during DSB repair by HR and thereby regulate genome stability.In the yeast Saccharomyces cerevisiae, mitotic and meiotic crossovers are suppressed by Sgs1 (26, 55), a member of the RecQ helicase family that includes five human proteins, three of which (BLM, WRN, and RECQ4) suppress tumors (25). RecQ helicases have conserved structures and interactions with type I topoisomerases (e.g., yeast Top3). Yeast Rmi1 is in complex with Sgs1-Top3 and may promote binding to branched DNAs or Top3 strand passage (10, 43). Yeast sgs1, top3, and rmi1 mutants show DNA damage hypersensitivity, genome instability, slow growth, poor sporulation, and hyperrecombination (10, 43). Sgs1...

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“…Crossovers were also estimated from just Ura + products (i.e., the ratio of Ura + His − : Ura + expressed as a percentage); this approach avoids uncertainties associated with loss of HIS3:telV by BIR or chromosome loss. Chromosome loss was determined directly as described [44], and BIR comprises non-loss, non-crossover His − products [16]. Gene conversion tract lengths were estimated as the fraction of Ura − recombinants among all (Ura + + Ura − ) recombinants (excluding BIR and chromosome loss products).…”

Section: Chromosomal Dsb Repair Assaysmentioning

confidence: 99%

“…Cells were cultured in YPGly as above, and harvested at various times after transfer to YPGal. Genomic DNA was prepared and DNA concentrations were measured by quantitative realtime PCR of NDC1 as described [44]. Equal quantities of non-denatured DNA were spotted on a nylon membrane using a BioRad dot blot apparatus locus and hybridized with a denatured 32 P-labeled 660 bp URA3 fragment amplified with primers 5′-CGCATATGTGGT GTTGAAGAA and 5′-TCTTTGTCGCTCTTCGCAAT.…”

Section: Analysis Of Single-strand Dna Resectionmentioning

confidence: 99%

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

et al. 2007

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The yeast Mre11-Rad50-Xrs2 (MRX) and Ku complexes regulate single-strand resection at DNA double-strand breaks (DSB), a key early step in homologous recombination (HR). A prior plasmid gap repair study showed that mre11 mutations, which slow single-strand resection, reduce gene conversion tract lengths and the frequency of associated crossovers. Here we tested whether mre11Delta or nuclease-defective mre11 mutations reduced gene conversion tract lengths during HR between homologous chromosomes in diploid yeast. We found that mre11 mutations reduced the efficiency of HR but did not reduce tract lengths or crossovers, despite substantially reduced end-resection at the test (ura3) locus. End-resection is increased in yku70Delta, but this change also had no effect on tract lengths. Thus, heteroduplex formation and tract lengths are not regulated by the extent of end-resection during DSB repair in a chromosomal context. In a plasmid-chromosome DSB repair assay, tract lengths were again similar in wild-type and mre11Delta, but they were reduced in mre11Delta in a gap repair assay. These results indicate that tract lengths are not affected by the extent of end processing when broken ends can invade nearby sites, perhaps because MRX coordination of the two broken ends is dispensable when ends invade nearby sites. Although HR outcome was largely unaffected in mre11 mutants, break-induced replication (BIR) and chromosome loss increased, suggesting that Mre11 function in mitotic HR is limited to early HR stages. Interestingly, yku70Delta suppressed BIR in mre11 mutants. BIR is also elevated in rad51 mutants, but yku70Delta did not suppress BIR in a rad51 background. These results indicate that Mre11 functions in Rad51-independent BIR, and that Ku functions in Rad51-dependent BIR.

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Analysis of Chromosome/Allele Loss in Genetically Unstable Yeast by Quantitative Real-Time PCR

Cited by 5 publications

References 14 publications

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

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

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

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

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