Recombinational substrates designed to study recombination between unique and repetitive sequences in vivo.

Fasullo, Michael; Davis, Ronald W.

doi:10.1073/pnas.84.17.6215

Cited by 100 publications

(91 citation statements)

References 23 publications

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“…Consequently, we investigated the epistatic relationship between the rad3-102 and rad27-null mutations with respect to USCR using an assay developed by Fasullo and Davis (1987). The assay monitors the frequency of recombination between 39 and 59 truncated copies of the HIS3 gene that share 300 bp of homologous sequence and are arranged tail to head at the TRP1 locus on chromosome IV.…”

Section: Methodsmentioning

confidence: 99%

“…Determination of unequal sister-chromatid recombination rates: The rates of unequal sister-chromatid recombination were determined as previously described (Fasullo and Davis 1987). At least five freshly dissected ABX761 segregants (of each genotype) containing the USCR construct and the his3-D200 allele were streaked out for single colonies on YPD.…”

Section: Methodsmentioning

confidence: 99%

“…The rad27-null mutation confers a slow-germination phenotype that is easily distinguishable from wild-type strains. ABX761, used for the unequal sister-chromatid recombination (USCR) assay, was constructed by transforming XbaI-digested PNN287, the generous gift of Michael Fasullo (Fasullo and Davis 1987), into ABX731-8C (MATa, his3-D200) by electroporation. Insertion of the plasmid at the targeted site was verified by Southern blot analysis (M. S. Navarro and A. M. Bailis, unpublished results).…”

Section: Methodsmentioning

confidence: 99%

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A Mutant Allele of the Transcription Factor IIH Helicase Gene, RAD3, Promotes Loss of Heterozygosity in Response to a DNA Replication Defect in Saccharomyces cerevisiae

Navarro

Bailis

2007

Genetics

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Increased mitotic recombination enhances the risk for loss of heterozygosity, which contributes to the generation of cancer in humans. Defective DNA replication can result in elevated levels of recombination as well as mutagenesis and chromosome loss. In the yeast Saccharomyces cerevisiae, a null allele of the RAD27 gene, which encodes a structure-specific nuclease involved in Okazaki fragment processing, stimulates mutation and homologous recombination. Similarly, rad3-102, an allele of the gene RAD3, which encodes an essential helicase subunit of the core TFIIH transcription initiation and DNA repairosome complexes confers a hyper-recombinagenic and hypermutagenic phenotype. Combining the rad27 null allele with rad3-102 dramatically stimulated interhomolog recombination and chromosome loss but did not affect unequal sister-chromatid recombination, direct-repeat recombination, or mutation. Interestingly, the percentage of cells with Rad52-YFP foci also increased in the double-mutant haploids, suggesting that rad3-102 may increase lesions that elicit a response by the recombination machinery or, alternatively, stabilize recombinagenic lesions generated by DNA replication failure. This net increase in lesions led to a synthetic growth defect in haploids that is relieved in diploids, consistent with rad3-102 stimulating the generation and rescue of collapsed replication forks by recombination between homologs.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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A Mutant Allele of the Transcription Factor IIH Helicase Gene, RAD3, Promotes Loss of Heterozygosity in Response to a DNA Replication Defect in Saccharomyces cerevisiae

Navarro

Bailis

2007

Genetics

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“…We examined the frequency of sister chromatid exchanges in mph1D cells in a system where recombination between sister chromatids carrying direct truncated his3 repeats, his3-D59 and his3-D39, gives His + colonies (Fasullo and Davis 1987). The results showed that mph1D cells (yGI012) have a 2.5-fold increase (3.0 6 0.3 3 10 À6 ) of unequal intersister exchanges per cell division when compared with wild-type cells (yNN301; 1.2 6 0.5 3 10 À6 ).…”

Section: Mph1 Regulates Crossovers In Allelic and Sister Chromatid Rementioning

confidence: 99%

“…Unequal SCE was measured using a system described by Fasullo and Davis (1987). The rates of spontaneous mitotic SCE were determined by the method of median using 11 independent colonies for each rate calculation.…”

Section: Unequal Sister Chromatid Exchangementioning

confidence: 99%

Yeast Mph1 helicase dissociates Rad51-made D-loops: implications for crossover control in mitotic recombination

Prakash¹,

Satory²,

Dray³

et al. 2009

Genes Dev.

232

342

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Eukaryotes possess mechanisms to limit crossing over during mitotic homologous recombination, thus avoiding possible chromosomal rearrangements. We show here that budding yeast Mph1, an ortholog of human FancM helicase, utilizes its helicase activity to suppress spontaneous unequal sister chromatid exchanges and DNA double-strand break-induced chromosome crossovers. Since the efficiency and kinetics of break repair are unaffected, Mph1 appears to channel repair intermediates into a noncrossover pathway. Importantly, Mph1 works independently of two other helicases-Srs2 and Sgs1-that also attenuate crossing over. By chromatin immunoprecipitation, we find targeting of Mph1 to double-strand breaks in cells. Purified Mph1 binds D-loop structures and is particularly adept at unwinding these structures. Importantly, Mph1, but not a helicase-defective variant, dissociates Rad51-made D-loops. Overall, the results from our analyses suggest a new role of Mph1 in promoting the noncrossover repair of DNA double-strand breaks.[Keywords: Genome instability; recombination; DNA helicase; crossing over; Fanconi anemia] Supplemental material is available at http://www.genesdev.org. Received September 8, 2008; revised version accepted November 12, 2008. DNA double-strand breaks (DSBs) that arise during DNA replication or are induced by DNA damaging agents, such as ionizing radiation, are frequently repaired by homologous recombination (HR). In yeast and other eukaryotes, the RAD52 epistasis group of proteins mediate homologous recombination (for reviews, see Paques and Haber 1999;Krogh and Symington 2004). In this process, DSB ends are resected by nucleases to create 39 ssDNA that becomes coated with the recombinase protein Rad51. The Rad51-ssDNA nucleoprotein filament then carries out a search for a homologous donor DNA sequence and promotes strand invasion of the donor molecule to form a D-loop (Sung and Klein 2006). After D-loop formation, there appear to be two alternative pathways that result in DSB repair. One pathway involves the formation of a double Holliday junction (dHJ) that can be resolved by symmetrical strand cleavage into either a crossover or noncrossover gene conversion (Szostak et al. 1983). An alternative mechanism, called synthesis-dependent strand annealing (SDSA), posits formation mostly of noncrossovers, and in most cases does not involve a dHJ intermediate (for review, see Paques and Haber 1999). In support of the SDSA model of gene conversion, we showed recently that both newly synthesized strands are inherited by the broken recipient DNA molecule (Ira et al. 2006). The choice between crossover and noncrossover is tightly regulated in both mitotic and meiotic cells. In meiotic S. cerevisiae cells the correct number of crossovers are required for proper chromosome segregation; the proportion of gene conversion accompanied by crossing over varies between 25% and 50% depending on the locus (Roeder 1995). In mitotic cells, the proportion of crossovers is much lower, ranging between <1% and 7 These authors c...

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An in vitro system for measuring genotoxicity mediated by human CYP3A4 in Saccharomyces cerevisiae

Fasullo

Freedland

John

et al. 2017

Environ and Mol Mutagen

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P450 activity is required to metabolically activate many chemical carcinogens, rendering them highly genotoxic. CYP3A4 is the most abundantly expressed P450 enzyme in the liver, accounting for most drug metabolism and constituting 50% of all hepatic P450 activity. CYP3A4 is also expressed in extrahepatic tissues, including the intestine. However, the role of CYP3A4 in activating chemical carcinogens into potent genotoxins is unclear. To facilitate efforts to determine whether CYP3A4, per se, can activate carcinogens into potent genotoxins, we expressed human CYP3A4 in the DNA-repair mutant (rad4 rad51) strain of budding yeast Saccharomyces cerevisiae and tested the novel, recombinant yeast strain for ability to report CYP3A4-mediated genotoxicity of a well-known genotoxin, aflatoxin B1 (AFB1). Yeast microsomes containing human CYP3A4, but not those that do not contain CYP3A4, were active in hydroxylation of diclofenac (DCF), a known CYP3A4 substrate drug, a result confirming CYP3A4 activity in the recombinant yeast strain. In cells exposed to AFB1, the expression of CYP3A4 supported DNA adduct formation, chromosome rearrangements, cell death, and expression of the large subunit of ribonucleotide reductase, Rnr3, a marker of DNA damage. Expression of CYP3A4 also conferred sensitivity in rad4 rad51 mutants exposed to colon carcinogen, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). These data confirm the ability of human CYP3A4 to mediate the genotoxicity of AFB1, and illustrate the usefulness of the CYP3A4-expressing, DNA-repair mutant yeast strain for screening other chemical compounds that are CYP3A4 substrates, for potential genotoxicity.

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Recombinational substrates designed to study recombination between unique and repetitive sequences in vivo.

Cited by 100 publications

References 23 publications

A Mutant Allele of the Transcription Factor IIH Helicase Gene, RAD3, Promotes Loss of Heterozygosity in Response to a DNA Replication Defect in Saccharomyces cerevisiae

A Mutant Allele of the Transcription Factor IIH Helicase Gene, RAD3, Promotes Loss of Heterozygosity in Response to a DNA Replication Defect in Saccharomyces cerevisiae

Yeast Mph1 helicase dissociates Rad51-made D-loops: implications for crossover control in mitotic recombination

An in vitro system for measuring genotoxicity mediated by human CYP3A4 in Saccharomyces cerevisiae

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