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

Paffett, Kimberly S.; Clikeman, Jennifer A.; Palmer, Sean; Nickoloff, Jac A.

doi:10.1016/j.dnarep.2005.03.003

Cited by 25 publications

(25 citation statements)

References 79 publications

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“…On the other hand, genome integrity has not been investigated in CaRAD51 deletion mutants. In S. cerevisiae, RAD51 overexpression has been shown to promote genome instability, probably by inhibiting the accurate repair of double-strand breaks by homologous recombination (48). Similarly, RAD51 overexpression in the mouse leads to an increase in genome instability, notably, LOH events (49).…”

Section: Discussionmentioning

confidence: 99%

A FACS-Optimized Screen Identifies Regulators of Genome Stability in Candida albicans

et al. 2015

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f Loss of heterozygosity (LOH) plays important roles in genome dynamics, notably, during tumorigenesis. In the fungal pathogen Candida albicans, LOH contributes to the acquisition of antifungal resistance. In order to investigate the mechanisms that regulate LOH in C. albicans, we have established a novel method combining an artificial heterozygous locus harboring the blue fluorescent protein and green fluorescent protein markers and flow cytometry to detect LOH events at the single-cell level. Using this fluorescence-based method, we have confirmed that elevated temperature, treatment with methyl methanesulfonate, and inactivation of the Mec1 DNA damage checkpoint kinase triggered an increase in the frequency of LOH. Taking advantage of this system, we have searched for C. albicans genes whose overexpression triggered an increase in LOH and identified four candidates, some of which are known regulators of genome dynamics with human homologues contributing to cancer progression. Hence, the approach presented here will allow the implementation of new screens to identify genes that are important for genome stability in C. albicans and more generally in eukaryotic cells.N ormally found as a harmless commensal organism, Candida albicans is also a major fungal pathogen of humans and is capable of causing serious and even life-threatening diseases when the immune system of the host is compromised (1). Although C. albicans is found mostly as a diploid organism, haploid and tetraploid forms have been observed in the laboratory and upon the passage of C. albicans in animal models of infection (2-4). The formation of tetraploids results from mating between diploids of opposite mating types that have undergone the so-called whiteopaque phenotypic switch (5). Meiosis is thought to have been lost in C. albicans, and the formation of haploids from diploids or diploids from tetraploids results from concerted chromosome loss (2, 6). The C. albicans genome is highly plastic, undergoing a number of important genome rearrangements, such as loss-ofheterozygosity (LOH) events, aneuploidies, and the formation of isochromosomes (7). In particular, LOH has been shown to occur during commensal carriage (8) and during systemic infection in a mouse model (9) and to contribute to the acquisition of antifungal resistance (10, 11). Stressful conditions such as high temperature, oxidative stress, or azole antifungal treatment trigger an increase in the frequency of LOH, as well as changes in the mechanisms leading to LOH; while azole treatment and temperature cause an increase in LOH due to chromosome loss and reduplication, oxidative stress results in an increase in gene conversion events (12). Yet, little is known about the molecular mechanisms that control LOH in C. albicans, despite the apparent importance of LOH in the biology of this species. Overall, it has been shown that knockout mutations in genes involved in base excision repair, nucleotide excision repair, and mismatch repair had little impact on the frequency of LOH in C. albican...

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Section: Discussionmentioning

confidence: 99%

A FACS-Optimized Screen Identifies Regulators of Genome Stability in Candida albicans

et al. 2015

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“…DNA-PK kinase and MRN nuclease, or overexpression of Rad51 or Rad52 recombination proteins, but not other Rad52 paralogs, downregulate the retroviral integration [112–117], while others have shown that damage sensors for NHEJ, including DNA-PK, are not critical [118]. The suppressive effects of Rad51/Rad52 overexpression could be due to a steric hindrance of integrase, a retroviral recombinase, by competition between functional domains shared by the two proteins [113, 119], since the overexpression of Rad51 is known to suppress DSB-induced HR [120, 121]. For instance, in cells infected with HIV-1 (human immunodeficiency virus 1), Rad51 proteins (a major component of HR) are recruited and promote viral DNA integration [122].…”

Section: Discussionmentioning

confidence: 99%

Cancer risk at low doses of ionizing radiation: artificial neural networks inference from atomic bomb survivors

Sasaki

Tachibana

Takeda

2013

Journal of Radiation Research

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Cancer risk at low doses of ionizing radiation remains poorly defined because of ambiguity in the quantitative link to doses below 0.2 Sv in atomic bomb survivors in Hiroshima and Nagasaki arising from limitations in the statistical power and information available on overall radiation dose. To deal with these difficulties, a novel nonparametric statistics based on the ‘integrate-and-fire’ algorithm of artificial neural networks was developed and tested in cancer databases established by the Radiation Effects Research Foundation. The analysis revealed unique features at low doses that could not be accounted for by nominal exposure dose, including (i) the presence of a threshold that varied with organ, gender and age at exposure, and (ii) a small but significant bumping increase in cancer risk at low doses in Nagasaki that probably reflects internal exposure to 239Pu. The threshold was distinct from the canonical definition of zero effect in that it was manifested as negative excess relative risk, or suppression of background cancer rates. Such a unique tissue response at low doses of radiation exposure has been implicated in the context of the molecular basis of radiation–environment interplay in favor of recently emerging experimental evidence on DNA double-strand break repair pathway choice and its epigenetic memory by histone marking.

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“…sgs1⌬ was replaced with hisG as described previously (4). A KanMX cassette was used to create srs2⌬ (52) and top3⌬ (this study). Helicase-defective sgs1 alleles were created at the natural SGS1 locus as follows.…”

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

Cited by 25 publications

References 79 publications

A FACS-Optimized Screen Identifies Regulators of Genome Stability in Candida albicans

A FACS-Optimized Screen Identifies Regulators of Genome Stability in Candida albicans

Cancer risk at low doses of ionizing radiation: artificial neural networks inference from atomic bomb survivors

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

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