Dataset Information

Role of the homologous recombination genes RAD51 and RAD59 in the resistance of Candida albicans to UV light, radiomimetic and anti-tumor compounds and oxidizing agents.

ABSTRACT: We have cloned and characterized the RAD51 and RAD59 orthologs of the pathogenic fungus Candida albicans. CaRad51 exhibited more than 50% identity with several other eukaryotes and the conserved the catalytic domain of a bacterial RecA. As compared to the parental strain, null strains of rad51 exhibited a filamentous morphology, had a decreased grow rate and exhibited a moderate sensitivity to UV light, oxidizing agents, and compounds that cause double-strand breaks (DSB), indicating a role in DNA repair. By comparison, the rad52 null had a higher percentage of filaments, a more severe growth defect and a greater sensitivity to DNA-damaging compounds. Null strains of rad59 showed a UV-sensitive phenotype but behaved similarly to the parental strain in the rest of the assays. As compared to Saccharomyces cerevisiae, C. albicans was much more resistant to bleomycin and the same was true for their respective homologous recombination (HR) mutants. These results indicate that, as described in S. cerevisiae, RAD52 plays a more prominent role than RAD51 in the repair of DSBs in C. albicans and suggest the existence of at least two Rad52-dependent HR pathways, one dependent and one independent of Rad51.

SUBMITTER: Garcia-Prieto F

PROVIDER: S-EPMC2852118 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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Role of the homologous recombination genes RAD51 and RAD59 in the resistance of Candida albicans to UV light, radiomimetic and anti-tumor compounds and oxidizing agents.

García-Prieto Fátima F Gómez-Raja Jonathan J Andaluz Encarnación E Calderone Richard R Larriba Germán G

Fungal genetics and biology : FG & B 20100303 5

We have cloned and characterized the RAD51 and RAD59 orthologs of the pathogenic fungus Candida albicans. CaRad51 exhibited more than 50% identity with several other eukaryotes and the conserved the catalytic domain of a bacterial RecA. As compared to the parental strain, null strains of rad51 exhibited a filamentous morphology, had a decreased grow rate and exhibited a moderate sensitivity to UV light, oxidizing agents, and compounds that cause double-strand breaks (DSB), indicating a role in D ...[more]

PMID: 20206282

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Project description:Most of our knowledge relating to molecular mechanisms of human fungal pathogenesis in Candida albicans relies on reverse genetics approaches, requiring strain engineering. DNA-mediated transformation of C. albicans has been described as highly mutagenic, potentially accentuated by the organism's genome plasticity, including the acquisition of genomic rearrangements, notably upon exposure to stress. The advent of CRISPR-Cas9 has vastly accelerated the process of genetically modifying strains, especially in diploid (such as C. albicans) and polyploid organisms. The effects of unleashing this nuclease within the genome of C. albicans are unknown, although several studies in other organisms report Cas9-associated toxicity and off-target DNA breaks. Upon the construction of a C. albicans strain collection, we took the opportunity to compare strains which were constructed using CRISPR-Cas9-free and CRISPR-Cas9-dependent transformation strategies, by quantifying and describing transformation-induced loss-of-heterozygosity and hyperploidy events. Our analysis of 57 strains highlights the mutagenic effects of transformation in C. albicans, regardless of the transformation protocol, but also underscores interesting differences in terms of genomic changes between strains obtained using different transformation protocols. Indeed, although strains constructed using the CRISPR-Cas9-free transformation method display numerous concomitant genomic changes randomly distributed throughout their genomes, the use of CRISPR-Cas9 leads to a reduced overall number of genome changes, particularly hyperploidies. Overall, in addition to facilitating strain construction by reducing the number of transformation steps, the CRISPR-Cas9-dependent transformation strategy in C. albicans appears to limit transformation-associated genome changes.IMPORTANCE Genome editing is essential to nearly all research studies aimed at gaining insight into the molecular mechanisms underlying various biological processes, including those in the opportunistic pathogen Candida albicans The adaptation of the CRISPR-Cas9 system greatly facilitates genome engineering in many organisms. However, our understanding of the effects of CRISPR-Cas9 technology on the biology of C. albicans is limited. In this study, we sought to compare the extents of transformation-induced genomic changes within strains engineered using CRISPR-Cas9-free and CRISPR-Cas9-dependent transformation methods. CRISPR-Cas9-dependent transformation allows one to simultaneously target both homologs and, importantly, appears less mutagenic in C. albicans, since strains engineered using CRISPR-Cas9 display an overall decrease in concomitant genomic changes.

Project description:Specification of the centromere location in most eukaryotes is not solely dependent on the DNA sequence. However, the non-genetic determinants of centromere identity are not clearly defined. While multiple mechanisms, individually or in concert, may specify centromeres epigenetically, most studies in this area are focused on a universal factor, a centromere-specific histone H3 variant CENP-A, often considered as the epigenetic determinant of centromere identity. In spite of variable timing of its loading at centromeres across species, a replication coupled early S phase deposition of CENP-A is found in most yeast centromeres. Centromeres are the earliest replicating chromosomal regions in a pathogenic budding yeast Candida albicans. Using a 2-dimensional agarose gel electrophoresis assay, we identify replication origins (ORI7-LI and ORI7-RI) proximal to an early replicating centromere (CEN7) in C. albicans. We show that the replication forks stall at CEN7 in a kinetochore dependent manner and fork stalling is reduced in the absence of the homologous recombination (HR) proteins Rad51 and Rad52. Deletion of ORI7-RI causes a significant reduction in the stalled fork signal and an increased loss rate of the altered chromosome 7. The HR proteins, Rad51 and Rad52, have been shown to play a role in fork restart. Confocal microscopy shows declustered kinetochores in rad51 and rad52 mutants, which are evidence of kinetochore disintegrity. CENP-ACaCse4 levels at centromeres, as determined by chromatin immunoprecipitation (ChIP) experiments, are reduced in absence of Rad51/Rad52 resulting in disruption of the kinetochore structure. Moreover, western blot analysis reveals that delocalized CENP-A molecules in HR mutants degrade in a similar fashion as in other kinetochore mutants described before. Finally, co-immunoprecipitation assays indicate that Rad51 and Rad52 physically interact with CENP-ACaCse4 in vivo. Thus, the HR proteins Rad51 and Rad52 epigenetically maintain centromere functioning by regulating CENP-ACaCse4 levels at the programmed stall sites of early replicating centromeres.

Project description:BackgroundRAD51-dependent homologous recombination (HR) is one of the most important pathways for repairing DNA double-strand breaks (DSBs), and its regulation is crucial to maintain genome integrity. Elp1 gene encodes IKAP/ELP1, a core subunit of the Elongator complex, which has been implicated in translational regulation. However, how ELP1 contributes to genome maintenance is unclear.MethodsTo investigate the function of Elp1, Elp1-deficient mouse embryonic fibroblasts (MEFs) were generated. Metaphase chromosome spreading, immunofluorescence, and comet assays were used to access chromosome abnormalities and DSB formation. Functional roles of Elp1 in MEFs were evaluated by cell viability, colony forming capacity, and apoptosis assays. HR-dependent DNA repair was assessed by reporter assay, immunofluorescence, and western blot. Polysome profiling was used to evaluate translational efficiency. Differentially expressed proteins and signaling pathways were identified using a label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomics approach.ResultsHere, we report that Elp1 depletion enhanced genomic instability, manifested as chromosome breakage and genotoxic stress-induced genomic DNA fragmentation upon ionizing radiation (IR) exposure. Elp1-deficient cells were hypersensitive to DNA damage and exhibited impaired cell proliferation and defective HR repair. Moreover, Elp1 depletion reduced the formation of IR-induced RAD51 foci and decreased RAD51 protein levels. Polysome profiling analysis revealed that ELP1 regulated RAD51 expression by promoting its translation in response to DNA damage. Notably, the requirement for ELP1 in DSB repair could be partially rescued in Elp1-deficient cells by reintroducing RAD51, suggesting that Elp1-mediated HR-directed repair of DSBs is RAD51-dependent. Finally, using proteome analyses, we identified several proteins involved in cancer pathways and DNA damage responses as being differentially expressed upon Elp1 depletion.ConclusionsOur study uncovered a molecular mechanism underlying Elp1-mediated regulation of HR activity and provides a novel link between translational regulation and genome stability.

Project description:BackgroundIn Saccharomyces cerevisiae, Rad59 is required for multiple homologous recombination mechanisms and viability in DNA replication-defective rad27 mutant cells. Recently, four rad59 missense alleles were found to have distinct effects on homologous recombination that are consistent with separation-of-function mutations. The rad59-K166A allele alters an amino acid in a conserved α-helical domain, and, like the rad59 null allele diminishes association of Rad52 with double-strand breaks. The rad59-K174A and rad59-F180A alleles alter amino acids in the same domain and have genetically similar effects on homologous recombination. The rad59-Y92A allele alters a conserved amino acid in a separate domain, has genetically distinct effects on homologous recombination, and does not diminish association of Rad52 with double-strand breaks.ResultsIn this study, rad59 mutant strains were crossed with a rad27 null mutant to examine the effects of the rad59 alleles on the link between viability, growth and the stimulation of homologous recombination in replication-defective cells. Like the rad59 null allele, rad59-K166A was synthetically lethal in combination with rad27. The rad59-K174A and rad59-F180A alleles were not synthetically lethal in combination with rad27, had effects on growth that coincided with decreased ectopic gene conversion, but did not affect mutation, unequal sister-chromatid recombination, or loss of heterozygosity. The rad59-Y92A allele was not synthetically lethal when combined with rad27, stimulated ectopic gene conversion and heteroallelic recombination independently from rad27, and was mutually epistatic with srs2. Unlike rad27, the stimulatory effect of rad59-Y92A on homologous recombination was not accompanied by effects on growth rate, cell cycle distribution, mutation, unequal sister-chromatid recombination, or loss of heterozygosity.ConclusionsThe synthetic lethality conferred by rad59 null and rad59-K166A alleles correlates with their inhibitory effect on association of Rad52 with double-strand breaks, suggesting that this may be essential for rescuing replication lesions in rad27 mutant cells. The rad59-K174A and rad59-F180A alleles may fractionally reduce this same function, which proportionally reduced repair of replication lesions by homologous recombination and growth rate. In contrast, rad59-Y92A stimulates homologous recombination, perhaps by affecting association of replication lesions with the Rad51 recombinase. This suggests that Rad59 influences the rescue of replication lesions by multiple recombination factors.

Dataset Information

Role of the homologous recombination genes RAD51 and RAD59 in the resistance of Candida albicans to UV light, radiomimetic and anti-tumor compounds and oxidizing agents.

Publications

Role of the homologous recombination genes RAD51 and RAD59 in the resistance of Candida albicans to UV light, radiomimetic and anti-tumor compounds and oxidizing agents.

Similar Datasets

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