Genetic interactions among homologous recombination mutants in Candida albicans

Andaluz

et al. 2018

Genes

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Candida albicans mutants deficient in homologous recombination (HR) are extremely sensitive to the alkylating agent methyl-methane-sulfonate (MMS). Here, we have investigated the role of HR genes in the protection and repair of C. albicans chromosomes by taking advantage of the heat-labile property (55 °C) of MMS-induced base damage. Acute MMS treatments of cycling cells caused chromosome fragmentation in vitro (55 °C) due to the generation of heat-dependent breaks (HDBs), but not in vivo (30 °C). Following removal of MMS wild type, cells regained the chromosome ladder regardless of whether they were transferred to yeast extract/peptone/dextrose (YPD) or to phosphate buffer saline (PBS); however, repair of HDB/chromosome restitution was faster in YPD, suggesting that it was accelerated by metabolic energy and further fueled by the subsequent overgrowth of survivors. Compared to wild type CAI4, chromosome restitution in YPD was not altered in a Carad59 isogenic derivative, whereas it was significantly delayed in Carad51 and Carad52 counterparts. However, when post-MMS incubation took place in PBS, chromosome restitution in wild type and HR mutants occurred with similar kinetics, suggesting that the exquisite sensitivity of Carad51 and Carad52 mutants to MMS is due to defective fork restart. Overall, our results demonstrate that repair of HDBs by resting cells of C. albicans is rather independent of CaRad51, CaRad52, and CaRad59, suggesting that it occurs mainly by base excision repair (BER).

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Role of Homologous Recombination Genes in Repair of Alkylation Base Damage by Candida albicans

Andaluz

et al. 2018

Genes

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“…Single and double mutant strains used in this work were generated from strain CAI4, a Ura - derivative of the reference strain SC5314 (Gillum et al 1984), by disrupting the indicated allele with the hisG-URA3-hisG cassette flanked by promoter and terminator regions of the target gene (Table S1). Transformants were verified by PCR and/or Southern blot analyses as previously described (Ciudad et al 2004; Bellido et al 2015). To isolate 5FOA R derivatives, a single colony from the indicated genetic background was re-isolated on an YPD plate and then streaked on a new YPD plate supplemented with 0.1% (w/v) 5FOA and 25 µg ml -1 uridine, since C. albicans ura3 mutants are fed with uridine.…”

Section: Methodsmentioning

confidence: 99%

Partner Choice in Spontaneous Mitotic Recombination in Wild Type and Homologous Recombination Mutants ofCandida albicans

G3 Genes|Genomes|Genetics

Hermosa

et al. 2019

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Candida albicans, the most common fungal pathogen, is a diploid with a genome that is rich in repeats and has high levels of heterozygosity. To study the role of different recombination pathways on direct-repeat recombination, we replaced either allele of the RAD52 gene (Chr6) with the URA-blaster cassette (hisG-URA3-hisG), measured rates of URA3 loss as resistance to 5-fluoroorotic acid (5FOAR) and used CHEF Southern hybridization and SNP-RFLP analysis to identify recombination mechanisms and their frequency in wildtype and recombination mutants. FOAR rates varied little across different strain backgrounds. In contrast, the type and frequency of mechanisms underlying direct repeat recombination varied greatly. For example, wildtype, rad59 and lig4 strains all displayed a bias for URA3 loss via pop-out/deletion vs. inter-homolog recombination and this bias was reduced in rad51 mutants. In addition, in rad51-derived 5FOAR strains direct repeat recombination was associated with ectopic translocation (5%), chromosome loss/truncation (14%) and inter-homolog recombination (6%). In the absence of RAD52, URA3 loss was mostly due to chromosome loss and truncation (80–90%), and the bias of retained allele frequency points to the presence of a recessive lethal allele on Chr6B. However, a few single-strand annealing (SSA)-like events were identified and these were independent of either Rad59 or Lig4. Finally, the specific sizes of Chr6 truncations suggest that the inserted URA-blaster could represent a fragile site.

“…Diploid W303 wild type and mutant derivatives used carry at least one normal RAD5 allele. All the strains were routinely grown in YPD medium or synthetic complete medium (Andaluz, Ciudad, Gomez-Raja, Calderone, & Larriba, 2006;Bellido, Andaluz, Gomez-Raja, Alvarez-Barrientos, & Larriba, 2015). Omission media were made using standard recipes.…”

Section: Chromosome Degradation and Restitution In Candida Albicansmentioning

confidence: 99%

“…Andaluz, Ciudad, Gomez-Raja, Calderone, & Larriba, 2006;Bellido, Andaluz, Gomez-Raja, Alvarez-Barrientos, & Larriba, 2015). Omission media were made using standard recipes.…”

mentioning

confidence: 99%

Role of homologous recombination genesRAD51,RAD52, andRAD59in the repair of lesions caused by γ-radiation to cycling and G2/M-arrested cells ofCandida albicans

Hermosa

et al. 2018

Cellular Microbiology

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We have analysed the role of homologous recombination (HR) genes on the repair of double-strand breaks induced by γ-ionising radiation in Candida albicans. Depletion of either CaRad51 or CaRad52 caused a dramatic drop in the number of survivors compared with wild type, whereas depletion of CaRad59 caused a moderate decrease. Besides, compared with Saccharomyces cerevisiae, C. albicans relies more on HR proteins for repair of ionising radiation lesions. Pulse-field electrokaryotypes of survivors identified genetic alterations mainly in the form of aneuploidy in HR mutants and chromosome length polymorphism and ectopic translocation in wild type. Increasing irradiation (4 to 80 krad) of both cycling and nocodazole-treated (G2/M-arrested) cells revealed a gradual loss of chromosomes, larger chromosomes being more affected than smaller ones. For cycling wild-type cells, shattered chromosomes were progressively restored following incubation in yeast extract, peptone, dextrose medium, but not in phosphate-buffered saline, and this accompanied by a moderate increase in colony-forming units, suggesting that repair was followed by replication of survivors. Irradiated G2/M arrested cells from wild type but not from HR mutants partially restored the chromosome ladder following incubation (4-8 hr) in yeast peptone dextrose-nocodazole. However, HR mutants showed a chromosome shattering pattern similar to wild type, an indication that lesions other than double-strand breaks, likely single-strand break, are responsible for their drastically reduced survivability.