SummaryWe have analysed the effect of RAD52 deletion in several aspects of the cell biology of Candida albicans . Cultures of rad52 D strains exhibited slow growth and contained abundant cells with a filamentous morphology. Filamentation with polarization of actin patches was accompanied by the induction of the hypha-specific genes (HSG) ECE1 , HWP1 and HGC1 . However, filament formation occurred in the absence of the transcription factors Efg1 and Cph1, even though disruption of EFG1 prevented expression of HSG. Therefore, expression of HSG genes accompanies but is dispensable for rad52 D filamentation. However, deletion of adenylate cyclase severely impaired filamentation, this effect being largely reverted by the addition of exogenous cAMP. Filaments resembled elongated pseudohyphae, but some of them looked like true hyphae. Following depletion of Rad52, many cells arrested at the G2/M phase of the cell cycle with a single nucleus suggesting the early induction of the DNA-damage checkpoint. Filaments formed later, preferentially from G2/M cells. The filamentation process was accompanied by the uncoupling of several landmark events of the cell cycle and was partially dependent on the action of the cell cycle modulator Swe1. Hyphae were still induced by serum, but a large number of rad52 cells myceliated in G2/M.

SummaryChromosomal rearrangements are common in both clinical isolates and spontaneous mutants of Candida albicans . It appears that many of these rearrangements are caused by translocations around the major sequence repeat (MSR) that is present in all chromosomes except chromosome 3, suggesting that homologous recombination (HR) may play an important role in the survival of this organism. In order to gain information on these processes, we have cloned the homologue of RAD52 , which in Saccharomyces cerevisiae is the only gene required for all HR events.

By testing the susceptibility to DNA damaging agents of several Candida albicans mutant strains derived from the commonly used laboratory strain, CAI4, we uncovered sensitivity to methyl methanesulfonate (MMS) in CAI4 and its derivatives, but not in CAF2-1. This sensitivity is not a result of URA3 disruption because the phenotype was not restored after URA3 reintroduction. Rather, we found that homozygosis of a short region of chromosome 3R (Chr3R), which is naturally heterozygous in the MMS-resistant-related strains CAF4-2 and CAF2-1, confers MMS sensitivity and modulates growth polarization in response to MMS. Furthermore, induction of homozygosity in this region in CAF2-1 or CAF4-2 resulted in MMS sensitivity. We identified 11 genes by SNP/comparative genomic hybridization containing only the a alleles in all the MMS-sensitive strains. Four candidate genes, SNF5, POL1, orf19.5854.1, and MBP1, were analyzed by generating hemizygous configurations in CAF2-1 and CAF4-2 for each allele of all four genes. Only hemizygous MBP1a/mbp1b::SAT1-FLIP strains became MMS sensitive, indicating that MBP1a in the homo-or hemizygosis state was sufficient to account for the MMS-sensitive phenotype. In yeast, Mbp1 regulates G1/S genes involved in DNA repair. A second region of homozygosis on Chr2L increased MMS sensitivity in CAI4 (Chr3R homozygous) but not CAF4-2 (Chr3R heterozygous). This is the first example of sign epistasis in C. albicans.KEYWORDS Candida albicans; LOH; MMS susceptibility; MBP1; growth polarization T HE opportunistic fungal pathogen Candida albicans is often isolated as a highly heterozygous diploid; the genome of the reference strain SC5314 has 67,500 single nucleotide polymorphisms (SNPs) (Jones et al. 2004;Braun et al. 2005;Muzzey et al. 2013). SNPs found within regulatory regions can affect transcription levels between the alleles (Staib et al. 2002). Even synonymous SNPs residing in open reading frames (ORFS) can result in differences in the rate and efficiency of messenger RNA (mRNA) translation since poorly used codons delay protein synthesis. These delays may lead to misfolding of the nascent protein or formation of mRNA secondary structures (reviewed in Larriba and Calderone 2008). Recent genome-wide analysis of cis elements on gene expression showed that allele-specific effects are often due to mRNA levels and/or translation efficiency (Muzzey et al. 2014).While the majority of SNPs reside in intergenic regions, more than half of open reading frames contain one or more SNPs. The vast majority (78%) of these are nonsynonymous, implying that a significant fraction of ORFs encode proteins that differ in one or more amino acids (Jones et al. 2004) that may affect crucial properties. Nonconservative amino acid substitutions within an enzyme's catalytic domain could result in an inactive allele (Gómez-Raja et al. 2008). However, many SNPs will cause only minor or insignificant alterations in protein properties.Mitotic recombination or less frequently, chromosome truncation or loss, will reveal ...

The virulence of Candida albicans mutants lacking one or both copies of RAD52, a gene involved in homologous recombination (HR), was evaluated in a murine model of hematogenously disseminated candidiasis. In this study, the virulence of the rad52⌬ mutant was dependent upon the inoculum concentration. Mice survived at a cell inoculum of 1 ؋ 10 6 , but there was a decrease in survival time at dosages of 1.5 ؋ 10 6 and especially at 3 ؋ 10 6 cells per animal. The heterozygote RAD52/rad52 behaved like wild type, whereas a reintegrant strain was intermediate in its ability to cause death compared to these strains and to the avirulent rad52/rad52 null at inocula of 1 ؋ 10 6 and 1.5 ؋ 10 6 cells. A double mutant, lig4/lig4/rad52/rad52, was avirulent at all inocula used. PCR analysis of the RAD52 and/or LIG4 loci showed that all strains recovered from animals matched the genotype of the inoculated strains. Analysis of the electrophoretical karyotypes indicated that the inoculated, reintegrant strain carried a large deletion in one copy of chromosome 6 (the shortest homologue, or Chr6b). Interestingly, truncated Chr6b was regenerated in all the strains recovered from moribund animals using the homologue as a template. Further, regeneration of Chr6b was paralleled by an increase in virulence that was still lower than that of wild type, likely because of the persistent loss of heterozygosity in the regenerated region. Overall, our results indicate that systemic candidiasis can develop in the absence of HR, but simultaneous elimination of both recombination pathways, HR and nonhomologous end-joining, suppresses virulence even at very high inocula.

Candida albicans can adapt and grow on sorbose plates by losing one copy of Chr5. Since rad52 mutants of Saccharomyces cerevisiae lose chromosomes at a high rate, we have investigated the ability of C. albicans rad52 to adapt to sorbose. Carad52-DeltaDelta mutants generate Sou(+) strains earlier than wild-type but the final yield is lower, probably because they die at a higher rate in sorbose. As other strains of C. albicans, CAF2 and rad52-DeltaDelta derivatives generate Sou(+) strains by a loss of one copy of Chr5 about 75% of the time. In addition, rad52 strains were able to produce Sou(+) strains by a fragmentation/deletion event in one copy of Chr5, consisting of loss of a region adjacent to the right telomere. Finally, both CAF2 and rad52-DeltaDelta produced Sou(+) strains with two apparent full copies of Chr5, suggesting that additional genomic changes may also regulate adaptation to sorbose.