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.

Electrophoretic karyotyping of the Candida albicans revealed a different migration pattern of ChR in three different stocks of the sequencing strain SC5314. In one stock, the high instability of ChR size prevented the migration of ChR as a compact band; ChR appeared, instead, as a smear. In some stocks, ChR and/or Ch1 ploidy diminished, suggesting mixed populations of disomic and monosomic cells. Similarly, some stocks of widely used derivatives CAI4 and BWP17 contained smearing of ChR. In addition, the most manipulated strain in the lineage of SC5314, the last derivative, BWP17, acquired an increase in the size of Ch7b and revealed an unusual property. BWP17 did not tolerate a well-established procedure of telomere-mediated fragmentation of a chromosome; the remaining intact homologue always duplicated. We suggest that some stocks of SC5314 are unstable and that BWP17 may not be appropriate for general studies. Instead of BWP17 or CAI4, we recommend using for general research CAF4-2, which is a relatively stable Ura − derivative, and which has been successfully used for more than a decade in our laboratory.

Summary RAD52 is required for almost all recombination events in S. cerevisiae. We took advantage of the heterozygosity of HIS4 in the C. albicans SC5314 lineage to study the role of Rad52 in the genomic stability of this important fungal pathogen. The rate of loss of heterozygosity (LOH) at HIS4 in rad52-ΔΔ strains was ~10−3, at least 100-fold higher than in Rad52+ strains. LOH of whole chromosome 4 or truncation of the homologue that carries the functional HIS4 allele was detected in all 80 rad52-ΔΔ His auxotrophs (GLH –GL lab His−) obtained from six independent experiments. Isolates that had undergone whole chromosome LOH, presumably due to loss of chromosome, carried two copies of the remaining homolog. Isolates with truncations carried centric fragments of broken chromosomes healed by de novo telomere addition. GLH strains exhibited variable degrees of LOH across the genome, including two strains that became homozygous for all the heterozygous markers tested. In addition, GLH strains exhibited increased chromosomal instability (CIN), which was abolished by reintroduction of RAD52. CIN of GLH isolates is reminiscent of genomic alterations leading to cancer in human cells, and support the mutator hypothesis in which a mutator mutation or CIN phenotype facilitate more mutations/aneuploidies.

We have cloned and characterized the RAD51 and RAD59 orthologues 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 S. 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.