DLH1, the Candida albicans homologue of the meiosis-specific DMC1, is not involved in DNA repair but catalyses spontaneous interhomologue recombination and might promote non-crossover events.

DLH1, the Candida albicans orthologue of the meiosis-specific recombinase DMC1 was expressed during the mitotic cycle. In contrast to rad51-ΔΔ that displayed reduced growth rate and severe susceptibility to DNA-damaging agents, dlh1-ΔΔ behaved as wild type (WT), rad51-ΔΔ being was epistatic to dlh1-ΔΔ. However, dlh1-ΔΔ showed an increased frequency of spontaneous loss-of-heterozygosity (LOH) at the HIS4/his4 (Chr4) locus. For both WT and dlh1-ΔΔ, His auxotrophs arose via Chr4 loss and interhomologue recombination whereas rad51-ΔΔ and rad51-ΔΔ dlh1-ΔΔ His− segregants were formed mainly by chromosome loss and truncation. A few rad51-ΔΔ, but not rad51-ΔΔ dlh1-ΔΔ, segregants showed interhomologue recombination. LOH events at the GAL1/URA3 locus (Chr1; URA3 substitutes one GAL1 allele) in WT and dlh1-ΔΔ involved mainly long tracts of DNA. A few short-tract LOH events were detected in WT but not in dlh1-ΔΔ, and this dlh1-ΔΔ phenotype was partially complemented by a WT DLH1 allele. Long-tract LOH events were also predominant in rad51-ΔΔ, but about half of them arose via chromosome truncation. We suggest that Dlh1, which conserves the Dmc1 lineage-specific amino acid residues, can promote strand invasion and might regulate in combination with Rad51 the length of the conversion tracts and the relative frequencies of mitotic non-crossovers in C. albicans.

Partner Choice in Spontaneous Mitotic Recombination in Wild Type and Homologous Recombination Mutants of Candida albicans.

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.

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

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).

Role of homologous recombination genes RAD51, RAD52, and RAD59 in the repair of lesions caused by γ-radiation to cycling and G2/M-arrested cells of Candida albicans.

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.

Candida tropicalis Isolates from Mexican Republic Exhibit High Susceptibility to Bleomycin and Variable Susceptibility to Hydrogen Peroxide.

Candida sp. are found as part of the commensal flora in humans but can cause invasive candidiasis in patients with severe underlying disease, especially cancer patients. These patients are frequently subjected to nonsurgical anticancer treatments such as ionizing radiation and anticancer drugs, which kill proliferating human cells by damaging DNA but also affect the microbiota of the patient. C. tropicalis, an emerging fungal pathogen, is associated with high mortality rates of cancer patients especially in tropical regions. In this study, we have investigated the in vitro susceptibility of 38 C. tropicalis clinical isolates from several Mexican hospitals to chronic treatments with several DNA damaging agents, including oxidizing compounds and anticancer drugs. C. tropicalis isolates displayed a high variability in their susceptibility to hydrogen peroxide (H2O2) while showing a high susceptibility to bleomycin (BLM), an anticancer drug that causes double-strand breaks in DNA. This contrasted with the moderate-to-high resistance exhibited by several C. albicans laboratory strains. At least for the C. tropicalis reference strain MYA3404, this susceptibility was hardly modified by the presence of serum. Our results open the possibility of using susceptibility to BLM to differentiate between C. tropicalis and C. albicans; however, analysis of a larger number of isolates is required. The use of BLM for prevention of C. tropicalis infections in neutropenic patients with cancer should be also evaluated. Finally, the variable susceptibility to H2O2 might be due to allelic variation of the histone acetyl-transferase complex which modulates the induction kinetics of H2O2-induced genes in C. tropicalis.

Phenotypic Consequences of a Spontaneous Loss of Heterozygosity in a Common Laboratory Strain of Candida albicans.

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.

The Candida albicans Ku70 modulates telomere length and structure by regulating both telomerase and recombination.

The heterodimeric Ku complex has been shown to participate in DNA repair and telomere regulation in a variety of organisms. Here we report a detailed characterization of the function of Ku70 in the diploid fungal pathogen Candida albicans. Both ku70 heterozygous and homozygous deletion mutants have a wild-type colony and cellular morphology, and are not sensitive to MMS or UV light. Interestingly, we observed complex effects of KU70 gene dosage on telomere lengths, with the KU70/ku70 heterozygotes exhibiting slightly shorter telomeres, and the ku70 null strain exhibiting long and heterogeneous telomeres. Analysis of combination mutants suggests that the telomere elongation in the ku70 null mutant is due mostly to unregulated telomerase action. In addition, elevated levels of extrachromosomal telomeric circles were detected in the null mutant, consistent with activation of aberrant telomeric recombination. Altogether, our observations point to multiple mechanisms of the Ku complex in telomerase regulation and telomere protection in C. albicans, and reveal interesting similarities and differences in the mechanisms of the Ku complex in disparate systems.

Loss and fragmentation of chromosome 5 are major events linked to the adaptation of rad52-DeltaDelta strains of Candida albicans to sorbose.

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.

Rad52 depletion in Candida albicans triggers both the DNA-damage checkpoint and filamentation accompanied by but independent of expression of hypha-specific genes.

We have analysed the effect of RAD52 deletion in several aspects of the cell biology of Candida albicans. Cultures of rad52Delta 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 rad52Delta 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.

Virulence and karyotype analyses of rad52 mutants of Candida albicans: regeneration of a truncated chromosome of a reintegrant strain (rad52/RAD52) in the host.

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 rad52Delta mutant was dependent upon the inoculum concentration. Mice survived at a cell inoculum of 1 x 10(6), but there was a decrease in survival time at dosages of 1.5 x 10(6) and especially at 3 x 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 x 10(6) and 1.5 x 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.

Homologous recombination in Candida albicans: role of CaRad52p in DNA repair, integration of linear DNA fragments and telomere length.

Chromosomal 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. CaRAD52 complemented poorly a rad52 mutant of S. cerevisiae. Two null Carad52Delta/Carad52Delta mutants were constructed by sequential deletion of both alleles and two reconstituted strains were obtained by reintegration of the gene. Characterization of these mutants indicated that HR plays an essential role in the repair of DNA lesions caused by both UV light and the radiomimetic compound methyl-methane-sulphonate (MMS), whereas the non-homologous end-joining pathway (NHEJ) is used only in the absence of Rad52p or after extensive DNA damage. Repair by HR is more efficient in exponentially growing than in stationary cells, probably because a larger number of cells are in late S or G2 phases of the cell cycle (and therefore, can use a sister chromatid as a substrate for recombinational repair), whereas stationary phase cells are mainly in G0 or G1, and only can be repaired using the chromosomal homologue. In addition, CaRad52p is absolutely required for the integration of linear DNA with long flanking homologous sequences. Finally, the absence of CaRad52p results in the lengthening of telomeres, even in the presence of an active telomerase, an observation not described in any other organism. This raises the possibility that both telomerase and homologous recombination may function simultaneously at C. albicans telomeres.

An evaluation of the role of LIG4 in genomic instability and adaptive mutagenesis in Candida albicans.

The non-homologous end-joining (NHEJ) pathway of DNA recombination is important for genomic stability in animal cells, since the absence of Ku70, Ku80, Lig4 or Xrcc4 results in non-reciprocal translocation and chromosome fragmentation. The role of LIG4 in the genomic instability of Candida albicans has been analyzed. We have found that both cell transformation and 5'-fluoroorotic acid selection steps used to obtain several lig4 mutants (LIG4/lig4 Ura(+); LIG4/lig4 Ura(-); lig4/lig4 Ura(+); lig4/lig4 Ura(-); and revertant lig4/LIG4 Ura(+)) resulted in significant alterations in chromosome R (ChrR). However, this effect is not specific for LIG4, since disruption of SHE9, a gene unrelated to recombination, also caused alterations in the mobility of ChrR. On the other hand, we could not detect reciprocal or non-reciprocal translocations between non-homologous chromosomes in several lig4 mutants. Furthermore, propagation of these mutants in rich medium did not cause other alterations in the mobility of ChrR. Adaptive mutagenesis of C. albicans, determined by the appearance of L-sorbose-utilizing mutants on L-sorbose plates, was also independent of the presence of Lig4 and occurred by monosomy of Chr5. Accordingly, the NHEJ pathway does not appear to be involved in the adaptive mutagenesis mediated by alterations in chromosome copy number.