BACKGROUND
the pathogenic yeast candida albicans is one of the most common causes of fungal infection in immune compromised patients.
there is a limited spectrum of antifungal drugs to which c. albicans is susceptible, which includes the azoles, amphotericin b and the echinocandins.
the azole drug fluconazole  is a commonly used drug to treat oropharyngeal candidiasis but resistance to this drug can develop rapidly in the clinical setting.
flc has long been used to treat cases of life-threatening invasive candidiasis, but the emergence of azole resistance has favored the use of the echinocandins in invasive disease  <cit> .
resistance to the azoles can develop through a number of mechanisms, including point mutations or overexpression of a number of resistance genes.
genes known to be involved in candida albicans resistance to flc include the drug efflux pumps encoded by cdr <dig>  cdr <dig> and mdr <dig>  the flc target encoded by erg <dig>   and the transcription factors that control the expression of these genes  <cit> .
studies of flc resistant clinical and laboratory derived isolates of candida albicans have shown that point mutations followed by loss of heterozygosity  events can further increase resistance  <cit> .
recent work has shown that gross chromosomal rearrangements that lead to aneuploidy and isochromosome formation contribute to flc resistance by amplification of erg <dig> and tac <dig> mutant alleles  <cit> .
this evidence suggests that the plasticity of the candida albicans genome provides a selective advantage in certain environmental conditions, such as exposure to antifungal drugs.
work to elucidate the mechanism that leads to these types of genome events in candida albicans has shown that certain dna repair mechanisms are not involved.
for example, mechanisms such as non-homologous end joining, base excision repair and nucleotide excision repair do not appear to contribute significantly to the development of flc resistance  <cit> .
however, there is some evidence suggesting a role for homologous recombination in flc resistance, as deletion of rad <dig>  rad <dig> or mre <dig> in candida albicans alters flc susceptibility  <cit> .
the role that homologous recombination plays in flc susceptibility and genome plasticity is not fully understood, although it is known that homologous recombination pathways preserve genome structure.
since the loh events seen in flc resistance candida albicans clinical isolates likely occurs through a recombination mediated event, homologous recombination pathways may be required for loh in clinical isolates, and defects in homologous recombination may inhibit the development of flc resistance through loh events.
to further understand the role that homologous recombination pathways play in genome maintenance and dna damage resistance in candida albicans, we have examined the phenotypes of two genes proposed to be involved in homologous recombination based on their homology to the saccharomyces cerevisiae genes.
in saccharomyces cerevisiae, two members of the snf <dig> family of chromatin remodelers, rad <dig> and rdh <dig> act in the repair of double strand dna breaks through homologous recombination  <cit> .
in vitro data suggest that rad <dig> and rdh <dig> act at stages of recombination involving strand displacement and d-loop formation  <cit> .
rad <dig> and rdh <dig> belong to the rad <dig> epistasis group, which contains genes required for repair of double strand breaks generated through spontaneous events or exogenous damage.
in humans, two rad <dig> homologues, hrad <dig> and rad54b are present, and mutation of these is associated with tumor formation  <cit> .
despite similar in vitro activities of the rad <dig> and rdh <dig> proteins, the saccharomyces cerevisiae mutants have different phenotypes with respect to mitotic and meiotic recombination  <cit>  and dna damage  <cit> .
the work presented here on candida albicans rad <dig> and rdh <dig> examines the role these genes play in dna damage sensitivity and in flc susceptibility in candida albicans.
we found that candida albicans rad <dig> is required for normal cell growth and in its absence cells had an aberrant cell cycle, misdivide the nucleus, and appeared to have a dna damage checkpoint arrest.
in contrast, we found no dna damage sensitivity or alteration of the cell cycle in rdh54Δ/rdh54Δ mutants.
we did not observe a changed growth response to flc, but merely observed slower growth of the rad54Δ/rad54Δ strain with or without flc.
interestingly, candida albicans rad <dig> and rdh <dig> appeared to have some functional overlap as we were unable to construct the double mutant rad54Δ/rad54Δ rdh54Δ/rdh54Δ.
RESULTS
identification of candida albicans homologues of saccharomyces cerevisiae rad <dig> and rdh54
to identify putative homologues of saccharomyces cerevisiae rad <dig> and rdh <dig>  the protein sequence from each orf was used for blast analysis.
for each protein, putative homologues encoded in the candida albicans genome were identified.
for rad <dig>  a blast score of  <dig> e- <dig> and 69% amino acid identity over the region of highest homology was obtained.
blast analysis of rdh <dig> identified a homologue with a score of  <dig> e- <dig> and with 45% amino acid identity.
the genes identified in the blast searches correspond to orf  <dig>  and  <dig> , respectively in the candida genome database maintained at stanford university .
expression profiling of candida albicans throughout the cell cycle has indicated that candida albicans rad <dig> expression peaks in s-phase and candida albicans rdh <dig> peaks approximately at the g1/s transition  <cit> .
candida albicans rad <dig> deletion results in a slow growth phenotype
to characterize the role of rad <dig> and rdh <dig> in candida albicans, deletion strains were made in the wildtype strain sc <dig> using the sat1-flp technique described in  <cit> .
homozygous null transformants were obtained for both genes, indicating that neither was essential for growth in candida albicans.
growth curves were performed in rich media  and revealed a growth defect in the rad54Δ/rad54Δ deletion mutant .
the rad <dig> reconstruction strain did not have this defect, and grew as well as wildtype.
the doubling times of each strain were calculated, and indicated that the heterozygous null mutants, the rdh54Δ/rdh54Δ mutant, and the rad <dig> reconstruction strain all have doubling times comparable to sc <dig>  whereas the rad54Δ/rad54Δ strain had an increased doubling time .
additionally, growth on solid media showed a decreased colony size in the rad54Δ/rad54Δ mutant when compared to the wildtype or reconstruction strains .
these results are similar to those obtained for other homologous recombination mutants in candida albicans, as previously reported for rad <dig> and rad <dig>  <cit> .
we attempted to construct the double mutant rad54Δ/rad54Δ rdh54Δ/rdh54Δ without success.
the rad54/rad54Δ rdh54Δ/rdh54Δ was fully viable and was identical to the single homozygous rdh54Δ/rdh54Δ mutant for all phenotypes assayed.
candida albicans rad <dig> deletion causes altered cell and colony morphology
growth of the rad54Δ/rad54Δ strain on ypd agar plates showed not only a decrease in colony size, but also a severe colony morphology defect.
the colonies had a wrinkled appearance in contrast to the larger, smooth colonies of the parental strain and the rdh54Δ/rdh54Δ mutant.
the heterozygous deletion mutants did not have altered colony morphology, and grew as smooth colonies as seen with the wildtype strain .
the altered colony morphology was rescued by reintroduction of candida albicans rad <dig> in the reconstruction strain .
cell morphology of the rad54Δ/rad54Δ strain was also abnormal, with over 20% of cells exhibiting an elongated morphology reminiscent of pseudohyphae .
these cells did not appear to be true pseudohyphae, as they had a highly aberrant and variable morphology, similar to that seen in candida albicans strains defective in cell cycle progression.
the numbers of cells with normal and abnormal morphology were quantitated and are shown in table  <dig> and figure 2c and 2d.
when compared to wildtype, log phase cultures of the rad54Δ/rad54Δ strain had far fewer normal budding yeast cells, and a large increase in the number of cells exhibiting the abnormal morphology shown in figure 2b.
the elongated pseudohyphal cells displayed an aberrant nuclear morphology with a preponderance of the pseudohyphal cells having an elongated single dapi staining body stuck in the neck between the two cell bodies .
additional nuclear morphologies included apparent anucleate cells , cells with a nucleus in each bud where one nucleus is elongated, and cells with multiple nuclei .
regarding the pseudohyphal cells, in the single nucleate cells, 9/ <dig> had an elongated single nucleus, and in the cells with two nuclei, 10/ <dig> had one or two elongated nuclei.
dapi staining of cells also showed additional defects in chromosome segregation in the rad54Δ/rad54Δ strain.
there was an increase in g <dig> doublet cells that have a single nucleus at the neck .
this morphology is suggestive of a dna damage checkpoint arrest in saccharomyces cerevisiae  <cit>  and could apply to candida albicans  <cit> .
these phenotypes were not seen in the rdh54Δ/rdh54Δ strain, showing that these two genes have different roles in vivo.
additionally, neither the wildtype strain nor the rad <dig> reintegration strain showed these aberrant nuclear morphologies.
sensitivity to dna damage is increased in the candida albicans rad54Δ/rad54Δ mutant
in saccharomyces cerevisiae, deletion of rdh <dig> and rad <dig> leads to increased sensitivity to dna damage.
the saccharomyces cerevisiae haploid rad54Δ is highly sensitive to methyl methanesulfonate   <cit> , but the saccharomyces cerevisiae rdh <dig> gene does not appear to have as strong of a role in haploid cells, as deletion of rdh <dig> only increases mms sensitivity in diploids at normal mms concentrations  <cit> .
to test the effect of deletion of candida albicans rad <dig> and rdh <dig> on mms and menadione sensitivity, spot dilution assays were performed on ypd agar plates containing a range of mms concentrations from  <dig> % to  <dig> %, or menadione concentrations from  <dig>  mm to  <dig>  mm.
figure 3a shows that the candida albicans rad54Δ/rad54Δ strain was exquisitely sensitive to mms when compared to the wildtype strain, as sensitive as the previously reported rad50Δ/rad50Δ, mre11Δ/mre11Δ and rad52Δ/rad52Δ strains  <cit> .
the rad54Δ/rad54Δ strain also had a moderate increase in sensitivity to oxidative damage from menadione , similar to that reported for rad50Δ/rad50Δ, mre11Δ/mre11Δ and rad52Δ/rad52Δ strains  <cit> .
the heterozygous deletion strains did not show increased mms or menadione sensitivity, nor did the rdh54Δ/rdh54Δ homozygous deletion strain.
restoration of one rad <dig> allele in the reconstruction strain restored the mms and menadione sensitivity to wildtype levels.
susceptibility to antifungal drugs is not altered in the candida albicans rad54Δ/rad54Δ and candida albicans rdh54Δ/rdh54Δ mutants
previous reports have linked genomic rearrangements with the development of flc resistance in clinical isolates of candida albicans  <cit> .
interestingly, defects in double strand break repair in laboratory generated candida albicans mutants were previously shown to result in decreased susceptibility to flc  <cit> .
to test whether the homologous recombination proteins rad <dig> and rdh <dig> affect susceptibility to flc, spot dilution assays were performed.
the rad54Δ/rad54Δ mutant did not show any alteration in susceptibility to flc, and this was corroborated by the e-test method.
the rdh54Δ/rdh54Δ mutant had wildtype level of susceptibility .
the rad54Δ/rad <dig> and rdh54Δ/rdh54Δ heterozygous mutants did not show increased susceptibility to flc, and the rad <dig> reconstruction strain also had flc susceptibility similar to the wildtype strain .
it appeared that better growing segregants arose at a higher frequency in the rad54Δ/rad54Δ mutant when plated on flc-containing plates .
this would be consistent with a higher spontaneous mutation rated noted for rad54Δ and other homologous recombination mutants in saccharomyces cerevisiae  <cit> .
susceptibility to other antifungals tested was also not altered for the mutants.
amphotericin b, 5-flucytosine and caspofungin were tested using the e-test method, and mic values are shown in table  <dig>
a mics were determined using standard e-test procedure on cas plates.
values
were read after  <dig> hours of growth.
discussion
these studies address the role of candida albicans rad <dig> and candida albicans rdh <dig> in dna damage and azole sensitivity.
this study shows that candida albicans rad <dig> and candida albicans rdh <dig> are not essential genes.
this is similar to deletion mutants of other homologous recombination genes such as mre <dig>  rad <dig> and rad <dig>  <cit> .
nonetheless, the rad54Δ/rad54Δ strain gave an aberrant colony morphology that suggested both a slower cell division time and checkpoint arrest to give lethal sectoring and a jagged colony edge.
in contrast, the rdh54Δ/rdh54Δ strain grew with wildtype morphology and kinetics.
determination of cell cycle division times verified the slow growth phenotype of the rad54Δ/rad54Δ strain while the heterozygous and reconstructed rad54Δ/rad <dig> strains grew with wildtype kinetics.
examination of individual cells corroborated the aberrant morphology and slower cell cycle time.
the rad54Δ/rad54Δ strain accumulated cells with a pseudohyphal shape during log phase growth.
dapi staining of cells showed that nuclear division was aberrant, with the pseudohyphal cells often having one elongated dapi-staining body.
additionally, the rad54Δ/rad54Δ strain had an excess of doublet  cells with a single nucleus at the bud neck.
this phenotype is suggestive of a checkpoint arrest and a defect in chromosome segregation.
interestingly, the aberrant morphology of the rad54Δ/rad54Δ strain also extends to growth on spider medium.
the rad54Δ/rad54Δ strain was defective in invasion of spider agar when compared to the wildtype and reconstructed strains , perhaps due to the altered morphology of the cells.
it is noted that this aberrant growth phenotype occurs in response to spontaneous damage.
while diploid homozygous homologous recombination mutants in saccharomyces cerevisiae grow slower than wildtype diploids, they do not show aberrant colony morphology.
the saccharomyces cerevisiae rad54Δ/rad54Δ rdh54Δ/rdh54Δ mutant shows an aberrant colony morphology similar to the candida albicans rad54Δ/rad54Δ strain but is more extreme  <cit> .
attempts to make a candida albicans rad54Δ/rad54Δ rdh54Δ/rdh54Δ double mutant were unsuccessful, suggesting that the double mutant may be lethal or grows extremely poorly.
homozygous deletions of rad <dig> in chicken dt <dig> cells  <cit> , mouse  <cit> , and drosophila  <cit>  have resulted in sensitivity to ionizing radiation, mms and crosslinking agents and defective meiosis, but have only a modest effect on cell growth, if discernible at all.
we assessed mms sensitivity to determine the importance of the homologous recombination genes in dna damage repair and found, similar to saccharomyces cerevisiae, that candida albicans rad <dig> is extremely important for mms damage repair and that candida albicans rdh <dig> had no discernible role in mms damage repair.
as flc susceptibility has been linked to homologous recombination deficiency in candida albicans, we determined the flc susceptibility of the rad54Δ/rad54Δ and rdh54Δ/rdh54Δ strains.
we also determined the susceptibility to several other antifungals by e-test analysis, to test whether genomic instability might result in altered susceptibility.
the rdh54Δ/rdh54Δ and rad54Δ/rad54Δ strains did not exhibit any significant altered susceptibility to any of the antifungals tested.
additionally, the rad54Δ/rad54Δ and rdh54Δ/rdh54Δ strains showed no significant increase in flc susceptibility above the reduced growth rate of the strain in the absence of flc, suggesting that at least in the rad54Δ/rad54Δ strain, despite the obvious defects in nuclear segregation and cell division, these do not contribute to flc resistance in the short term.
it is possible that long term exposure to flc might reveal a role for genomic instability and flc resistance.
it is also possible that the rad54Δ/rad54Δ mutant is buffered by the presence of the wild type rdh <dig> genes as regards flc resistance, however the inability to recover the double mutant precludes a direct test of this hypothesis.
we noted that strains segregated colonies of varying size on flc and menadione plates.
such colonies could be candidates for segregants with mutations or genome rearrangements, but nature of the change and the rate of such segregants has not been determined.
CONCLUSIONS
the results reported here support a role for homologous recombination genes rad <dig> and rdh <dig> in dna repair under nondamaging conditions.
the nuclear morphology defects in the rad54Δ/rad54Δ mutants show that rad <dig> performs an essential role during mitotic growth and that in its absence, cells arrest in g <dig>  despite the presence of rdh <dig>  the viability of the single mutant rad54Δ/rad54Δ and the inability to construct the double mutant rad54Δ/rad54Δ rdh54Δ/rdh54Δ suggests that rdh <dig> can partially compensate for rad <dig> during mitotic growth, but that the two proteins have unique roles that contribute to cell viability.

 the pathogenic yeast candida albicans is one of the most common causes of fungal infection in immune compromised patients. there is a limited spectrum of antifungal drugs to which c. albicans is susceptible, which includes the azoles, amphotericin b and the echinocandins. the azole drug fluconazole is a commonly used drug to treat oropharyngeal candidiasis but resistance to this drug can develop rapidly in the clinical setting. flc has long been used to treat cases of life-threatening invasive candidiasis, but the emergence of azole resistance has favored the use of the echinocandins in invasive disease. resistance to the azoles can develop through a number of mechanisms, including point mutations or overexpression of a number of resistance genes. genes known to be involved in candida albicans resistance to flc include the drug efflux pumps encoded by cdr cdr and mdr the flc target encoded by erg and the transcription factors that control the expression of these genes. studies of flc resistant clinical and laboratory derived isolates of candida albicans have shown that point mutations followed by loss of heterozygosity events can further increase resistance. recent work has shown that gross chromosomal rearrangements that lead to aneuploidy and isochromosome formation contribute to flc resistance by amplification of erg and tac mutant alleles. this evidence suggests that the plasticity of the candida albicans genome provides a selective advantage in certain environmental conditions, such as exposure to antifungal drugs. work to elucidate the mechanism that leads to these types of genome events in candida albicans has shown that certain dna repair mechanisms are not involved. for example, mechanisms such as non-homologous end joining, base excision repair and nucleotide excision repair do not appear to contribute significantly to the development of flc resistance. however, there is some evidence suggesting a role for homologous recombination in flc resistance, as deletion of rad rad or mre in candida albicans alters flc susceptibility. the role that homologous recombination plays in flc susceptibility and genome plasticity is not fully understood, although it is known that homologous recombination pathways preserve genome structure. since the loh events seen in flc resistance candida albicans clinical isolates likely occurs through a recombination mediated event, homologous recombination pathways may be required for loh in clinical isolates, and defects in homologous recombination may inhibit the development of flc resistance through loh events. to further understand the role that homologous recombination pathways play in genome maintenance and dna damage resistance in candida albicans, we have examined the phenotypes of two genes proposed to be involved in homologous recombination based on their homology to the saccharomyces cerevisiae genes. in saccharomyces cerevisiae, two members of the snf family of chromatin remodelers, rad and rdh act in the repair of double strand dna breaks through homologous recombination. in vitro data suggest that rad and rdh act at stages of recombination involving strand displacement and d-loop formation. rad and rdh belong to the rad epistasis group, which contains genes required for repair of double strand breaks generated through spontaneous events or exogenous damage. in humans, two rad homologues, hrad and rad54b are present, and mutation of these is associated with tumor formation. despite similar in vitro activities of the rad and rdh proteins, the saccharomyces cerevisiae mutants have different phenotypes with respect to mitotic and meiotic recombination and dna damage. the work presented here on candida albicans rad and rdh examines the role these genes play in dna damage sensitivity and in flc susceptibility in candida albicans. we found that candida albicans rad is required for normal cell growth and in its absence cells had an aberrant cell cycle, misdivide the nucleus, and appeared to have a dna damage checkpoint arrest. in contrast, we found no dna damage sensitivity or alteration of the cell cycle in rdh54Δ/rdh54Δ mutants. we did not observe a changed growth response to flc, but merely observed slower growth of the rad54Δ/rad54Δ strain with or without flc. interestingly, candida albicans rad and rdh appeared to have some functional overlap as we were unable to construct the double mutant rad54Δ/rad54Δ rdh54Δ/rdh54Δ.  identification of candida albicans homologues of saccharomyces cerevisiae rad and rdh54 to identify putative homologues of saccharomyces cerevisiae rad and rdh the protein sequence from each orf was used for blast analysis. for each protein, putative homologues encoded in the candida albicans genome were identified. for rad a blast score of e- and 69% amino acid identity over the region of highest homology was obtained. blast analysis of rdh identified a homologue with a score of e- and with 45% amino acid identity. the genes identified in the blast searches correspond to orf and, respectively in the candida genome database maintained at stanford university. expression profiling of candida albicans throughout the cell cycle has indicated that candida albicans rad expression peaks in s-phase and candida albicans rdh peaks approximately at the g1/s transition. candida albicans rad deletion results in a slow growth phenotype to characterize the role of rad and rdh in candida albicans, deletion strains were made in the wildtype strain sc using the sat1-flp technique described in. homozygous null transformants were obtained for both genes, indicating that neither was essential for growth in candida albicans. growth curves were performed in rich media and revealed a growth defect in the rad54Δ/rad54Δ deletion mutant. the rad reconstruction strain did not have this defect, and grew as well as wildtype. the doubling times of each strain were calculated, and indicated that the heterozygous null mutants, the rdh54Δ/rdh54Δ mutant, and the rad reconstruction strain all have doubling times comparable to sc whereas the rad54Δ/rad54Δ strain had an increased doubling time. additionally, growth on solid media showed a decreased colony size in the rad54Δ/rad54Δ mutant when compared to the wildtype or reconstruction strains. these results are similar to those obtained for other homologous recombination mutants in candida albicans, as previously reported for rad and rad. we attempted to construct the double mutant rad54Δ/rad54Δ rdh54Δ/rdh54Δ without success. the rad54/rad54Δ rdh54Δ/rdh54Δ was fully viable and was identical to the single homozygous rdh54Δ/rdh54Δ mutant for all phenotypes assayed. candida albicans rad deletion causes altered cell and colony morphology growth of the rad54Δ/rad54Δ strain on ypd agar plates showed not only a decrease in colony size, but also a severe colony morphology defect. the colonies had a wrinkled appearance in contrast to the larger, smooth colonies of the parental strain and the rdh54Δ/rdh54Δ mutant. the heterozygous deletion mutants did not have altered colony morphology, and grew as smooth colonies as seen with the wildtype strain. the altered colony morphology was rescued by reintroduction of candida albicans rad in the reconstruction strain. cell morphology of the rad54Δ/rad54Δ strain was also abnormal, with over 20% of cells exhibiting an elongated morphology reminiscent of pseudohyphae. these cells did not appear to be true pseudohyphae, as they had a highly aberrant and variable morphology, similar to that seen in candida albicans strains defective in cell cycle progression. the numbers of cells with normal and abnormal morphology were quantitated and are shown in table and figure 2c and 2d. when compared to wildtype, log phase cultures of the rad54Δ/rad54Δ strain had far fewer normal budding yeast cells, and a large increase in the number of cells exhibiting the abnormal morphology shown in figure 2b. the elongated pseudohyphal cells displayed an aberrant nuclear morphology with a preponderance of the pseudohyphal cells having an elongated single dapi staining body stuck in the neck between the two cell bodies. additional nuclear morphologies included apparent anucleate cells, cells with a nucleus in each bud where one nucleus is elongated, and cells with multiple nuclei. regarding the pseudohyphal cells, in the single nucleate cells, 9/ had an elongated single nucleus, and in the cells with two nuclei, 10/ had one or two elongated nuclei. dapi staining of cells also showed additional defects in chromosome segregation in the rad54Δ/rad54Δ strain. there was an increase in g doublet cells that have a single nucleus at the neck. this morphology is suggestive of a dna damage checkpoint arrest in saccharomyces cerevisiae and could apply to candida albicans. these phenotypes were not seen in the rdh54Δ/rdh54Δ strain, showing that these two genes have different roles in vivo. additionally, neither the wildtype strain nor the rad reintegration strain showed these aberrant nuclear morphologies. sensitivity to dna damage is increased in the candida albicans rad54Δ/rad54Δ mutant in saccharomyces cerevisiae, deletion of rdh and rad leads to increased sensitivity to dna damage. the saccharomyces cerevisiae haploid rad54Δ is highly sensitive to methyl methanesulfonate, but the saccharomyces cerevisiae rdh gene does not appear to have as strong of a role in haploid cells, as deletion of rdh only increases mms sensitivity in diploids at normal mms concentrations. to test the effect of deletion of candida albicans rad and rdh on mms and menadione sensitivity, spot dilution assays were performed on ypd agar plates containing a range of mms concentrations from % to %, or menadione concentrations from mm to mm. figure 3a shows that the candida albicans rad54Δ/rad54Δ strain was exquisitely sensitive to mms when compared to the wildtype strain, as sensitive as the previously reported rad50Δ/rad50Δ, mre11Δ/mre11Δ and rad52Δ/rad52Δ strains. the rad54Δ/rad54Δ strain also had a moderate increase in sensitivity to oxidative damage from menadione, similar to that reported for rad50Δ/rad50Δ, mre11Δ/mre11Δ and rad52Δ/rad52Δ strains. the heterozygous deletion strains did not show increased mms or menadione sensitivity, nor did the rdh54Δ/rdh54Δ homozygous deletion strain. restoration of one rad allele in the reconstruction strain restored the mms and menadione sensitivity to wildtype levels. susceptibility to antifungal drugs is not altered in the candida albicans rad54Δ/rad54Δ and candida albicans rdh54Δ/rdh54Δ mutants previous reports have linked genomic rearrangements with the development of flc resistance in clinical isolates of candida albicans. interestingly, defects in double strand break repair in laboratory generated candida albicans mutants were previously shown to result in decreased susceptibility to flc. to test whether the homologous recombination proteins rad and rdh affect susceptibility to flc, spot dilution assays were performed. the rad54Δ/rad54Δ mutant did not show any alteration in susceptibility to flc, and this was corroborated by the e-test method. the rdh54Δ/rdh54Δ mutant had wildtype level of susceptibility. the rad54Δ/rad and rdh54Δ/rdh54Δ heterozygous mutants did not show increased susceptibility to flc, and the rad reconstruction strain also had flc susceptibility similar to the wildtype strain. it appeared that better growing segregants arose at a higher frequency in the rad54Δ/rad54Δ mutant when plated on flc-containing plates. this would be consistent with a higher spontaneous mutation rated noted for rad54Δ and other homologous recombination mutants in saccharomyces cerevisiae. susceptibility to other antifungals tested was also not altered for the mutants. amphotericin b, 5-flucytosine and caspofungin were tested using the e-test method, and mic values are shown in table a mics were determined using standard e-test procedure on cas plates. values were read after hours of growth. discussion these studies address the role of candida albicans rad and candida albicans rdh in dna damage and azole sensitivity. this study shows that candida albicans rad and candida albicans rdh are not essential genes. this is similar to deletion mutants of other homologous recombination genes such as mre rad and rad. nonetheless, the rad54Δ/rad54Δ strain gave an aberrant colony morphology that suggested both a slower cell division time and checkpoint arrest to give lethal sectoring and a jagged colony edge. in contrast, the rdh54Δ/rdh54Δ strain grew with wildtype morphology and kinetics. determination of cell cycle division times verified the slow growth phenotype of the rad54Δ/rad54Δ strain while the heterozygous and reconstructed rad54Δ/rad strains grew with wildtype kinetics. examination of individual cells corroborated the aberrant morphology and slower cell cycle time. the rad54Δ/rad54Δ strain accumulated cells with a pseudohyphal shape during log phase growth. dapi staining of cells showed that nuclear division was aberrant, with the pseudohyphal cells often having one elongated dapi-staining body. additionally, the rad54Δ/rad54Δ strain had an excess of doublet cells with a single nucleus at the bud neck. this phenotype is suggestive of a checkpoint arrest and a defect in chromosome segregation. interestingly, the aberrant morphology of the rad54Δ/rad54Δ strain also extends to growth on spider medium. the rad54Δ/rad54Δ strain was defective in invasion of spider agar when compared to the wildtype and reconstructed strains, perhaps due to the altered morphology of the cells. it is noted that this aberrant growth phenotype occurs in response to spontaneous damage. while diploid homozygous homologous recombination mutants in saccharomyces cerevisiae grow slower than wildtype diploids, they do not show aberrant colony morphology. the saccharomyces cerevisiae rad54Δ/rad54Δ rdh54Δ/rdh54Δ mutant shows an aberrant colony morphology similar to the candida albicans rad54Δ/rad54Δ strain but is more extreme. attempts to make a candida albicans rad54Δ/rad54Δ rdh54Δ/rdh54Δ double mutant were unsuccessful, suggesting that the double mutant may be lethal or grows extremely poorly. homozygous deletions of rad in chicken dt cells, mouse, and drosophila have resulted in sensitivity to ionizing radiation, mms and crosslinking agents and defective meiosis, but have only a modest effect on cell growth, if discernible at all. we assessed mms sensitivity to determine the importance of the homologous recombination genes in dna damage repair and found, similar to saccharomyces cerevisiae, that candida albicans rad is extremely important for mms damage repair and that candida albicans rdh had no discernible role in mms damage repair. as flc susceptibility has been linked to homologous recombination deficiency in candida albicans, we determined the flc susceptibility of the rad54Δ/rad54Δ and rdh54Δ/rdh54Δ strains. we also determined the susceptibility to several other antifungals by e-test analysis, to test whether genomic instability might result in altered susceptibility. the rdh54Δ/rdh54Δ and rad54Δ/rad54Δ strains did not exhibit any significant altered susceptibility to any of the antifungals tested. additionally, the rad54Δ/rad54Δ and rdh54Δ/rdh54Δ strains showed no significant increase in flc susceptibility above the reduced growth rate of the strain in the absence of flc, suggesting that at least in the rad54Δ/rad54Δ strain, despite the obvious defects in nuclear segregation and cell division, these do not contribute to flc resistance in the short term. it is possible that long term exposure to flc might reveal a role for genomic instability and flc resistance. it is also possible that the rad54Δ/rad54Δ mutant is buffered by the presence of the wild type rdh genes as regards flc resistance, however the inability to recover the double mutant precludes a direct test of this hypothesis. we noted that strains segregated colonies of varying size on flc and menadione plates. such colonies could be candidates for segregants with mutations or genome rearrangements, but nature of the change and the rate of such segregants has not been determined.  the results reported here support a role for homologous recombination genes rad and rdh in dna repair under nondamaging conditions. the nuclear morphology defects in the rad54Δ/rad54Δ mutants show that rad performs an essential role during mitotic growth and that in its absence, cells arrest in g despite the presence of rdh the viability of the single mutant rad54Δ/rad54Δ and the inability to construct the double mutant rad54Δ/rad54Δ rdh54Δ/rdh54Δ suggests that rdh can partially compensate for rad during mitotic growth, but that the two proteins have unique roles that contribute to cell viability. 
the nuclear morphology defects in the rad54Δ/rad54Δ mutants show that rad performs an essential role during mitotic growth and that in its absence, cells arrest in g the viability of the single mutant rad54Δ/rad54Δ and the inability to construct the double mutant rad54Δ/rad54Δ rdh54Δ/rdh54Δ suggests that rdh can partially compensate for rad during mitotic growth. this study addresses the role of the homologous recombination factors rad and rdh in cell growth, dna damage and flc resistance in candida albicans. the data presented here support a role for homologous recombination in cell growth and dna damage sensitivity, as candida albicans rad54Δ/rad54Δ mutants were hypersensitive to mms and menadione, and had an aberrant cell and nuclear morphology. in contrast, mutation of the related gene rdh did not contribute significantly to dna damage resistance and cell growth, and deletion of either candida albicans rad or candida albicans rdh did not alter flc susceptibility. together, these results support a role for homologous recombination in genome stability under nondamaging conditions. the fungal pathogen candida albicans is frequently seen in immune suppressed patients, and resistance to one of the most widely used antifungals, fluconazole, can evolve rapidly. in recent years it has become clear that plasticity of the candida albicans genome contributes to drug resistance through loss of heterozygosity at resistance genes and gross chromosomal rearrangements that amplify gene copy number of resistance associated genes. the candida albicans rad54Δ/rad54Δ mutant was defective in invasion of spider agar, presumably due to the altered cellular morphology.
 the fungal pathogen candida albicans is frequently seen in immune suppressed patients, and resistance to one of the most widely used antifungals, fluconazole, can evolve rapidly. in recent years it has become clear that plasticity of the candida albicans genome contributes to drug resistance through loss of heterozygosity at resistance genes and gross chromosomal rearrangements that amplify gene copy number of resistance associated genes. this study addresses the role of the homologous recombination factors rad and rdh in cell growth, dna damage and flc resistance in candida albicans.  the data presented here support a role for homologous recombination in cell growth and dna damage sensitivity, as candida albicans rad54Δ/rad54Δ mutants were hypersensitive to mms and menadione, and had an aberrant cell and nuclear morphology. the candida albicans rad54Δ/rad54Δ mutant was defective in invasion of spider agar, presumably due to the altered cellular morphology. in contrast, mutation of the related gene rdh did not contribute significantly to dna damage resistance and cell growth, and deletion of either candida albicans rad or candida albicans rdh did not alter flc susceptibility.  together, these results support a role for homologous recombination in genome stability under nondamaging conditions. the nuclear morphology defects in the rad54Δ/rad54Δ mutants show that rad performs an essential role during mitotic growth and that in its absence, cells arrest in g the viability of the single mutant rad54Δ/rad54Δ and the inability to construct the double mutant rad54Δ/rad54Δ rdh54Δ/rdh54Δ suggests that rdh can partially compensate for rad during mitotic growth. 