Centromeres are critically important for chromosome stability and integrity. Most eukaryotes have regional centromeres that include long tracts of repetitive DNA packaged into pericentric heterochromatin. Neocentromeres, new sites of functional kinetochore assembly, can form at ectopic loci because no DNA sequence is strictly required for assembly of a functional kinetochore. In humans, neocentromeres often arise in cells with gross chromosome rearrangements that rescue an acentric chromosome. Here, we studied the properties of centromeres in Candida albicans, the most prevalent fungal pathogen of humans, which has small regional centromeres that lack pericentric heterochromatin. We functionally delimited centromere DNA on Chromosome 5 (CEN5) and then replaced the entire region with the counter-selectable URA3 gene or other marker genes. All of the resulting cen5Δ::URA3 transformants stably retained both copies of Chr5, indicating that a functional neocentromere had assembled efficiently on the homolog lacking CEN5 DNA. Strains selected to maintain only the cen5Δ::URA3 homolog and no wild-type Chr5 homolog also grew well, indicating that neocentromere function is independent of the presence of any wild-type CEN5 DNA. Two classes of neocentromere (neoCEN) strains were distinguishable: “proximal neoCEN” and “distal neoCEN” strains. Neocentromeres in the distal neoCEN strains formed at loci about 200–450 kb from cen5Δ::URA3 on either chromosome arm, as detected by massively parallel sequencing of DNA isolated by CENP-ACse4p chromatin immunoprecipitation (ChIP). In the proximal neoCEN strains, the neocentromeres formed directly adjacent to cen5Δ::URA3 and moved onto the URA3 DNA, resulting in silencing of its expression. Functional neocentromeres form efficiently at several possible loci that share properties of low gene density and flanking repeated DNA sequences. Subsequently, neocentromeres can move locally, which can be detected by silencing of an adjacent URA3 gene, or can relocate to entirely different regions of the chromosome. The ability to select for neocentromere formation and movement in C. albicans permits mechanistic analysis of the assembly and maintenance of a regional centromere.

Candida albicans strains tolerate aneuploidy, historically detected as karyotype alterations by pulsed-field gel electrophoresis and more recently revealed by array comparative genome hybridization, which provides a comprehensive and detailed description of gene copy number. Here, we first retrospectively analyzed 411 expression array experiments to predict the frequency of aneuploidy in different strains. As expected, significant levels of aneuploidy were seen in strains exposed to stress conditions, including UV light and/or sorbose treatment, as well as in strains that are resistant to antifungal drugs. More surprisingly, strains that underwent transformation with DNA displayed the highest frequency of chromosome copy number changes, with strains that were initially aneuploid exhibiting ϳ3-fold more copy number changes than strains that were initially diploid. We then prospectively analyzed the effect of lithium acetate (LiOAc) transformation protocols on the stability of trisomic chromosomes. Consistent with the retrospective analysis, the proportion of karyotype changes was highly elevated in strains carrying aneuploid chromosomes. We then tested the hypothesis that stresses conferred by heat and/or LiOAc exposure promote chromosome number changes during DNA transformation procedures. Indeed, a short pulse of very high temperature caused frequent gains and losses of multiple chromosomes or chromosome segments. Furthermore, milder heat exposure over longer periods caused increased levels of loss of heterozygosity. Nonetheless, aneuploid chromosomes were also unstable when strains were transformed by electroporation, which does not include a heat shock step. Thus, aneuploid strains are particularly prone to undergo changes in chromosome number during the stresses of DNA transformation protocols.

Dihydropyrimidine dehydrogenase (DPD; DPYD gene) variants have emerged as reliable predictors of adverse toxicity to the chemotherapy agent 5-fluorouracil (5-FU). The intronic DPYD variant rs75017182 has been recently suggested to promote alternative splicing of DPYD. However, both the extent of alternative splicing and the true contribution of rs75017182 to DPD function remain unclear. In the present study we quantified alternative splicing and DPD enzyme activity in rs75017182 carriers utilizing healthy volunteer specimens from the Mayo Clinic Biobank. Although the alternatively spliced transcript was uniquely detected in rs75017182 carriers, canonically spliced DPYD levels were only reduced by 30% (p=2.8×10−6) relative to controls. Similarly, DPD enzyme function was reduced by 35% (p=0.025). Carriers of the well-studied toxicity-associated variant rs67376798 displayed similar reductions in DPD activity (31% reduction). The modest effects on splicing and function suggest that rs75017182 may have clinical utility as a predictor of 5-FU toxicity similar to rs67376798.

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.