Mitotic Recombination and Adaptive Genomic Changes in Human Pathogenic Fungi

Abstract

Genome rearrangements and ploidy alterations are important for adaptive change in the pathogenic fungal species Candida and Cryptococcus, which propagate primarily through clonal, asexual reproduction. These changes can occur during mitotic growth and lead to enhanced virulence, drug resistance, and persistence in chronic infections. Examples of microevolution during the course of infection were described in both human infections and mouse models. Recent discoveries defining the role of sexual, parasexual, and unisexual cycles in the evolution of these pathogenic fungi further expanded our understanding of the diversity found in and between species. During mitotic growth, damage to DNA in the form of double-strand breaks (DSBs) is repaired, and genome integrity is restored by the homologous recombination and non-homologous end-joining pathways. In addition to faithful repair, these pathways can introduce minor sequence alterations at the break site or lead to more extensive genetic alterations that include loss of heterozygosity, inversions, duplications, deletions, and translocations. In particular, the prevalence of repetitive sequences in fungal genomes provides opportunities for structural rearrangements to be generated by non-allelic (ectopic) recombination. In this review, we describe DSB repair mechanisms and the types of resulting genome alterations that were documented in the model yeast Saccharomyces cerevisiae. The relevance of similar recombination events to stress- and drug-related adaptations and in generating species diversity are discussed for the human fungal pathogens Candida albicans and Cryptococcus neoformans.

Keywords: Candida; Cryptococcus; DSB repair; adaptation; asexual reproduction; gene rearrangements; genome diversity; microevolution; mitotic recombination; pathogenic fungi.

Figure 1

Double-strand break (DSB) repair mechanisms in Saccharomyces cerevisiae. Solid lines correspond to single DNA strands and dotted lines correspond to newly synthesized DNA; 3′ ends are indicated by half arrowheads. General mechanistic details are described in the text. (a) Unresected DNA ends are joined by non-homologous end-joining (NHEJ) while resection commits repair to a process that involves invasion of a donor repair template. During the resolution of the double Holliday junction (HJ) intermediates of the DSB repair pathway, the paired open and filled triangles reflect positions of junction cleavage and ligation. Cleavage of both junctions in either the horizontal or vertical orientation (open and closed triangles, respectively) yields non-crossover (NCO) products; cleavage in different orientations results in crossovers (COs). In break-induced replication (BIR), the invading end is extended to the end of the repair template and then is used as the template for synthesis of the complementary strand. Both new strands are on the repaired molecule, and the donor is unchanged. (b) Homology between direct repeats is exposed by end resection, and the annealing of the complementary strands generates tails that must be removed before ligation. Single-strand annealing (SSA) requires more homology than microhomology-mediated end-joining (MMEJ).

Figure 2

Allelic recombination and loss of heterozygosity (LOH). Replicated sister chromatids are attached at their centromeres (ovals/circles), and lines represent double-stranded DNA. The lightning bolt indicates the position of the initiating DSB, which defines the recipient molecule during repair. Black and red letters indicate heterozygous donor and recipient alleles, respectively. Thin vertical and diagonal arrows indicate segregation of sister chromatids into daughter cells, and regions of LOH are highlighted in gray boxes.

Figure 3

Rearrangements generated by ectopic interactions within or between chromosomes. Replicated sister chromatids of non-homologous chromosomes (one red and the other black) are attached at their centromeres (ovals/circles), and each line represents double-stranded DNA. Filled yellow arrows correspond to repeated sequences, and numbered lines with double arrowheads indicate the various types of ectopic interactions that can occur. The outcome of each type of CO-resolved interaction is indicated.

Figure 4

Crossover outcomes between direct and inverted repeats. (a) A CO between direct repeats deletes the region between the repeats and leaves one repeat on the chromosome. A CO between inverted repeats flips the orientation of the region between the repeats. (b) Unequal COs between direct repeats on the same arm of sister chromatids (or homologs) alter the number of repeats. If the repeats are separated by unique sequence, the intervening region is deleted in one product and duplicated in the other. An unequal CO between inverted repeats on different chromosome arms results in isochromosomes.

Figure 5

Generation of complex rearrangements. (a) Each line corresponds to a single DNA strand, and inverted repeats are indicated by gray and yellow arrows. Intra-strand pairing between closely/directly apposed inverted repeats generates a cruciform, the base of which resembles a Holliday junction. Cleavage creates hairpin-capped fragments, and replication through the hairpins results in large acentric and dicentric inverted chromosome fragments. The acentric fragment is unstable, while the dicentric fragment can undergo multiple rounds of bridge–breakage–fusion during subsequent cell divisions. (b) Lines correspond to duplex DNA, and colored triangles indicate regions of microhomology, with the relative orientation of each indicated. The lightning bolt reflects the position of the initiating DSB, and the dotted arrows indicate DNA synthesis. The numbers indicate switches to a different repair template via pairing with a region of microhomology. The final product contains multiple rearrangements.