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Identification of Recessive Lethal Alleles in the Diploid Genome of a Candida albicans Laboratory Strain Unveils a Potential Role of Repetitive Sequences in Buffering Their Deleterious Impact - PubMed

  • ️Tue Jan 01 2019

Identification of Recessive Lethal Alleles in the Diploid Genome of a Candida albicans Laboratory Strain Unveils a Potential Role of Repetitive Sequences in Buffering Their Deleterious Impact

Timea Marton et al. mSphere. 2019.

Abstract

The heterozygous diploid genome of Candida albicans is highly plastic, with frequent loss of heterozygosity (LOH) events. In the SC5314 laboratory strain, while LOH events are ubiquitous, a chromosome homozygosis bias is observed for certain chromosomes, whereby only one of the two homologs can occur in the homozygous state. This suggests the occurrence of recessive lethal allele(s) (RLA) preventing large-scale LOH events on these chromosomes from being stably maintained. To verify the presence of an RLA on chromosome 7 (Chr7), we utilized a system that allows (i) DNA double-strand break (DSB) induction on Chr7 by the I-SceI endonuclease and (ii) detection of the resulting long-range homozygosis. I-SceI successfully induced a DNA DSB on both Chr7 homologs, generally repaired by gene conversion. Notably, cells homozygous for the right arm of Chr7B were not recovered, confirming the presence of RLA(s) in this region. Genome data mining for RLA candidates identified a premature nonsense-generating single nucleotide polymorphism (SNP) within the HapB allele of C7_03400c whose Saccharomycescerevisiae ortholog encodes the essential Mtr4 RNA helicase. Complementation with a wild-type copy of MTR4 rescued cells homozygous for the right arm of Chr7B, demonstrating that the mtr4K880* RLA is responsible for the Chr7 homozygosis bias in strain SC5314. Furthermore, we observed that the major repeat sequences (MRS) on Chr7 acted as hot spots for interhomolog recombination. Such recombination events provide C. albicans with increased opportunities to survive DNA DSBs whose repair can lead to homozygosis of recessive lethal or deleterious alleles. This might explain the maintenance of MRS in this species.IMPORTANCECandida albicans is a major fungal pathogen, whose mode of reproduction is mainly clonal. Its genome is highly tolerant to rearrangements, in particular loss of heterozygosity events, known to unmask recessive lethal and deleterious alleles in heterozygous diploid organisms such as C. albicans By combining a site-specific DSB-inducing system and mining genome sequencing data of 182 C. albicans isolates, we were able to ascribe the chromosome 7 homozygosis bias of the C. albicans laboratory strain SC5314 to an heterozygous SNP introducing a premature STOP codon in the MTR4 gene. We have also proposed genome-wide candidates for new recessive lethal alleles. We additionally observed that the major repeat sequences (MRS) on chromosome 7 acted as hot spots for interhomolog recombination. Maintaining MRS in C. albicans could favor haplotype exchange, of vital importance to LOH events, leading to homozygosis of recessive lethal or deleterious alleles that inevitably accumulate upon clonality.

Keywords: Candida albicans; homologous recombination; loss of heterozygosity; major repeat sequences; recessive lethal alleles.

Copyright © 2019 Marton et al.

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Figures

FIG 1
FIG 1

Coupling of a DNA double-strand-break-inducing system and a FACS-optimized LOH reporter system on Chr7. (A) Illustration of strains bearing the BFP/GFP LOH reporter system on the right arm of Chr7 associated with an I-SceI-inducible DNA DSB system on Chr7B (CEC5062) or Chr7A (CEC5061). Upon addition of ATc, the mega-endonuclease I-SceI is expressed and will generate a DNA DSB at its target sequence (I-SceI TS linked to the URA3 marker) on Chr7. Theoretically, when the DNA DSB is repaired by break-induced replication or mitotic crossover, doubly fluorescent cells harboring the I-SceI-TS on BFP-bearing Chr7A will become mono-GFP, while doubly fluorescent cells harboring the I-SceI-TS on GFP-bearing Chr7B will become mono-BFP. The centromere (oval), MRS (pink arrow), and candidate RLA (diamond) on Chr7 are shown. (B) Average frequencies of 5-FOAR colonies obtained after 8 h of I-SceI overexpression and recovery from three independent experiments (n = 3) (plus standard deviations [S.D.] [error bars]) alongside fold changes observed between YPD and ATc conditions. (C) Percentage of molecular mechanisms leading to LOH among 5-FOAR colonies in induced condition (n = 32). (D) Histogram representing average frequency (n = 6) (plus S.D.) of appearance of monofluorescent cells in the presence (ATc) and absence (YPD) of expression of I-SceI and hence induced DNA DSBs on Chr7B (CEC5062) or Chr7A (CEC5061). Fold changes between uninduced and induced conditions for each monofluorescent population are indicated. (E) Characterization of monofluorescence-sorted individuals (n = 32) from induced CEC5062 (I-SceI TS on HapB) and CEC5061 (I-SceI TS on HapA) strains. A subset of sorted individuals was characterized for fluorescence and auxotrophy status in addition to SNP-RFLP for haplotype appointment in order to profile homozygosis of the right arm of Chr7 and determine the molecular mechanism used for I-SceI-induced DNA DSB repair. In the stacked histogram, white and black hatched portions represent missorted cells (false-positive results), while green and blue portions represent the proportions of properly FACS-sorted monofluorescent individuals. The presence of stripes on the green or blue bars indicate that those individuals are not fully homozygous for one given haplotype from the I-SceI TS to the BFP/GFP LOH reporter system on the right arm of Chr7. Abbreviations of molecular mechanisms are as follows; gene conversion (GC), break-induced replication (BIR), mitotic crossover (MCO), gene conversion with crossover (GC with CO).

FIG 2
FIG 2

Complementation with a functional MTR4 allele restores viability of Chr7B homozygous cells. (A) Schematic alignment of the wild-type MTR4 and mtr4K880*-encoded proteins. MTR4 encodes an ATP-dependent RNA-helicase, and the nonsense mutation in the mtr4K880* allele leads to loss of the DSH C-terminal domain shared by DEAD box helicases. aa, amino acids. (B) Illustration of the CEC5075 complemented strain possessing the functional MTR4 allele at the RPS1 locus on Chr1. Centromeres are indicated by ovals. (C) Comparison of the average fold increase of the monofluorescent populations upon DNA DSB induction (ATc/YPD) in both parental (CEC5061) and complemented (CEC5075) strains (n = 6) (plus S.D.), significance was determined using a bilateral t test (P value). (D) Characterization of monofluorescence-sorted individuals (n = 32) from induced CEC5061 (I-SceI TS on HapA) and MTR4-complemented CEC5075 strains. See the legend to Fig. 1E for explanation.

FIG 3
FIG 3

Upon DNA DSB, major repeat sequences are a source of interhomolog recombination. (A) Three subunits compose the MRS, which are hot spots for mitotic crossover. (B) Engineering of strains where the I-SceI TS is located on HapB upstream (CEC5072) or downstream (CEC5078) of mrs-7b. Illustration of the right arm of Chr7 upon interhomolog recombination at mrs-7b followed by the predominant monofluorescence profiles upon DNA DSB repair leading to long-range LOH events. (C) Histogram showing average fold changes (n = 6) (plus S.D.) of monofluorescent populations upon I-SceI induction (ATc versus YPD). While no significant difference in augmentation of both populations is detected when the I-SceI TS is placed upstream of mrs-7b (CEC5072), a significant difference is observed when the I-SceI TS is placed downstream of mrs-7b (CEC5078). Significance was determined using a bilateral t test (P value).

FIG 4
FIG 4

Schematic representation of the localization of premature STOP-inducing SNPs in the C. albicans SC5314 genome. Representation of 70 heterozygous SNPs (vertical bars) inducing a premature STOP codon distributed on all eight chromosomes of the reference strain SC5314. SNPs inducing premature STOP codons that are never found in the homozygous state in the library of 182 genomes of clinical C. albicans isolates are represented in orange, while confirmed recessive lethal and deleterious alleles are identified in red. Details regarding each SNP can be found in Table S1 using the assigned numbers in this figure.

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