scholarly journals Analysis of Repair Mechanisms following an Induced Double-Strand Break Uncovers Recessive Deleterious Alleles in theCandida albicansDiploid Genome

mBio ◽  
2016 ◽  
Vol 7 (5) ◽  
Author(s):  
Adeline Feri ◽  
Raphaël Loll-Krippleber ◽  
Pierre-Henri Commere ◽  
Corinne Maufrais ◽  
Natacha Sertour ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Genome Sequencing ◽  
Strand Break ◽  
Double Strand Break ◽  
Clonal Reproduction ◽  
Strong Increase ◽  
Recessive Lethal ◽  
Independent Events ◽  
Deleterious Alleles ◽  
Strain Sc5314

ABSTRACTThe diploid genome of the yeastCandida albicansis highly plastic, exhibiting frequent loss-of-heterozygosity (LOH) events. To provide a deeper understanding of the mechanisms leading to LOH, we investigated the repair of a unique DNA double-strand break (DSB) in the laboratoryC. albicansSC5314 strain using the I-SceI meganuclease. Upon I-SceI induction, we detected a strong increase in the frequency of LOH events at an I-SceI target locus positioned on chromosome 4 (Chr4), including events spreading from this locus to the proximal telomere. Characterization of the repair events by single nucleotide polymorphism (SNP) typing and whole-genome sequencing revealed a predominance of gene conversions, but we also observed mitotic crossover or break-induced replication events, as well as combinations of independent events. Importantly, progeny that had undergone homozygosis of part or all of Chr4 haplotype B (Chr4B) were inviable. Mining of genome sequencing data for 155C. albicansisolates allowed the identification of a recessive lethal allele in theGPI16gene on Chr4B unique toC. albicansstrain SC5314 which is responsible for this inviability. Additional recessive lethal or deleterious alleles were identified in the genomes of strain SC5314 and two clinical isolates. Our results demonstrate that recessive lethal alleles in the genomes ofC. albicansisolates prevent the occurrence of specific extended LOH events. While these and other recessive lethal and deleterious alleles are likely to accumulate inC. albicansdue to clonal reproduction, their occurrence may in turn promote the maintenance of corresponding nondeleterious alleles and, consequently, heterozygosity in theC. albicansspecies.IMPORTANCERecessive lethal alleles impose significant constraints on the biology of diploid organisms. Using a combination of an I-SceI meganuclease-mediated DNA DSB, a fluorescence-activated cell sorter (FACS)-optimized reporter of LOH, and a compendium of 155 genome sequences, we were able to unmask and identify recessive lethal and deleterious alleles in isolates ofCandida albicans, a diploid yeast and the major fungal pathogen of humans. Accumulation of recessive deleterious mutations upon clonal reproduction ofC. albicanscould contribute to the maintenance of heterozygosity despite the high frequency of LOH events in this species.

scholarly journals Identification of Recessive Lethal Alleles in the Diploid Genome of aCandida albicansLaboratory Strain Unveils a Potential Role of Repetitive Sequences in Buffering Their Deleterious Impact

mSphere ◽  
2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Timea Marton ◽  
Adeline Feri ◽  
Pierre-Henri Commere ◽  
Corinne Maufrais ◽  
Christophe d’Enfert ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Hot Spots ◽  
Laboratory Strain ◽  
Chromosome 7 ◽  
Recessive Lethal ◽  
Content Type ◽  
Repeat Sequences ◽  
Deleterious Alleles ◽  
Strain Sc5314 ◽  
The Right

ABSTRACTThe heterozygous diploid genome ofCandida albicansis 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 whoseSaccharomycescerevisiaeortholog encodes the essential Mtr4 RNA helicase. Complementation with a wild-type copy ofMTR4rescued cells homozygous for the right arm of Chr7B, demonstrating that themtr4K880*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 provideC. albicanswith 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 albicansis 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 asC. albicans. By combining a site-specific DSB-inducing system and mining genome sequencing data of 182 C. albicansisolates, we were able to ascribe the chromosome 7 homozygosis bias of theC. albicanslaboratory strain SC5314 to an heterozygous SNP introducing a premature STOP codon in theMTR4gene. 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 inC. albicanscould favor haplotype exchange, of vital importance to LOH events, leading to homozygosis of recessive lethal or deleterious alleles that inevitably accumulate upon clonality.

scholarly journals Marker Recycling in Candida albicans through CRISPR-Cas9-Induced Marker Excision

mSphere ◽  
2017 ◽  
Vol 2 (2) ◽  
Author(s):  
Manning Y. Huang ◽  
Aaron P. Mitchell
Keyword(s):  
Candida Albicans ◽  
Genetic Manipulation ◽  
Strand Break ◽  
Double Strand Break ◽  
Selection Marker ◽  
Content Type ◽  
Marker Recycling ◽  
Marker Excision ◽  
Selection For ◽  
Selection Markers

ABSTRACT It is critical to be able to alter genes in order to elucidate their functions. These alterations often rely upon markers that allow selection for a rare cell in a population that has incorporated a piece of DNA. The number of alterations that can be accomplished is thus limited by the number of selection markers that are available. This limitation is circumvented by marker recycling strategies, in which a marker is eliminated after its initial use. Then, the marker can be used again. In this report, we describe a new marker recycling strategy that is enabled by recently developed CRISPR-Cas9 technology. We describe here a new approach to marker recycling, a controlled sequence of steps in which a genetic marker is selected and then lost. Marker recycling is important for genetic manipulation, because it allows a single selection marker to be used repeatedly. Our approach relies upon the ability of the CRISPR-Cas9 system to make a targeted double-strand break in DNA and the expectation that a double-strand break within a selection marker may promote recombination between directly repeated sequences that flank the marker. We call the approach CRISPR-Cas9-induced marker excision (CRIME). We tested the utility of this approach with the fungal pathogen Candida albicans, which is typically diploid. We used two selection markers, modified to include flanking direct repeats. In a proof-of-principle study, we created successive homozygous deletions in three genes through use of the two markers and had one of the markers available in the final strain for further selection and recycling. This strategy will accelerate the creation of multiple-mutant strains in C. albicans. CRISPR-Cas9 systems have been applied to many organisms, so the genetic design principles described here may be broadly applicable. IMPORTANCE It is critical to be able to alter genes in order to elucidate their functions. These alterations often rely upon markers that allow selection for a rare cell in a population that has incorporated a piece of DNA. The number of alterations that can be accomplished is thus limited by the number of selection markers that are available. This limitation is circumvented by marker recycling strategies, in which a marker is eliminated after its initial use. Then, the marker can be used again. In this report, we describe a new marker recycling strategy that is enabled by recently developed CRISPR-Cas9 technology.

scholarly journals Role of DNA Mismatch Repair and Double-Strand Break Repair in Genome Stability and Antifungal Drug Resistance in Candida albicans

2007 ◽  
Vol 6 (12) ◽  
pp. 2194-2205 ◽  
Author(s):  
Melanie Legrand ◽  
Christine L. Chan ◽  
Peter A. Jauert ◽  
David T. Kirkpatrick
Keyword(s):  
Hydrogen Peroxide ◽  
Drug Resistance ◽  
Dna Repair ◽  
Candida Albicans ◽  
Mismatch Repair ◽  
Genome Instability ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Break Repair ◽  
Break Repair

ABSTRACT Drug resistance has become a major problem in the treatment of Candida albicans infections. Genome changes, such as aneuploidy, translocations, loss of heterozygosity, or point mutations, are often observed in clinical isolates that have become resistant to antifungal drugs. To determine whether these types of alterations result when DNA repair pathways are eliminated, we constructed yeast strains bearing deletions in six genes involved in mismatch repair (MSH2 and PMS1) or double-strand break repair (MRE11, RAD50, RAD52, and YKU80). We show that the mre11Δ/mre11Δ, rad50Δ/rad50Δ, and rad52Δ/rad52Δ mutants are slow growing and exhibit a wrinkly colony phenotype and that cultures of these mutants contain abundant elongated pseudohypha-like cells. These same mutants are susceptible to hydrogen peroxide, tetrabutyl hydrogen peroxide, UV radiation, camptothecin, ethylmethane sulfonate, and methylmethane sulfonate. The msh2Δ/msh2Δ, pms1Δ/pms1Δ, and yku80Δ/yku80Δ mutants exhibit none of these phenotypes. We observed an increase in genome instability in mre11Δ/mre11Δ and rad50Δ/rad50Δ mutants by using a GAL1/URA3 marker system to monitor the integrity of chromosome 1. We investigated the acquisition of drug resistance in the DNA repair mutants and found that deletion of mre11Δ/mre11Δ, rad50Δ/rad50Δ, or rad52Δ/rad52Δ leads to an increased susceptibility to fluconazole. Interestingly, we also observed an elevated frequency of appearance of drug-resistant colonies for both msh2Δ/msh2Δ and pms1Δ/pms1Δ (MMR mutants) and rad50Δ/rad50Δ (DSBR mutant). Our data demonstrate that defects in double-strand break repair lead to an increase in genome instability, while drug resistance arises more rapidly in C. albicans strains lacking mismatch repair proteins or proteins central to double-strand break repair.

scholarly journals Aberrant Double-Strand Break Repair inrad51 Mutants of Saccharomyces cerevisiae

2000 ◽  
Vol 20 (24) ◽  
pp. 9162-9172 ◽  
Author(s):  
Leslie E. Kang ◽  
Lorraine S. Symington
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Strand Break ◽  
Double Strand Break ◽  
Single Strand ◽  
Double Strand Break Repair ◽  
Inverted Repeats ◽  
Independent Events ◽  
Single Strand Annealing ◽  
Break Repair ◽  
Strand Annealing

ABSTRACT A number of studies of Saccharomyces cerevisiae have revealed RAD51-independent recombination events. These include spontaneous and double-strand break-induced recombination between repeated sequences, and capture of a chromosome arm by break-induced replication. Although recombination between inverted repeats is considered to be a conservative intramolecular event, the lack of requirement for RAD51 suggests that repair can also occur by a nonconservative mechanism. We propose a model forRAD51-independent recombination by one-ended strand invasion coupled to DNA synthesis, followed by single-strand annealing. The Rad1/Rad10 endonuclease is required to trim intermediates formed during single-strand annealing and thus was expected to be required forRAD51-independent events by this model. Double-strand break repair between plasmid-borne inverted repeats was less efficient inrad1 rad51 double mutants than in rad1 andrad51 strains. In addition, repair events were delayed and frequently associated with plasmid loss. Furthermore, the repair products recovered from the rad1 rad51 strain were primarily in the crossover configuration, inconsistent with conservative models for mitotic double-strand break repair.

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