High-Resolution Genome-Wide Analysis of Irradiated (UV and γ-Rays) Diploid Yeast Cells Reveals a High Frequency of Genomic Loss of Heterozygosity (LOH) Events

Abstract

In diploid eukaryotes, repair of double-stranded DNA breaks by homologous recombination often leads to loss of heterozygosity (LOH). Most previous studies of mitotic recombination in Saccharomyces cerevisiae have focused on a single chromosome or a single region of one chromosome at which LOH events can be selected. In this study, we used two techniques (single-nucleotide polymorphism microarrays and high-throughput DNA sequencing) to examine genome-wide LOH in a diploid yeast strain at a resolution averaging 1 kb. We examined both selected LOH events on chromosome V and unselected events throughout the genome in untreated cells and in cells treated with either γ-radiation or ultraviolet (UV) radiation. Our analysis shows the following: (1) spontaneous and damage-induced mitotic gene conversion tracts are more than three times larger than meiotic conversion tracts, and conversion tracts associated with crossovers are usually longer and more complex than those unassociated with crossovers; (2) most of the crossovers and conversions reflect the repair of two sister chromatids broken at the same position; and (3) both UV and γ-radiation efficiently induce LOH at doses of radiation that cause no significant loss of viability. Using high-throughput DNA sequencing, we also detected new mutations induced by γ-rays and UV. To our knowledge, our study represents the first high-resolution genome-wide analysis of DNA damage-induced LOH events performed in any eukaryote.

Phenotypic Consequences of a Spontaneous Loss of Heterozygosity in a Common Laboratory Strain of Candida albicans

“…The remaining MMSresistant strains (P27 and P29) were a mixture of heterozygous and homozygous (allele b) ORFs/DNA tracks ( Figure 4, Figure 5B, Figure S4, and Table 1). While our data cannot unambiguously identify the pathways that yielded the P strains, we propose the following mechanisms (Lee et al 2009;Lee and Petes 2010;St Charles et al 2012). The allelic configurations in MMS-sensitive strains (P21, P32, and P41) are consistent with interhomolog reciprocal exchange between orf19.5854.1 and CEN3, followed by cosegregation of the chromatids carrying the a alleles into the same daughter cell ( Figure 6, A and B).…”

Homologous recombination rescues ssDNA gaps generated by nucleotide excision repair and reduced translesion DNA synthesis in yeast G2 cells

“…The requirement for HR in the absence of TLS could be caused by the generation of DSBs, possibly through overlapping repair regions as previously suggested (32,33) or somehow during the generation of the ssDNAs. Using our previously described circular chromosome assay that can detect random DSBs efficiently at frequencies of only a few DSBs per yeast genome (18,23), we investigated whether TLS deficiency leads to increased DSBs during the processing of UV damage.…”

Understanding the origins of UV-induced recombination through manipulation of sister chromatid cohesion

“…Recently, this issue was elegantly tackled by the Petes lab. 17 Through an examination of the pattern of UV-induced LOH, they deduced how and when recombination is triggered (Fig. 2).…”

“…They concluded that for cells irradiated at G 1 , recombination is also initiated in G 1 (or before the damaged locus is replicated) and occurs through a DSB mechanism. 17 Their approach involved first the identification of daughter colonies using a color-based, colony-sectoring assay. Then, they addressed LOH across the genome by following 55,000 polymorphic sites of heterozygosity.…”