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.

Figure 1

Pathways of DSB repair by homologous recombination. In this figure, we show accepted models of DSB repair by homologous recombination. DNA strands from two different homologs are shown in red and blue; light red and blue lines indicate newly synthesized DNA. Regions of the duplex that have strands of different colors represent heteroduplexes. These pathways are described in detail in the text. (A) Synthesis-dependent strand annealing (SDSA) pathway. Following processing of the DSB, the 3′ end of the left end of the broken DNA molecule invades the other duplex. Following DNA synthesis, the invading strand is displaced and hybridizes to the right end of the broken chromosome. This pathway results in conversion events unassociated with crossovers. (B) Double-strand break repair (DSBR) pathway. In this pathway, a double Holliday junction (dHJ) is formed. Resolution of these junctions by resolvase cleavage can result in two different crossover products (middle) and two different noncrossover products (bottom right). These products have two regions of heteroduplex located in trans. Alternatively, the dHJ can be dissolved by the action of topoisomerases/helicases, resulting in a noncrossover product with heteroduplexes located in cis. (C) Break-induced replication (BIR) pathway. One of the broken ends invades the homologous chromosome and duplicates sequences from the point of invasion to the telomere. The net result of BIR events is an apparent long terminal gene conversion event.

Figure 2

Genetic system used to select mitotic crossovers and associated conversions on the left arm of chromosome V. The starting diploid strain PG311 has the ochre-suppressible can1-100 on one copy of chromosome V (shown in red) and the SUP4-o gene (encoding an ochre suppressor tRNA gene) at an allelic position on the other homolog (shown in black). The strain is homozygous for the ochre-suppressible ade2-1 allele. Strains with an unsuppressed ade2-1 mutation form red colonies. The starting diploid strain is canavanine-sensitive and forms pink colonies. (A) Reciprocal crossover without an associated gene conversion initiated by a single DSB in G2. This type of event produces a canavanine-resistant red/white sectored colony (Barbera and Petes 2006). The transition from heterozygous markers to LOH is identical in the two sectors. (B) Reciprocal crossover with an associated conversion event initiated by a single DSB in G2. If a DSB forms on one of the black chromatids, a conversion associated with the crossover may occur. This event will also result in a canavanine-resistant red/white sectored colony in which the transitions between heterozygous markers and LOH are different in the two sectors. The region of conversion is indicated by the blue rectangle. (C) Reciprocal crossover and conversion resulting from a DSB formed in G1. A black chromosome with a DSB is replicated to form two sister chromatids that are broken at the same place. One chromatid is repaired to yield a reciprocal crossover and an associated conversion; the second is repaired to yield a conversion without a crossover. The resulting red and black sectors will have a 4:0 conversion event, a region in which both sectors are homozygous for SNPs derived from the red chromatid (included within the blue rectangle).

Figure 3

Production of hybrid conversion tracts by repair of two broken sister chromatids. The black chromosome is broken in G1 and replicated to yield two broken sister chromatids. (A) Production of a 3:1/4:0 hybrid tract. If the two DSBs are repaired to yield conversion tracts that have the same centromere-proximal boundary, but different centromere-distal boundaries, a 3:1/4:0 hybrid will be generated (shown in the blue rectangle). (B) Production of a 3:1/4:0/3:1 hybrid tract. If one conversion event is extended beyond the other at both the centromere-proximal and centromere-distal boundaries, a 3:1/4:0/3:1 tract will be formed.

Figure 4

Analysis of a spontaneous reciprocal crossover (PG311-2A) on the left arm of chromosome V by SNP microarrays. Most of the details concerning this figure are explained in the text. In brief, DNA samples isolated from the red and white sectors were labeled with one fluorescent nucleotide and DNA from a heterozygous control strain was labeled with a different fluorescent nucleotide. The samples were competitively hybridized to the SNP array, and we measured the ratio of hybridization of the probes to SNP-specific oligonucleotides. The red and blue colors indicate hybridization to the W303a- and YJM789-specific oligonucleotides, respectively. CEN5 is located approximately at SGD coordinate 152000. (A) Low-resolution depiction of the samples derived from the red and white sectors. In the boxed region, the red sector has a region of LOH whereas the white sector is heterozygous at the same position. This pattern indicates a 3:1 conversion associated with the crossover. Centromere-distal to the conversion event, the red and white sectors are homozygous for the W303a- and YJM789-specific SNPs, respectively. (B) High-resolution depiction of the samples derived from the red and white sectors. Each blue and red square represents hybridization to a single oligonucleotide on the array; the converted region is boxed.

Figure 5

Mapping of crossovers and associated conversion events on the left arm of chromosome V in untreated cells and cells treated with γ-rays or UV by SNP microarrays. Each red/white sectored canavanine-resistant colony is given a number and is depicted as a pair of lines with the upper line representing the red sector and the lower line the white sector. The colored segments signify heterozygosity for the markers (green), homozygosity for the YJM789-derived SNPs (black), or homozygosity for the W303a-derived SNPs (red). The green arrows show that the markers are heterozygous from the position at which the green segment begins to the end of the chromosome, and the red and black arrows indicate that the markers are homozygous for the W303a- or the YJM789-derived SNPs, respectively, from the point at which the segment begins to the telomere of the chromosome. Internal regions of heterozygosity and homozygosity are shown as line segments without arrows and are drawn approximately to scale. The numbers at the top of the figure are SGD coordinates. and the region between CEN5 and the can1-100/SUP4-o markers is ∼120 kb in length. (A) Spontaneous crossovers and conversions. (B) γ-Ray-induced crossovers and conversions. (C) UV-induced crossovers and conversions.

Figure 6

Genomic locations of unselected recombination events and de novo mutations in untreated cells and in cells treated with UV or γ-rays as determined by SNP microarrays and HTS. The horizontal black lines depict each chromosome and are proportional to the chromosome length except for chromosome XII. The solid circles depict the centromere of each chromosome. Short horizontal bars above each chromosome depict conversion events unassociated with crossovers and the length of each bar is approximately proportional to the length of the conversion tract. All conversion tracts are shown as single solid lines without regard to the complexity of the event (e.g., transitions between 4:0 and 3:1). Single arrowheads depict reciprocal crossovers and double arrowheads depict BIR events. Asterisks located on the chromosome indicate the approximate positions of mutations induced by UV or γ-rays; two of the mutations (located at SGD coordinates 171529 and 301552 on X) are in regions of LOH. Events observed in untreated cells, in cells treated with UV, and in cells treated with γ-rays are shown in green, red, and blue, respectively. None of the events selected on the left arm of V are shown.

Figure 7

SNP array analysis of a gene conversion event unassociated with a crossover. In cells treated with γ-rays, one of the canavanine-resistant red/white sectored colonies (43RW) had an unselected gene conversion event on chromosome IV. As shown at low (A) and high (B) resolution, both sectors had an LOH region in which YJM789-derived SNPs became homozygous (0:4 conversion event). The depiction of the SNP array data is the same as in Figure 4. The length of the conversion tract is ∼3 kb. CEN4 is located approximately at SGD coordinate 450000.

Figure 8

Analysis of the same recombination event by both SNP arrays and HTS. This figure shows the analysis of the unselected recombination event on chromosome II in the red sector of the UV-induced sectored colony 8. Our standard SNP array analysis (top, A and B) showed a single transition between heterozygosity and homozygosity at about SGD coordinate 452000. (Bottom, A and B) HTS data for the same genomic sample. For the HTS data, the y-axis represents the frequency of YJM789 SNP (blue) or W303a SNP (red) “reads” for the experimental sample when assembled to the PSL2 genome. For heterozygous regions, there should be approximately equal frequencies of the two types of SNPs. It is clear from the high-resolution depictions of the HTS data that there is a short LOH region (boxed in B) located near SGD coordinate 450000 that was not detected by the SNP arrays. This region was not detected because oligonucleotides containing these SNPs were not present on the array. In the low-resolution depiction of the HTS data, within the LOH region, there is a small region near SGD coordinate 800000 in which SNPs appear to be heterozygous. These signals are artifacts on the basis of “reads” from the repeated diverged MAL and MPH genes that were incorrectly mapped by the genome analysis software to chromosome II. CEN2 is located near SGD coordinate 238000.