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. 2008 Jul;179(3):1179-95.
doi: 10.1534/genetics.108.089250. Epub 2008 Jun 18.

A genetic screen for increased loss of heterozygosity in Saccharomyces cerevisiae

Marguerite P Andersen  1 , Zara W Nelson, Elizabeth D Hetrick, Daniel E Gottschling
Affiliations

A genetic screen for increased loss of heterozygosity in Saccharomyces cerevisiae

Marguerite P Andersen et al. Genetics. 2008 Jul.

Abstract

Loss of heterozygosity (LOH) can be a driving force in the evolution of mitotic/somatic diploid cells, and cellular changes that increase the rate of LOH have been proposed to facilitate this process. In the yeast Saccharomyces cerevisiae, spontaneous LOH occurs by a number of mechanisms including chromosome loss and reciprocal and nonreciprocal recombination. We performed a screen in diploid yeast to identify mutants with increased rates of LOH using the collection of homozygous deletion alleles of nonessential genes. Increased LOH was quantified at three loci (MET15, SAM2, and MAT) on three different chromosomes, and the LOH events were analyzed as to whether they were reciprocal or nonreciprocal in nature. Nonreciprocal LOH was further characterized as chromosome loss or truncation, a local mutational event (gene conversion or point mutation), or break-induced replication (BIR). The 61 mutants identified could be divided into several groups, including ones that had locus-specific effects. Mutations in genes involved in DNA replication and chromatin assembly led to LOH predominantly via reciprocal recombination. In contrast, nonreciprocal LOH events with increased chromosome loss largely resulted from mutations in genes implicated in kinetochore function, sister chromatid cohesion, or relatively late steps of DNA recombination. Mutants of genes normally involved in early steps of DNA damage repair and signaling produced nonreciprocal LOH without an increased proportion of chromosome loss. Altogether, this study defines a genetic landscape for the basis of increased LOH and the processes by which it occurs.

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Figures

F<sc>igure</sc> 1.—
Figure 1.—
Loss of MET15 function serves as a robust assay for LOH. (A) In the presence of Pb+2 ion, colonies formed by cells with a functional copy of MET15 appear normal cream colored, while cells lacking a functional MET15 allele are black. LOH events in heterozygous cells (MET15/met15Δ) that become homozygous (met15/met15), result in a black colony. (B) A portion of a 96-well plate from the LOH screen is shown. Liquid cultures of MET15/met15Δ deletion mutants were applied in a 96-well format on Pb+2-containing agar medium. Upon growth and color development, LOH events at MET15 appeared as black sectors.
F<sc>igure</sc> 2.—
Figure 2.—
The multiple heterozygous markers (MHM) strain enables quantitative and qualitative study of LOH on three chromosomes. All chromosomes are shown with the left arm up. Chromosomes III, IV, and XII were each marked with two pairs of selectable heterozygous markers. One locus (on the right arm of each chromosome) is marked for quantitative measurement of LOH and a second locus is marked to facilitate qualitative analysis of the LOH events. (A) The naturally heterozygous MAT locus on the right arm of chromosome III was used for quantitative measurement of LOH. To enable detection of chromosome loss, one homolog of chromosome III was marked with HIS3 and GFP at the HIS4 locus on the left arm. (B) Chromosome IV contains heterozygous markers on the right arm only. The ADE2 marker, at the SAM2 locus of one homolog, was used for quantitative measurement of LOH. The URA3 marker inserted at the SAM2 locus of the homologous chromosome was used to distinguish between reciprocal and nonreciprocal events. HphMX and NatMX are located distal to SAM2 in a noncoding region. These markers were used to distinguish between single and multilocus LOH events. (C) MET15, on the right arm of chromosome XII, was used for quantitative measurement of LOH. The opposite homolog is marked with TRP1 to distinguish between reciprocal and nonreciprocal LOH. The LEU3 locus contains the LEU2 marker on one homolog to distinguish between single and multilocus LOH events.
F<sc>igure</sc> 3.—
Figure 3.—
Distinguishing between types of chromosome XII LOH events in the MHM strain. Phenotypes were determined for both halves of half-sectored colonies. Analysis of the TRP1 marker in the white half of the colony was used to determine whether MET15 LOH events were reciprocal (A) or nonreciprocal (B). The dark half of the colony was always Met− and Trp+. (A) Following a reciprocal LOH event (due to a crossover), the white half of the colony is Trp−. (B) The white half of the colony resulting from a nonreciprocal LOH event is Trp+. Analysis of distal markers, by growth phenotype on media lacking leucine, was used to distinguish between local LOH events (local gene conversion) and multilocus LOH events (BIR, nondisjunction or chromosome truncation). The black half of a colony resulting from a local gene conversion LOH event is Leu+, whereas in any other nonreciprocal LOH it is Leu−. [Note: in the absence of leucine, a leu3Δ allele yields slow growth (+/−) while leu2Δ does not grow (−).]
F<sc>igure</sc> 4.—
Figure 4.—
Deletion alleles can have locus-specific effects on LOH. Each circular node corresponds to a mutant with increased LOH at MET15. Connecting lines are drawn between nodes (deletion mutants) and loci (MET15, SAM2, and MAT) indicating which deletion mutants have increased LOH at these loci. For the MET15 and SAM2 loci, the criteria for increased LOH was a twofold or greater increase in the rate of LOH relative to wild type and P ≤ 0.01 (Fisher's exact test). The criteria for increased LOH at the MAT locus was a twofold or greater increase in the frequency of LOH relative to wild type and P ≤ 0.05 (Mann–Whitney test). Nodes are shaded to represent the level of increased LOH as indicated. Where appropriate, nodes are divided into multiple parts to indicate different levels of increased LOH for each locus.
F<sc>igure</sc> 5.—
Figure 5.—
Deletion alleles can affect the proportion of reciprocal LOH events at the MET15 locus. The percentages of reciprocal LOH events at MET15 in mutants with at least a fivefold increase in LOH at MET15 are shown. Phenotypic analysis was used to determine whether LOH events were reciprocal or nonreciprocal as described in Figure 3.
F<sc>igure</sc> 6.—
Figure 6.—
Deletion alleles can affect the proportion of reciprocal LOH events at the SAM2 locus. The percentages of reciprocal LOH events at SAM2 in mutants with at least a fivefold increase in LOH at SAM2 are shown. The analysis was carried out in the same manner as for Figure 5 and is outlined in supplemental Figure 1.
F<sc>igure</sc> 7.—
Figure 7.—
The LOH and chromosome loss assay for chromosome III. Heterozygous markers on both arms of chromosome III were used to distinguish between loss of a chromosome III homolog and other types of LOH. A quantitative mating assay was used to measure LOH at the MAT locus on the right arm. Diploid cells that lose the MATa allele, an LOH event, are able to mate with a MATa haploid tester strain. Those cells that can mate are screened for GFP expression. When the entire chromosome is lost in the LOH event at MAT, colonies do not express GFP. When LOH affects only the right arm of the chromosome, such as in a recombination event, colonies are GFP+.
F<sc>igure</sc> 8.—
Figure 8.—
Deletion alleles differentially affect the type of LOH event that occurs at the MAT locus. Mutants with at least a fourfold increase in the frequency of LOH at the MAT locus are shown. The median frequency of LOH at MAT is represented by the total height of the bars (both open and solid). The solid portion of the bar represents the median frequency of LOH due to chromosome III loss. Phenotypic analysis used to determine LOH and loss events is outlined in Figure 7.
F<sc>igure</sc> 9.—
Figure 9.—
Deletion alleles of genes involved in common processes lead to similar types of increased LOH. A schematic classification of mutants is shown. Mutants were categorized by whether they had increased LOH that occurred predominantly via reciprocal or nonreciprocal processes at the MET15 locus. Mutants with predominantly nonreciprocal MET15 LOH were then classified on the basis of proportions of chromosome III loss. The gene names boxed together participate in a common biochemical process or exist in a biochemically defined complex, as explained in the text.
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