Genome-wide mapping of spontaneous genetic alterations in diploid yeast cells

Abstract

Genomic alterations including single-base mutations, deletions and duplications, translocations, mitotic recombination events, and chromosome aneuploidy generate genetic diversity. We examined the rates of all of these genetic changes in a diploid strain of Saccharomyces cerevisiae by whole-genome sequencing of many independent isolates (n = 93) subcloned about 100 times in unstressed growth conditions. The most common alterations were point mutations and small (<100 bp) insertion/deletions (n = 1,337) and mitotic recombination events (n = 1,215). The diploid cells of most eukaryotes are heterozygous for many single-nucleotide polymorphisms (SNPs). During mitotic cell divisions, recombination can produce derivatives of these cells that have become homozygous for the polymorphisms, termed loss-of-heterozygosity (LOH) events. LOH events can change the phenotype of the cells and contribute to tumor formation in humans. We observed two types of LOH events: interstitial events (conversions) resulting in a short LOH tract (usually less than 15 kb) and terminal events (mostly cross-overs) in which the LOH tract extends to the end of the chromosome. These two types of LOH events had different distributions, suggesting that they may have initiated by different mechanisms. Based on our results, we present a method of calculating the probability of an LOH event for individual SNPs located throughout the genome. We also identified several hotspots for chromosomal rearrangements (large deletions and duplications). Our results provide insights into the relative importance of different types of genetic alterations produced during vegetative growth.

Keywords: chromosome rearrangements; loss of heterozygosity; mutations; spontaneous mitotic recombination.

Fig. 1.

LOH patterns resulting from gene conversion and cross-overs. Red and blue lines represent the homolog pairs. (A) Terminal LOH resulting from a cross-over in G2 of the cell cycle. Both daughter cells have terminal LOH, although one cell is homozygous for red SNPs and the other is homozygous for blue SNPs. (B) Terminal LOH resulting from break-induced replication in G2. Terminal LOH is observed in only one of the two daughter cells. (C) Interstitial LOH resulting from repair of a DSB in G2. DSB repair usually produces a region of gene conversion, but only a fraction of these conversions is associated with cross-overs (28). This type of conversion event results in an interstitial LOH event (right daughter cell). (D) Terminal LOH and gene conversions resulting from repair of a G1-associated DSB. Spontaneous cross-overs are often initiated by a DSB in G1 that is replicated to produce two sister chromatids broken at the same place. If one DSB is repaired by conversion unassociated with a cross-over and the second by conversion associated with a cross-over, the left cell will have an interstitial blue region of LOH and a terminal red LOH region. The right cell will have only a terminal blue region of LOH.

Fig. 2.

Patterns of deletions and duplications resulting from recombination between nontandem repeats. (A) Chromosomal region that is a hotspot for deletions and duplications on chromosome XIII. Both deletions and duplications are a consequence of homologous recombination between YMLWTy1-1 and YMLWTy1-2. (B) Heterozygous deletion between YMLWTy1-1 and YMLWTy1-2. Red and blue dots reflect the number of “reads” of W303-specific and YJM789-specific SNPs, respectively. The number of reads of W303-specific and YJM789-specific SNPs are divided by the average number of W303-specific plus the YJM789-specific reads for all SNPs in the genome, resulting in the RC (ratio of coverage) of W303-specific and YJM789-specific SNPs. Thus, SNPs represented in zero, one, or two copies in the genome have RC values of about 0, about 0.5, and about 1, respectively. The deleted region corresponds to the region containing the RRN11, CAT2, and VSP71 genes on the W303-derived chromosome. (C) Heterozygous duplication between YMLWTy1-1 and YMLWTy1-2. (D) Terminal deletion on chromosome V and terminal duplication on chromosome XIII in isolate Spo11-188. The breakpoint of the deletion is at YERCTy1-1, and the duplication breakpoint is at YMRCTy1-4. As shown in SI Appendix, Fig. S1 B and C, pairs of terminal deletions and duplications likely reflect translocations formed by recombination between repeats on nonhomologous chromosomes. This translocation was confirmed by other methods as described in the text.

Fig. 3.

Examples of interstitial and terminal LOH events as determined by DNA sequence analysis. As in Fig. 2, we show the RC (ratio of coverage) for each SNP, with W303- and YJM789-derived SNPs depicted as red and blue dots, respectively. (A) Interstitial LOH event. There is a ∼10-kb region in which there are no YJM789-derived SNPs and a double dose of W303-derived SNPs, as expected for the gene conversion event. (B) Terminal LOH event. The RC values of SNPs are about 0.5 until coordinate 1035000. Distal to that coordinate, the isolate has no sequences derived from W303 and a double dose of counts from the YJM789 homolog. This pattern is expected for a reciprocal cross-over or a BIR event.

Fig. 4.

Distribution of interstitial LOH events (gene conversions) along the chromosomes. The vertical lines of different colors indicate the number of times that SNPs were included in gene conversion tracts. The ovals represent centromeres, and the gray lines show chromosomes.

Fig. 5.

Distribution of terminal LOH breakpoints along the chromosomes. For each terminal LOH event, we defined a “window” of 20 kb that was centered on the middle of the interval between the heterozygous SNP and the homozygous SNP that define the LOH breakpoint. As in Fig. 4, the vertical lines of different colors indicate the number of times that SNPs were included in these windows. The hotspot for cross-overs on chromosome XII is misleading because the SGD (Saccharomyces Genome Database) coordinates show the 100-repeat rRNA gene cluster as a 2-repeat cluster. As discussed in the text, the rRNA gene cluster does not have more cross-overs than expected based on its physical length.

Fig. 6.

Numbers of different classes of genomic alterations summed over 93 subcultured WYspo11 isolates. A total of 2,628 events were observed.

Fig. 7.

Simplified form of the double-strand break-repair model. In this figure, recombination is initiated on the blue chromatid, and the broken ends are resected 5′ to 3′. The broken end invades the homologous chromatid, forming a D-loop. Two possible outcomes of the strand invasion are shown in A and B. It should be emphasized that recent studies of patterns of heteroduplex formation during meiotic and mitotic recombination indicate that additional steps (branch migration, “patchy” mismatch repair, and strand switching) are required to explain some recombination events. (A) Synthesis-dependent strand annealing (SDSA). Following strand invasion, the end of the invading strand is used as a primer for DNA synthesis, resulting in a longer D-loop. The invading strand is then extruded, pairing with the other broken end. The resulting heteroduplex may contain mismatches that can be repaired to produce a conversion event (enclosed in a rectangle) unassociated with a cross-over. (B) Formation of a double Holliday junction. Following strand invasion and DNA synthesis primed from the invading strand, the D-loop pairs with the second broken end. The resulting junctions can be cleaved in a variety of ways as indicated by the numbered arrows. Cleavage at positions 5, 6, 7, and 8 results in a region of conversion without an associated cross-over. Cleavage at positions 2, 4, 5, and 6 results in a conversion tract associated with a cross-over.

Fig. 8.

Number of times SNPs underwent LOH at different sites along the yeast chromosomes. The gray and green colors indicate T-LOH and I-LOH, respectively. Red triangles show the positions of centromeres. Numbers on the y axis are the numbers of LOH events at each SNP, and numbers on the x axis are SGD coordinates (in kb). Black dots at the ends of the chromosome indicate the expected level of LOH at the ends of the chromosomes resulting from cross-overs (as calculated in Dataset S7).