Remarkably Long-Tract Gene Conversion Induced by Fragile Site Instability in Saccharomyces cerevisiae

Abstract

Replication stress causes breaks at chromosomal locations called common fragile sites. Deletions causing loss of heterozygosity (LOH) in human tumors are strongly correlated with common fragile sites, but the role of gene conversion in LOH at fragile sites in tumors is less well studied. Here, we investigated gene conversion stimulated by instability at fragile site FS2 in the yeast Saccharomyces cerevisiae In our screening system, mitotic LOH events near FS2 are identified by production of red/white sectored colonies. We analyzed single nucleotide polymorphisms between homologs to determine the cause and extent of LOH. Instability at FS2 increases gene conversion 48- to 62-fold, and conversions unassociated with crossover represent 6-7% of LOH events. Gene conversion can result from repair of mismatches in heteroduplex DNA during synthesis-dependent strand annealing (SDSA), double-strand break repair (DSBR), and from break-induced replication (BIR) that switches templates [double BIR (dBIR)]. It has been proposed that SDSA and DSBR typically result in shorter gene-conversion tracts than dBIR. In cells under replication stress, we found that bidirectional tracts at FS2 have a median length of 40.8 kb and a wide distribution of lengths; most of these tracts are not crossover-associated. Tracts that begin at the fragile site FS2 and extend only distally are significantly shorter. The high abundance and long length of noncrossover, bidirectional gene-conversion tracts suggests that dBIR is a prominent mechanism for repair of lesions at FS2, thus this mechanism is likely to be a driver of common fragile site-stimulated LOH in human tumors.

Keywords: BIR; fragile site; gene conversion; homologous recombination; loss of heterozygosity.

Figure 1

Diploids for analysis of gene-conversion events near fragile site FS2. Four diploid strains were used to detect events that result in LOH near fragile site FS2. The MS71-derived homolog of chromosome III is shown in white and the YJM789-derived homolog of chromosome III is shown in pink. Ty1 elements are represented by black arrows. All diploids are homozygous for ade2-1. All diploids contain fragile site FS2 on the MS71-derived homolog of chromosome III. Both experimental diploids are homozygous for the GAL-POL1 construct that permits induction of replication stress by low levels of polymerase α. Both control diploids are homozygous for the POL1 gene under its native promoter. Experimental and control diploid #1 are hemizygous for SUP4-o inserted 150 bp centromere-distal to FS2. Experimental and control diploid #2 have full-length ADE2 inserted 150 bp centromere-distal to FS2, and a 5′ deletion allele of ade2 inserted in the corresponding location on the opposite homolog.

Figure 2

Sectored colonies result from events that cause LOH near FS2. In our experimental system, LOH resulting from point mutation, gene conversion, RCO, BIR, and chromosome loss cause sectored colony formation (Rosen et al. 2013). In this figure the MS71-derived homolog of chromosome III is shown in white and the YJM789-derived homolog of chromosome III is shown in pink. (A) In experimental diploid #1 and control diploid #1: any gene-conversion event in which the homolog that does not contain SUP4-o is used as a template for copying, and which results in LOH at the SUP4-o locus at the time of plating, produces a pink/red sectored colony. (B) In experimental diploid #2 and control diploid #2: any gene-conversion event in which the homolog that does not contain the full-length ADE2 is used as a template for copying, and which results in LOH at the ADE2 locus at the time of plating, produces a white/red sectored colony. (C) An RCO event is diagrammed in diploid #1. In all diploids, a crossover that occurs centromere-proximal to the marker gene (SUP4-o or ADE2) will produce a sectored colony. (D) A BIR event is diagrammed in diploid #1. In all diploids, BIR that is initiated by a lesion on the chromosome III homolog carrying the fragile site and in which invasion of the opposite homolog occurs at a location centromere-proximal to the marker gene (SUP4-o or ADE2) will produce a sectored colony. (E) A chromosome-loss event is diagrammed in diploid #1. In all diploids, loss of the chromosome III homolog carrying the fragile site will produce a sectored colony.

Figure 3

Use of SNPs to map the location of mitotic recombination events. SNPs between the two homologs of chromosome III that alter restriction sites were used to evaluate the type of event responsible for sectoring and to map the location of each event. Experimental diploid #1 is shown. The gray chromosome represents the MS71-derived homolog and the red chromosome represents the YJM789-derived homolog. Centromeres are represented by large ovals and SNP sites by small ovals. FS2 is indicated by yellow bands and SUP4-o is represented by a gray rectangle. This strain is homozygous for the ochre-suppressible ade2-1 mutation. (A) A BIR event that is stimulated by a lesion at FS2 is shown. The YJM789-derived homolog is used as a template for repair. After chromosome segregation in mitosis, the light pink cell remains heterozygous at all SNPs, while the red cell is homozygous for the YJM789 form of all SNPs distal to the invasion site. (B) A gene-conversion tract associated with RCO that occurs due to repair of a lesion at FS2 in S or G₂ phase is shown. The crossover location is indicated by a black X. Transfer of genetic information from the YJM789-derived homolog during repair resulting in 3:1 gene conversion is shown in the yellow box. After chromosome segregation in mitosis, the light pink cell is homozygous for the MS71 version of SNPs distal to the crossover, while the red cell is homozygous for the YJM789 form of SNPs distal to the crossover, and SNPs within the region of gene conversion are homozygous in the red cell but heterozygous in the light pink cell. If a crossover occurs at a more centromere-proximal location such that the SUP4-o gene is not included in a 3:1 conversion tract (not shown here), then two copies of SUP4-o could be segregated into the same cell during mitosis; resulting in red/white sectoring instead of red/light pink sectoring. (C) A noncrossover gene-conversion tract stimulated by a lesion at FS2 in S or G₂ phase is shown. Transfer of genetic information from the YJM789-derived homolog during repair resulting in 3:1 gene conversion of SNPs shown in the yellow box. After chromosome segregation in mitosis, the SNPs within the region of gene conversion are homozygous in the red cell but heterozygous in the light pink cell. Both the red cell and the light pink cell are heterozygous for SNPs centromere-distal to the gene-conversion tract.

Figure 4

LOH and gene-conversion events on chromosome III are increased under conditions of replication stress. Experimental diploids #1 and #2 contain the POL1 gene under control of the GAL1/10 promoter, therefore growth of these strains in no-galactose medium results in replication stress. Control diploids #1 and #2 have the POL1 gene under its native promoter, therefore growth of these strains in no-galactose medium does not cause replication stress. − indicates no replication stress, + indicates a low level of replication stress, and +++ indicates high levels of replication stress. (A) Total frequency of all events causing LOH on the right arm of chromosome III per mitotic division. The combined total frequency of LOH includes colonies observed due to BIR, chromosome loss, gene conversion unassociated with crossover, and RCO. Error bars represent 95% C.I. (B) Frequency of gene conversion on the right arm of chromosome III resulting in LOH during mitosis. Error bars represent 95% C.I. Gal, galactose; GC, gene conversion.

Figure 5

Gene conversions are stimulated by instability at fragile site FS2 during S phase. The MS71-derived homolog of chromosome III is shown in gray and the YJM789-derived homolog of chromosome III is shown in red. SNP markers used to map events are shown by ● and ▾ on the chromosome diagrams. ▾ indicates a restriction site exists, ● indicates lack of the site. Numbers are the approximate chromosome coordinate in kb. On the chromosome diagrams, ovals represent the centromere and black arrows represent Ty1 elements. The yellow band extending through all parts of the figure highlights the position of the fragile site and the marker gene. Gene-conversion tracts are represented by horizontal lines. Line color indicates which homolog was copied during gene conversion. Thin horizontal lines indicate 3:1 conversion tracts and thick lines indicate 4:0 tracts. Lines with an X at the end indicate crossover-associated gene conversions; we did not observe any crossover events without associated gene conversion. Lines lacking an X at the end are gene conversions that are unassociated with crossover. The initiation and termination of each gene-conversion tract are depicted in the middle between the closest flanking SNPs, but the actual location can be anywhere between the flanking SNPs. Tracts for each strain are shown in three groupings: crossover-associated tracts, noncrossover tracts with one endpoint at FS2, and noncrossover tracts that extend on both sides of FS2. (A) Locations of 54 gene conversions collected in experimental diploid #1 under replication stress (no galactose). These gene-conversion events were collected in two ways: 20 events were collected among the colonies in Table 1, and 34 events were collected among another set of 28,083 colonies. (B) Locations of 22 gene conversions collected in experimental diploid #1 in high galactose. (C) Location of the one gene conversion collected in control diploid #1 in no galactose. (D) Locations of the 25 gene conversions collected in experimental diploid #2 under replication stress (no galactose).

Figure 6

Bidirectional noncrossover gene-conversion tracts are longer than unidirectional noncrossover tracts. (A) Comparison of tract length distribution, divided by type of tract, strain, and treatment. Bidirectional noncrossover tracts are those that extend on both sides of fragile site FS2. Unidirectional noncrossover tracts are those that begin at the fragile site FS2 and extend only centromere-distally. All gene-conversion tracts from experimental diploids #1 and #2 are plotted. Each round dot represents one tract. The length of each gene conversion was calculated as the average between the minimum possible size (distance from the first to last SNP included in the tract) and the maximum possible size (distance between the two nearest outside SNPs). (B) All noncrossover-associated gene-conversion tracts, both uni- and bidirectional (n = 83), binned by 5-kb length increments. GC, gene conversion.

Figure 7

Models of repair by homologous recombination at yeast fragile site FS2. (A) The right arm of one homolog of yeast chromosome III is shown. Drawing is not to scale. The centromere is depicted by a gray oval and the two Ty1 elements at fragile site FS2 are depicted by black arrows. The closest highly-active origins are ARS 310, located ∼1 kb centromere-proximal to the fragile site; and ARS 315, located ∼55 kb centromere-distal. (B) Under conditions of replication stress resulting from low levels of polymerase α, extended ssDNA on the lagging strand at the replication fork, which (C) permits intrastrand base pairing between the two Ty1 elements forming a hairpin secondary structure. (D) If replication continues, leaving a single-strand gap on the lagging strand, this could be repaired by template switching, which can result in gene conversion in regions of hDNA, highlighted in yellow. (E) If nuclease cleavage at the fragile site collapses the replication fork, this will result in a hairpin-capped double-strand break. (F) The arrival of a replication fork from a centromere-distal ARS will result in a two-end double-strand break. Homologous recombination repair pathways at the double-strand break can result in gene conversion in regions of hDNA, highlighted in yellow. (G) Prior to convergence with a fork from a distal ARS, a one-end double-strand break exists after nuclease cleavage. A one-end double-strand break is typically repaired by BIR. The invading 3′ strand during BIR can be displaced and reinvade at a region of homology and establish a second BIR process, called dBIR. In the dBIR mechanism, gene conversion results from the amount of DNA synthesis completed prior to the template switch, highlighted in yellow. HJ, Holliday junction.