High-resolution mapping of two types of spontaneous mitotic gene conversion events in Saccharomyces cerevisiae

Abstract

Gene conversions and crossovers are related products of the repair of double-stranded DNA breaks by homologous recombination. Most previous studies of mitotic gene conversion events have been restricted to measuring conversion tracts that are <5 kb. Using a genetic assay in which the lengths of very long gene conversion tracts can be measured, we detected two types of conversions: those with a median size of ∼6 kb and those with a median size of >50 kb. The unusually long tracts are initiated at a naturally occurring recombination hotspot formed by two inverted Ty elements. We suggest that these long gene conversion events may be generated by a mechanism (break-induced replication or repair of a double-stranded DNA gap) different from the short conversion tracts that likely reflect heteroduplex formation followed by DNA mismatch repair. Both the short and long mitotic conversion tracts are considerably longer than those observed in meiosis. Since mitotic crossovers in a diploid can result in a heterozygous recessive deleterious mutation becoming homozygous, it has been suggested that the repair of DNA breaks by mitotic recombination involves gene conversion events that are unassociated with crossing over. In contrast to this prediction, we found that ∼40% of the conversion tracts are associated with crossovers. Spontaneous mitotic crossover events in yeast are frequent enough to be an important factor in genome evolution.

Keywords: DNA damage repair; gene conversion; loss of heterozygosity; mitotic recombination; yeast.

Figure 1

Pathways of repair of DNA breaks by homologous recombination. Two recombining double-stranded DNA molecules are shown as paired red and blue lines. Dashed lines indicate DNA synthesis. Recombination events are initiated by a DSB, followed by 5′ to 3′ processing of the broken ends. All pathways are initiated by invasion of one end of the broken chromosome into the unbroken chromosome, followed by formation of a D loop caused by DNA synthesis from the invading 3′ strand. (A) Synthesis-dependent strand annealing (SDSA). Following DNA synthesis, the invading strand disassociates from the template and reassociates with the other broken end. The net result is a heteroduplex on one side of the position of the original DSB with flanking markers in the original configuration (NCO). The region of the heteroduplex is boxed. Repair of mismatches within the heteroduplex can result in a gene conversion (two blue strands) or a restoration event (two red strands). (B) Double Holliday junction (dHJ). The non-invading broken end anneals with the D loop forming two junctions. Depending on the mode of cleavage of the junctions, this structure can be resolved as a noncrossover (left) or a crossover (center). Alternatively, the structure can be dissolved without junction cleavage (right). (C) Break-induced replication (BIR). The end from the left portion of the broken chromosome sets up a moving D loop that replicates the intact chromosome by conservative replication. The right portion of the broken chromosome is lost.

Figure 2

Genetic system for the detection of gene conversion and crossover events on chromosome IV. Conversion and crossover events are shown as occurring between replicated chromatids. Centromeres are depicted as ovals, and red and blue lines indicate the YJM789- and the W303-1A-derived homologs, respectively. The hphMX4 and the SUP4-o genes are located on only one of the two homologs. Recombination events are selected on plates containing 5-FOA that selects for loss of the wild-type URA3 gene. Diploid cells with zero, one, and two copies of SUP4-o form red, pink, and white colonies, respectively. (A) Gene conversion without an associated crossover (NCO, class IA). In this event, wild-type URA3 sequences are replaced by mutant sequences as indicated by the horizontal arrow. As shown by the pair of arrows, one type of segregation will result in a Trp⁺ Hyg^R 5-FOA^R pink colony with the flanking markers in the original coupling arrangement. (B) Conversion with an associated crossover (CO, class IB). Following the crossover, if the recombinant chromatids cosegregate (left), a Trp⁺ Hyg^R 5-FOA^R pink colony will be observed as in A. If one recombinant and one nonrecombinant chromatid cosegregate (right), a Trp⁺ Hyg^R 5-FOA^R red colony will be formed. (C) Crossover centromere-proximal to the hphMX4 marker. In this event, the 5-FOA^R derivative is generated without a conversion. Following the crossover, if one of the recombinant chromosomes cosegregates with the nonrecombinant YJM789-derived homolog, a Trp⁺ Hyg^S 5-FOA^R red colony will be generated.

Figure 3

Microarray analysis of the extent of a gene conversion event in EY7-40. The strain EY7-40 has the phenotype indicative of a gene conversion event (class I, 5-FOA^R Trp⁺ Hyg^R pink). DNA was isolated and hybridized to a SNP-specific microarray, and the ratio of hybridization (EY7-40 vs. DNA from a control heterozygous strain) to W303-1A-specific and YJM789-specific SNPs was measured. The red lines or boxes show hybridization to YJM789-specific SNPs, and the blue lines or diamonds indicate hybridization to W303-1A-specific SNPs. (A) Low-resolution analysis. The values on the x-axis are SGD coordinates in base pairs. The URA3 insertion is located between bases 1013217 and 1013218. The ratio was calculated in a moving window of 9 SNPs. (B) High-resolution analysis. In this depiction, each square and diamond shows the hybridization signal to a specific oligonucleotide on the microarray. (C) Schematic depiction of the conversion event showing the transitions between heterozygous and homozygous SNPs (green indicating heterozygous SNPs and red showing the region homozygous for the YJM789-derived SNPs). The “a” transition is located between coordinates 1008881 and 1009116, and the “b” transition is between 1013370 and 1013909 (Table S4).

Figure 4

Map locations of gene conversion events. Our mapping of 54 conversion events that include the ura3-e mutation is summarized. The blue and red lines indicate the homologs derived from W303-1A and YJM789, respectively. The lengths of independent conversion events are shown by horizontal lines labeled with the number of the EY7 isolate. Green and purple lines indicate NCO conversions and CO conversions, respectively. The vertical black lines at the bottom of the figure show the distribution of SNPs on the microarray, and the vertical orange lines show the location of SNPs examined by SPA.

Figure 5

Histogram of gene conversion tract lengths. Based on the analysis of Table S4, the 54 conversion events appear to have two distinct size distributions. All of the events with tracts lengths >25 kb include HS4.

Figure 6

Meiotic analysis of the coupling of markers flanking a mitotic gene conversion event. To distinguish whether the conversion was unassociated (NCO) or associated (CO) with a crossover, we examined class I strains by tetrad analysis. We analyzed the patterns of segregation for the centromere-proximal hphMX4 marker and a SNP located centromere-distal to the conversion tract. The red and blue lines signify chromosome regions derived from the YJM789-derived and W303-1A-derived homologs, respectively. (A) Meiotic segregation patterns expected for class I strains with an NCO conversion. If there is no meiotic crossover (NCO), we expect a PD tetrad: two Hyg^R SNP^W spores and two Hyg^S SNP^Y spores. A single crossover (SCO) between the hphMX4 marker and the diagnostic SNP would produce a tetratype tetrad (TT): one Hyg^R SNP^W spore, one Hyg^R SNP^Y spore, one Hyg^S SNP^W spore, and one Hyg^S SNP^Y spore. Finally, a four-stranded double-crossover (DCO) between the hphMX4 marker and the diagnostic SNP would produce an NPD: two Hyg^R SNP^Y spores and two Hyg^S SNP^W spores. (B) Meiotic segregation patterns expected for class I strains with a CO conversion. Similar segregation patterns to those observed in A are expected, but the coupling of the markers in the PD and NPD tetrads would be reversed from those observed in A.

Figure 7

Generation of a long gene conversion tract by a double BIR event. We show two double-stranded recombining DNA molecules with the centromere shown as a circle. Dotted lines show DNA synthesis. The conversion tract is formed by two BIR events in the following steps: step 1—a DSB occurs on the blue chromosome (thick arrow) with 5′ to 3′ resection of the broken ends; step 2—the left end invades the red chromosome and initiates DNA synthesis (dotted red line); step 3—synthesis continues in a moving D-loop mode with second strand synthesis occurring on the displaced strand; step 4—the chromosome involved in the BIR event disengages from the red chromosome and invades the centromere-distal fragment of the blue chromosome to begin a second BIR event; and step 5—the BIR event continues to the end of the chromosome, and the acentric blue chromosome fragment is lost.