A fine-structure map of spontaneous mitotic crossovers in the yeast Saccharomyces cerevisiae

Abstract

Homologous recombination is an important mechanism for the repair of DNA damage in mitotically dividing cells. Mitotic crossovers between homologues with heterozygous alleles can produce two homozygous daughter cells (loss of heterozygosity), whereas crossovers between repeated genes on non-homologous chromosomes can result in translocations. Using a genetic system that allows selection of daughter cells that contain the reciprocal products of mitotic crossing over, we mapped crossovers and gene conversion events at a resolution of about 4 kb in a 120-kb region of chromosome V of Saccharomyces cerevisiae. The gene conversion tracts associated with mitotic crossovers are much longer (averaging about 12 kb) than the conversion tracts associated with meiotic recombination and are non-randomly distributed along the chromosome. In addition, about 40% of the conversion events have patterns of marker segregation that are most simply explained as reflecting the repair of a chromosome that was broken in G1 of the cell cycle.

Figure 1. Detection of mitotic recombination events in a diploid heterozygous for the can1 gene.

The two homologues are depicted in G2 with the duplicated chromatids held together at the centromere (shown as ovals). A) Following a reciprocal crossover (RCO), one daughter cell is homozygous for the recessive can1 allele and is canavanine resistant, whereas the other daughter cell is homozygous for the wild-type allele and is canavanine sensitive. Note that only one of the two possible chromosome disjunction patterns is shown; the other pattern does not lead to the markers becoming homozygous. B) Break-induced replication (BIR) is a fundamentally non-reciprocal process. In this depiction, the black chromatid is broken and the broken end invades the red chromatid, duplicating all the sequences to the end of the chromatid. The net result of this process is one Can^R can1/can1 cell and one Can^S can1/CAN1 cell.

Figure 2. Intragenic mitotic gene conversion associated with crossing over.

The two heteroalleles (a1 and a2) are shown as rectangles with the position of the mutation indicated by a horizontal line within the rectangle. In this diagram, wild-type genetic information is transferred (indicated by a short horizontal arrow) from the centromere-distal part of the a1 allele to the centromere-distal part of the a2 allele, resulting in a wild-type A gene. The horizontal rectangle shows the region of gene conversion (three of the chromatids having wild-type sequences at the distal end of the gene and one having mutant sequences). The wild-type and mutant alleles of the centromere-distal marker are shown as white and black rectangles, respectively.

Figure 3. A diploid strain that allows the selection of both products of an RCO.

The SUP4-o gene encodes a tRNA that suppresses both the can1-100 and ade2-1 alleles. Strains that have these mutations in the absence of the suppressor are canavanine resistant, adenine auxotrophs, and form red colonies (because of the accumulation of a pigmented precursor to adenine). In the presence of the suppressor, the strains are canavanine sensitive, adenine prototrophs, and form white colonies. If there is an RCO between the centromere and the can1-100/SUP4-o markers, two Can^R cells will be produced; subsequent divisions of these cells will result in a red/white Can^R sectored colony.

Figure 4. Segregation patterns of heterozygous markers after RCO.

In this figure, the heterozygous markers are depicted as circles. Only seven of the 34 heterozygous markers in the CEN5-CAN1 interval are shown. The red and black colors represent markers derived from the W303A- and YJM789-related chromosomes, respectively. A) Following an RCO that had no associated conversion, both sectors are heterozygous for all markers centromere-proximal to the exchange. Distal to the exchange, the white sector is homozygous for the YJM789 markers and the red sector is homozygous for the W303A markers. B) This diagram shows a conversion event (indicated by the arrow) in which one of the black markers is lost and one of the red markers is duplicated. For this marker (boxed with the horizontal rectangle), three of the chromatids have the red marker and one has the black marker. Proximal to the conversion and associated crossover, the markers in both sectors are heterozygous; distal to the conversion/crossover boundary, the markers are homozygous with the same patterns observed in Figure 4A.

Figure 5. Mapping of RCOs and associated gene conversion tracts in the CEN5-CAN1 interval.

Thirty-four markers were used to map events in 74 independent red/white Can^R colonies; both sectors were analyzed by methods described in the text. The positions of the markers are shown by circles and X's on the two chromosomes, with the circle indicating that the diagnostic restriction site exists and the X indicating that the site does not exist. The numbers associated with the markers represent approximate SGD coordinates in kb. CEN5 is located at about SGD coordinate 152,000, and CAN1 is located at about position 33,000. Green X's show the positions of RCOs that are not associated with a gene conversion tract. Thin horizontal lines show the extent of “normal” 3∶1 gene conversion tracts and thick lines show 4∶0 conversions. The color indicates whether the markers donated in the conversion event were derived from the homologue with the YJM789 (black) or W303A (red) markers. For example, a thin red line indicates that one sector was homozygous for the markers derived from W303A and the other sector was heterozygous for these markers. For most of the conversion tracts, the crossover maps adjacent to the tract. For those tracts with an arrow above the tract, the crossover occurred within the conversion tract. The tracts in brackets have markers in the unexpected association as discussed in the text. In addition, for two of the tracts, the position of the crossover was separated from the conversion tract; these events are shown with a dotted line connecting the tract and the crossover.

Figure 6. Mapping of mitotic crossovers in meiosis-deficient derivatives of PSL101 (MD457 and PG311) and meiotic crossovers and conversions in PSL101.

The depictions of crossovers and conversions are the same as in Figure 5. A) Analysis of crossovers and conversions in 14 sectored colonies derived from MD457, an isogenic spo11/spo11 derivative of PSL101. B) Analysis of crossovers and conversions in 15 sectored colonies derived from PG311, an isogenic MATa/MATαΔ::NAT derivative of PSL101. C) Meiotic crossovers and conversion in PSL101. The diploid was sporulated and the segregation of markers in the spores was examined. Conversion tracts that were unassociated with crossovers are indicated by a horizontal line with a superimposed oval. Multiple events within one tetrad are shown with a connecting dotted line. Two conversion events that include the can1-100/SUP4-o marker are not shown.

Figure 7. Distribution of mitotic recombination events in the CEN5-CAN1 interval.

This figure is a summary of the distribution of mitotic recombination events in the strains PSL100, PS101, MD457, and PG311. A) For each marker, we summed the conversion events that include the marker over all of the strains. Both simple and complex conversion events were used in this analysis. B) For each interval, we summed the conversion tracts that end in the interval and the crossovers within the interval. We then divided that sum by the length of the interval in kb.

Figure 8. Mechanism to generate a 4∶0 conversion event.

The recombination event initiates by a DSB in G1 on the black chromosome. The broken chromosome is replicated to yield two broken black chromatids. In gene conversion events initiated by a DSB, the broken chromatid is the recipient of information . Repair of the first broken chromosome is associated with a conversion event in which the red marker is duplicated, and there is an associated crossover. Repair of the second broken chromatid could occur by an interaction with the sister chromatid (as shown) or with one of the two non-sister chromatids. This repair event would produce a second gene conversion, resulting in the 4∶0 class of event. If the first repair event had a longer conversion tract than the second, a hybrid 4∶0/3∶1 conversion tract would be formed.

Figure 9. Generation of long conversion tracts by repair of mismatches within a heteroduplex or by gap repair.

A) Conversion by mismatch repair. Conversion is initiated by a DSB, followed by 5′ to 3′ resection of the broken ends (step 1). The 3′ strand on one of the broken ends invades the other homologue and the invading strand is used as a primer for DNA synthesis (step 2); the newly-synthesized strand is shown as a dashed line. The broken end that is not used in the initial interaction undergoes more extensive resection. The single strand displaced by DNA synthesis pairs with the extensively-resected end, resulting in a long heteroduplex (step 3). The mismatches within the heteroduplex are converted in the same direction (excision of the black strand) to generate a long continuous conversion tract (step 4). The intermediate with double Holliday junctions is cleaved (cleavage sites indicated by arrows) to generate a conversion event associated with a crossover (step 5). B) Conversion by gap repair. Both strands of the broken ends resulting from the DSB are degraded to yield a gapped molecule (step 1). One of the ends invades the homologous chromosome and initiates DNA synthesis (step 2). The strand displaced by DNA synthesis pairs with the other broken end (step 3), and there is a second round of DNA synthesis (step 4). The intermediate is processed by cleaving the Holliday junctions as in Figure 9A (step 5).