Fragile Site Instability in <i>Saccharomyces cerevisiae</i> Causes Loss of Heterozygosity by Mitotic Crossovers and Break-Induced Replication

<div><p>Loss of heterozygosity (LOH) at tumor suppressor loci is a major contributor to cancer initiation and progression. Both deletions and mitotic recombination can lead to LOH. Certain chromosomal loci known as common fragile sites are susceptible to DNA lesions under replication stress, and replication stress is prevalent in early stage tumor cells. There is extensive evidence for deletions stimulated by common fragile sites in tumors, but the role of fragile sites in stimulating mitotic recombination that causes LOH is unknown. Here, we have used the yeast model system to study the relationship between fragile site instability and mitotic recombination that results in LOH. A naturally occurring fragile site, FS2, exists on the right arm of yeast chromosome III, and we have analyzed LOH on this chromosome. We report that the frequency of spontaneous mitotic BIR events resulting in LOH on the right arm of yeast chromosome III is higher than expected, and that replication stress by low levels of polymerase alpha increases mitotic recombination 12-fold. Using single-nucleotide polymorphisms between the two chromosome III homologs, we mapped the locations of recombination events and determined that FS2 is a strong hotspot for both mitotic reciprocal crossovers and break-induced replication events under conditions of replication stress.</p></div

Instability at fragile site FS2 stimulates LOH by mitotic recombination and chromosome loss.

a<p>All diploids result from mating a MS71-derived haploid with a YJM789-derived haploid. Each haploid is isogenic with its parent strain, except for changes introduced by transformation (described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003817#pgen.1003817.s003" target="_blank">Table S1</a>). MS71 is a <i>LEU2</i> derivative of AMY125 (<i>MATα ade5-1 leu2-3 trp1-289 ura3-52 his7-2</i>) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003817#pgen.1003817-Kokoska1" target="_blank">[64]</a>. YJM789 is a derivative of a clinical yeast isolate (<i>MAT</i>a <i>ura3 gal2 ho::hisG</i>) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003817#pgen.1003817-Wei1" target="_blank">[42]</a>. All diploids except AMC324 and AMC331 are homozygous <i>GAL-POL1</i>.</p>b<p>The frequency of reciprocal crossovers was calculated as 2*(number of crossover events/total colonies).</p>c<p>Only BIR events that result from a break on the chromosome containing the <i>SUP4-o</i> allele can be detected as a red/light pink sectored colony. Therefore, in Y332, Y382, AMC324, and AMC331, we report BIR resulting from breaks on the MS71-derived chromosome III. In AMC310, we report BIR resulting from breaks on the YJM789-derived chromosome III.</p>d<p>Only loss of the chromosome containing the <i>SUP4-o</i> allele can be detected as a red/light pink sectored colony. Therefore, in Y332, Y382, AMC324, and AMC331, we report loss of the MS71-derived chromosome III. In AMC310, we report loss of the YJM789-derived chromosome III.</p>e<p>The number in parenthesis is the 95% confidence interval. The number in brackets is the fold change from Y332 in high galactose. No local gene conversion at the <i>SUP4-o</i> locus was observed in any strain; therefore this category is not included in the table.</p

Fragile site FS2 is a hotspot for initiation of BIR events resulting in LOH.

<p>The number of events initiated at each SNP is shown, and the two SNPs flanking fragile site FS2 are highlighted with a yellow box. The SNP indicated where each event maps is the first homozygous SNP in the stretch of homozygous SNPs. The BIR initiation site can be anywhere between the last heterozygous SNP and the first homozygous SNP. (A) The two homologs of the right arm of chromosome III are shown. The gray homolog is MS71-derived and contains fragile site FS2 and the <i>SUP4-o</i> allele. The red chromosome represents the YJM789-derived homolog. Large ovals represent the centromere. Black arrows on the chromosome diagrams indicate Ty1 elements. SNP markers used to map events are shown by circles and triangles on the chromosome diagrams. Triangles indicate a restriction site exists, circles indicate lack of the site. Numbers are the approximate chromosome coordinate in kb. The 66 BIR event initiation sites collected in Experimental Diploid #1 under low galactose conditions that cause replication stress are shown in the graph above the chromosome diagram. All BIR events in this diploid had three copies of the YJM789-derived SNPs, implying that the initiating lesion occurred on the gray, FS2-containing chromosome. (B) The 14 BIR event initiation sites collected in Experimental Diploid #1 under high galactose conditions that do not cause replication stress. (C) The 28 BIR event initiation sites collected in Experimental Diploid # 2 under low galactose conditions that cause replication stress. In this diploid, <i>SUP4-o</i> is located on the red, YJM789-derived chromosome. All BIR events in this diploid had three copies of the MS71-derived SNPs, implying that the initiating lesion occurred on the red, non-FS2-containing chromosome.</p

Mechanisms for fragile site stimulated mitotic crossovers and BIR events.

<p>In cells with low levels of polymerase alpha, slowed DNA replication likely results in larger regions of single-stranded DNA on the lagging strand, which allows intra-strand pairing at FS2 to form a hairpin. Here, the MS71-derived homolog of chromosome III is depicted in gray, with the two inverted Ty1 elements of FS2 as black arrows. Stalling could be followed by either cleavage at the secondary structure, resulting in fork collapse, or replication may proceed and leave a single-strand gap at the secondary structure. Fork collapse to a one-ended double-strand break is expected to primarily stimulate repair by BIR. Here, the homologous YJM789-derived chromosome III is shown in red as a repair template, resulting in LOH. If a replication from a nearby origin converges with the collapsed fork, a double-strand break will form. This lesion can be repaired by invasion of the red, YJM789-derived homolog, and capture of the second end, as shown. Crossover resolution of the double Holliday junction can result in LOH after mitotic chromosome segregation. If replication stalling results in formation of a single-strand gap rather than a break, template switching may be used to fill the gap. Template switch to the red, YJM789-derived homolog forming a double Holliday junction is shown. Crossover resolution followed by mitotic chromosome segregation can result in LOH.</p

Lesions at fragile site FS2 during S-phase initiate mitotic crossovers resulting in LOH.

<p>(A) Number of events at each SNP in Experimental Diploids #1 and #2 under replication stress. We summed the number of conversion tracts (both 3∶1 and 4∶0 events) crossing each SNP in both experimental diploids, and crossovers un-associated with a gene conversion were added to the sum of the closest centromere-distal SNP. Numbers are the approximate chromosome coordinate in kb for each SNP. (B) Locations of 41 crossovers and associated gene conversions collected in Experimental Diploid #1 under replication stress. Chromosome diagrams for Experimental Diploid #1 are the same as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003817#pgen-1003817-g004" target="_blank">Figure 4</a>. Crossover events are shown below the chromosomes. Black X's indicate crossovers that did not have gene conversion associated at the SNPs tested. A number in parenthesis indicates how many crossover events were at the site, if more than one. Thin horizontal lines indicate 3∶1 conversion tracts and thick lines indicate 4∶0 tracts. Dotted lines indicate a non-adjacent conversion tract. Line color shows which chromosome was copied in gene conversion. These crossover events were collected in two ways; 29 crossover events were collected among the colonies in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003817#pgen-1003817-t001" target="_blank">Table 1</a>, and 12 crossover events were collected among another set of 14,792 colonies. (C) Locations of the 15 crossovers and associated gene conversions collected in Experimental Diploid #2 under replication stress. In this diploid, SUP4-o is located on the red, YJM789-derived homolog of chromosome III. (D) Locations of 5 crossovers and associated gene conversions in Experimental Diploid #1 in high galactose, which permits abundant production of polymerase alpha. These crossover events were collected in two ways; four crossover events were collected among the 30,543 colonies in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003817#pgen-1003817-t001" target="_blank">Table 1</a>, and one crossover event was collected among another set of 4,792 colonies.</p

Detection of mitotic events by sectored colony formation.

<p>Mitotic events that result in loss of heterozygosity (LOH) at the <i>SUP4-o</i> locus in Experimental Diploid #1 are shown. Only chromosome III is depicted; white represents the MS71-derived homolog and pink represents the YJM789-derived homolog. The <i>SUP4-o</i> gene is approximately 159 kb from the centromere on the MS71-derived chromosome III homolog. This diploid is homozygous <i>ade2-1</i>/<i>ade2-1</i>. This mutation is suppressible by the <i>SUP4-o</i> tRNA, therefore all starting diploids are light pink in color. (A) A reciprocal crossover between the centromere of chromosome III and the <i>SUP4-o</i> locus that occurs at the time of plating results in red/white sectoring. Only half of crossover events are detected as a sectored colony, due to the pattern of sister chromatid segregation. The segregation pattern that results in sectoring is shown. (B) A break-induced replication (BIR) event that is initiated by damage centromere-proximal to <i>SUP4-o</i>, and in which the homolog that does not contain <i>SUP4-o</i> is used as a template for copying, produces a red/light pink sectored colony. (C) A local gene conversion event at the <i>SUP4-o</i> locus in which genetic information is copied from the homolog that does not contain <i>SUP4-o</i> results in red/light pink sectoring. A point mutation at the <i>SUP4-o</i> locus (not shown) will also produce a red/light pink sectored colony. (D) Damage on the homolog containing <i>SUP4-o</i> that results in chromosome loss, or deletion of the right arm of the chromosome (not shown), produces a red/light pink sectored colony.</p

Use of SNPs to map the location of mitotic recombination events.

<p>Single nucleotide polymorphisms (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 stars and <i>SUP4-o</i> is represented by a gray rectangle. This strain is homozygous for the ochre-suppressible <i>ade2-1</i> 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 reciprocal crossover that occurs to repair a lesion at FS2 in S phase or G2 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 of one SNP is shown in the yellow box. After chromosome segregation in mitosis, the white 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 the SNP within the region of gene conversion is homozygous in the red cell but heterozygous in the white cell.</p

Locations of crossovers and BIR events in Control Diploids.

<p>For each diploid, the two homologs of the right arm of chromosome III are shown. The gray homolog is MS71-derived and the red homolog is YJM789-derived. Large ovals represent the centromere. Black arrows on the chromosome diagrams indicate Ty1 elements. SNP markers used to map events are shown by circles and triangles on the chromosome diagrams. Triangles indicate a restriction site exists, circles indicate lack of the site. Numbers are the approximate chromosome coordinate in kb. Crossover and BIR events are shown below the chromosomes. Black X's indicate crossovers that did not have gene conversion associated at the SNPs tested. Thin horizontal lines indicate 3∶1 conversion tracts associated with crossovers, and dotted lines indicate a non-adjacent conversion tract. Line color shows which chromosome was copied in gene conversion. BIR events are shown by arrowheads. The flat vertical edge of the arrowhead indicates the site at which the BIR was initiated; all BIR events extended to the end of the chromosome. The SNP indicated where each BIR event maps is the first homozygous SNP in the stretch of homozygous SNPs. The BIR initiation site can be anywhere between the last heterozygous SNP and the first homozygous SNP. The red color of the arrowheads indicates that the YJM789-derived homolog was the template for copying, implying that the initiating lesion occurred on the MS71-derived homolog. (A) Five crossover and eight BIR events mapped in Control Diploid #1, grown in high galactose. Both fragile site FS2 and the <i>SUP4-o</i> allele are located on the MS71-derived homolog, and this diploid is homozygous for the <i>POL1</i> gene under its native promoter. (B) One crossover and nine BIR events mapped in Control Diploid #2, grown in high galactose. This diploid is homozygous for the <i>POL1</i> gene under its native promoter, and the <i>NAT</i> gene replaces both Ty1 elements of fragile site FS2. (C) Three crossover and four BIR events mapped in Control Diploid #3 under replication stress caused by low levels of polymerase alpha. Fragile site FS2 has been inactivated in this diploid by expansion of the space between the two Ty1 elements of the fragile site.</p

Diploids used for analysis of mitotic recombination events.

<p>Five diploid strains were used to detect events that result in loss of heterozygosity (LOH) at the <i>SUP4-o</i> locus. Only chromosome III is depicted. The white chromosome represents the MS71-derived homolog and the pink chromosome represents the YJM789-derived homolog. Experimental Diploids #1 and #2 both contain fragile site FS2, and are homozygous for the <i>GAL-POL1</i> construct that permits induction of replication stress by low levels of polymerase alpha. Experimental Diploids #1 and #2 differ in which homolog of chromosome III carries the single copy of <i>SUP4-o</i> that is present in the strain. Control Diploid #1 is isogenic to Experimental Diploid #1 except that the <i>POL1</i> gene is under its native promoter. Control Diploid #2 is isogenic to Control Diploid #1 except that the entire FS2 region has been replaced by the <i>NAT</i> drug resistance gene. Control Diploid #3 is isogenic to Experimental Diploid #1 except that fragile site FS2 has been stabilized by insertion of the <i>NAT</i> gene between the pair of inverted Ty1 elements at this site.</p