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

Abstract

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.

Genome-wide high-resolution mapping of chromosome fragile sites in Saccharomyces cerevisiae

“…Of the 43 deletions/duplications, 20 were within the tandem cluster of CUP1 genes, 8 involved the two pairs of Ty elements on chromosome III discussed above, and 3 occurred between inverted pairs of Ty elements located on chromosome IV near SGD coordinates 880 kb and 990 kb. We note that the two pairs of Ty elements on chromosome III were previously shown to be hotspots for chromosome rearrangements (11) and BIR events (18) in strains with low levels of DNA polymerase-α. These observations suggest that large (>1 kb) deletions and duplications in yeast primarily reflect homologous recombination between directly oriented repeats located on sister chromatids.…”

“…incubated in the absence of galactose (18). Eleven red/white sectored colonies were examined for LOH events by microarrays, and the coordinates for the detected LOH events are given in Dataset S9 (depictions in Dataset S10).…”

“…If low levels of DNA polymerase-α increase the frequency of S-associated DSBs, this model also predicts an increase in the frequency of unequal sister-chromatid recombination events; this increase was observed. Rosen et al (18) showed that low levels of DNA polymerase-α also result primarily in 3:1 conversion events (90%) relative to 4:0 events (10%) on chromosome III.…”

“…We found that the low levels of polymerase-α elevated the frequency of chromosome loss and chromosome rearrangements on chromosome III about 100-fold, and most of the chromosome rearrangements involved an inverted pair of retrotransposons located on the right arm of III (fragile site FS2) (11). Rosen et al (18) mapped mitotic recombination events between homologous copies of chromosome III in strains with low levels of DNA polymerase-α and showed that DSBs formed at FS2 initiated many of these exchanges. Low levels of DNA polymerase-α also stimulated high levels of mitotic and meiotic recombination in the tandemly repeated ribosomal RNA genes (19).…”

Comprehensive Analyses of White-Handed Gibbon Chromosomes Enables Access to 92 Evolutionary Conserved Breakpoints Compared to the Human Genome

“…Furthermore, they are reported for several species like Drosophila [von Grotthuss et al, [Alekseyev and Pevzner, 2010], yeast [Rosen et al, 2013], or birds [Gordon et al, 2007]. We compared ∼ 230 common known human CFSs and rare FSs [Mrasek et al, 2010] together with 47 recently molecularly defined fragile site (online suppl.…”