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.
Highlights
Cancer cells contain a variety of genomic changes that result in altered gene expression affecting cell growth
Loss of heterozygosity (LOH) at tumor-suppressor genes contributes to cancer, and deletions resulting in loss of heterozygosity (LOH) are frequently observed in tumor cells at certain chromosomal regions known as common fragile sites
We report that yeast chromosome III has a high frequency of spontaneous mitotic recombination that involves the homologous chromosome
Summary
Cancer cells contain a variety of genomic changes that result in altered gene expression affecting cell growth. Human common fragile sites have been extensively investigated for their contribution to genomic changes that cause tumor initiation and progression. Common fragile sites are large genomic regions of 250 kb–1 Mb that are unstable under conditions that partially inhibit DNA replication (reviewed in [1]). Treatment with aphidicolin, which inhibits DNA polymerases [2,3], or hydroxyurea, which inhibits ribonucleotide reductase and results in unbalanced nucleotide pools [4], both cause replication stress that induces instability at fragile sites. Several mechanisms have been proposed to explain why breaks form in human common fragile sites, including secondary structure formation within single-stranded DNA (ssDNA) at stalled replication forks [5,6], paucity of replication origins [7,8], replication fork pausing between early- and late-replicating regions [9,10], and collision between RNA and DNA polymerases [11]. The mutations at common fragile sites appear to often be early drivers of tumorogenesis rather than later ‘‘passenger’’ events [12,13,14]
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