Evidence‐Based Design of a Fragile Site in Budding Yeast

Abstract

Common fragile sites (CFSs) are seen as breaks on metaphase chromosomes when cells are grown under replication stress. Our mammalian cell study demonstrates that CFSs overlap copy number variant (CNV) hotspots characteristic of human diseases including autism, schizophrenia, and cancer. These at‐risk regions are characterized by late replication and large transcription units, supporting hypotheses that CFSs result from a replication‐transcription conflict. However, mammalian systems fall short of explaining what the repair mechanisms and intermediates are at these sites and whether transcription unit length is correlative or causative of prolonged replication. We have used budding yeast in an attempt to prospectively design a fragile site/CNV hotspot to elucidate the roles of transcription and repair in the formation and resolution of chromosome breaks. We examined genome‐wide replication data for expansive replicons that are free of Ty elements, repetitive sequences, and essential genes. We selected loci on chromosomes XII and XV as candidates for higher genomic instability under replication stress, especially when replication and transcription are manipulated. A URA3‐CAN1 reporter was used to ablate a late‐firing origin and detect locus‐specific deletion rates using fluctuation analysis. We are delineating the effects of these large replicons lacking late‐firing origins and hydroxyurea, a replication inhibitor and inducer of CNVs in mammalian cells, on CNV formation in yeast. This yeast model will provide insight into the mechanisms that create CNV breakpoint junctions with a goal of establishing a high‐throughput assay for agents that induce CNV formation. Source of Funds: R25 GM086262, R21 ES022311.