Genetic Screens in Yeast to Identify BRCA1 Modifiers

摘要

Mutations in the BRCA1 checkpoint gene results in aneuploidy and an increased risk of breast cancer. The yeast RAD9 protein has similar functions and sequence motifs as BRCA1 and we proposed to identify haploinsufficient mutations at a second locus that alters the chromosome loss rate of our rad9-/- diploid strains. We created a rad9-delta/delta strain both a qualitative (sectoring colonies) and quantitative assay (canavanine resistance) sensitive enough to detect the increase of heterozygous mutations on chromosome loss rates. We analyzed 30,000 insertional mutants and obtained 400 independent insertions that reproducibly alter CF loss rate. By FA, 40% of this group show statistically significant increase (3-10 fold) in mutation rate. We identified the insertion site for 300 mutants and gene ontology analysis of the 225 independent insertions reveals a statistically significant over-representation for Cell Cycle process and Chromosome location and under-representation of the Protein Synthesis process. Recreation of precise gene deletions in wild-type and rad9-delta/delta backgrounds verifies the instability phenotype and distinguishes heterozygous mutations that are specific modifiers of rad9 mutant strains from those that cause genomic instability independently.