We are using yeast to investigate how the DNA helicase mutation mcm4Chaos3 interferes with the early events leading to DNA replication and how these replication defects may lead to its role in cancer development. Mcm4Chaos3 encodes a mutation in a subunit of the MCM helicase, an essential complex required for unwinding double stranded DNA during replication. Mcm4Chaos3 mice exhibit genomic instability and more than 80% of female mice homozygous for Mcm4Chaos3 developed mammary tumors (Shima et. al 2007). Further work in yeast identified an origin-specific minichromosome loss phenotype, suggesting the mcm4Chaos3 mutation may have particular sequence requirements at origins, where DNA replication initiates. To determine the basis of origin sequence specificity in mcm4Chaos3 function, we had previously performed a plasmid maintenance competition assay using a mutARS317-seq library, containing 500+ plasmids with random single mutations within the ARS317 origin sequence. This assay identified 5 origin sequence variants that performed better in mcm4Chaos3 yeast than wild type ARS317. To investigate mcm4Chaos3 interaction with ARS317 variants, I first measured the loss rate for wild type ARS317 plasmids in mcm4chaos3 vs wild type yeast. As predicted, wild type cells maintained the plasmids better than the mcm4Chaos3 mutants (16.8% loss rate/generation compared to 5% in wild type). I am currently characterizing the 5 sequence variants, and based on the competition assay data, am predicting to see differences in plasmid loss rates across the ARS317 variants. Understanding the cause for this origin specificity could help us develop a greater understanding of the mechanics involved in DNA replication, genome stability, and cancer-causing mutations.