PROJECT SUMMARY Somatic copy number alterations (SCNAs) are an established hallmark of cancer and result from errors in the mechanisms that maintain genome stability. The presence of SCNAs across most cancer types highlights prevention of SCNAs as a potential strategy to prevent the generation and propagation of cancer. Prevention of SCNAs requires the maintenance of genome stability, which is dependent on the normal functioning of centromeres. Centromeres, which are repetitive DNA domains, are the site of assembly of the kinetochore, a multi-protein complex that couples centromeres with microtubules and regulates chromosome segregation. Interestingly, the repeat-rich architecture of centromeres makes them prone to genomic rearrangements, with 40-60% of genomic rearrangements in cancer cells occurring at centromeres. Hence, centromeres maintain genome stability and their own genetic integrity by associating with kinetochore proteins. Cancer initiation and progression always involve some form of genome instability that leads to SCNA, hence the mechanisms through which centromere DNA/kinetochore protein interactions contribute to genome stability are an understudied but critical component of cancer research. The goal of this project is to determine how changes at the centromere-kinetochore interface impact genome stability, with the long-term goal being to uncover new mechanisms of genome instability leading to cancer initiation and progression. This proposal is novel because it is the first to test the mechanistic effect of centromere DNA and associated protein diversity on genome stability. I hypothesize that centromere variation can influence centromere/kinetochore association and subsequently impact genome stability. Aim 1 will determine whether CENP-A, a centromere-specific histone H3 variant, differentially associates with divergent centromere satellite repeats within species, indicating differential ability of centromere DNA repeats to associate with CENP-A to maintain their stability. Aim 2 will determine the dependence of kinetochore protein function on centromere sequence compatibility. This will be done by observing the function of human Shugoshin 1, a kinetochore protein that associates with centromere DNA to maintain centromeric cohesin during mitosis, with mouse centromere satellite repeats. Together, these aims will highlight pathways that are dependent on centromere sequence for their function, implicating centromere-dependent mechanisms to maintain genome stability. My goals for training are (i) to gain an experimental skill set that will enable me to use a cell culture system to test computationally generated hypotheses and (ii) to improve my scientific communication skills. These will contribute to my career development to become an independent academic scientist investigating the role of evolutionary genetic processes in human cancer incidence and evolution. My graduate program and institution with a successful training record, ...