Genomic Instability at Single Cell Resolution

Abstract

All organisms maintain the integrity of their genome through highly precise DNA replication and repair. Errors in these mechanisms can lead to genetic instability that results in cellular dysfunction or malignancy. Modern sequencing technology has enabled the development of methods to interrogate these processes during individual cell divisions. Our lab previously devised a single cell resolution approach that suggested that Saccharomyces cerevisiae cells with abrogated replicative fidelity exhibit multiple mutator states, a phenomenon termed ‘mutator volatility’, but the data was also consistent with a hypothesis in which replication errors segregated unequally during cell division. The research reported in my dissertation uses an expanded approach in chapter two to confirm that mutator volatility exists in strong mutators. It also shows that unequal inheritance of replication errors occurs due to the sequential processes of semiconservative DNA replication and chromosome segregation. Using computational modeling, I show that unequal segregation may dramatically expand heterogeneity in the mutation burden in human tumors. In chapter three, I model the strongest human mutator allele in cancer (POLE-P286R) and find an even greater volatility. Although this mutator phenotype depends in yeast on the S-phase checkpoint, the volatility does not. In the fourth chapter, I present a novel single cell resolution method to detect loss of heterozygosity (LOH) in yeast and then use this method to investigate if LOH increases with replicative age. These findings highlight the numerous insights that may be gleaned by studying the processes that give rise to genome instability at the level of single cells.