| Aberrant changes to the genome structure underlie numerous human diseases such as cancers. The functional characterization of genes and proteins that maintain chromosome stability will be important in understanding disease etiology and developing therapeutics. I took a multi-faceted approach to identify and characterize genes involved in the maintenance of genome stability.;As biological pathways involved in genome maintenance are highly conserved in evolution, results from model organisms can greatly facilitate functional discovery in humans. In S. cerevisiae, I identified 47 essential gene depletions with elevated levels of spontaneous DNA damage foci and 92 depletions that caused elevated levels of chromosome rearrangements. Of these, a core subset of 15 DNA replication genes demonstrated both phenotypes when depleted. Analysis of rearrangement breakpoints revealed enrichment at yeast fragile sites, Ty retrotransposons, early origins of replication and replication termination sites. Together, this highlighted the integral role of DNA replication in genome maintenance.;In light of my findings in S. cerevisiae, I identified a list of 153 human proteins that interact with the nascent DNA at replication forks, using a DNA pull down strategy (iPOND) in human cell lines. As a complementary approach for identifying human proteins involved in genome maintenance, I used the BioID technique to discern in vivo proteins proximal to the human BLM-TOP3A-RMI1-RMI2 genome stability complex, which has an emerging role in DNA replication progression. I uncovered 45 proximal proteins to the wildtype complex and catalogued the gains and losses of proximal proteins following the expression of three RMI1 mutants and in conditions of DNA replication stress. Altogether, I used a multi-faceted approach to identify a replication-enriched dataset of genes and proteins that collectively safeguard the genome from mutations, which will be invaluable for functional characterization studies in the future. |
