PROJECT SUMMARY DNA replication is a highly conserved process essential for all life. Since eukaryotic genomes are very large, they are replicated in parallel from thousands of replication origins. Proper regulation of replication initiation is essential for maintaining genome stability. Oncogene activation dysregulates replication initiation and enables rapid cell proliferation in cancer. Key regulators of replication initiation are over-expressed in cancer, and mutations in these proteins cause rare genetic disorders. Yet, the molecular mechanisms that underlie replication initiation remain poorly understood, especially in multicellular organisms, for two main reasons: (1) replication initiation involves transient protein-protein and protein-DNA interactions that are very challenging to study using existing biochemical, genetic, and structural methods; and (2) the most comprehensive studies of replication initiation have been done in yeast, however key regulators of replication initiation are poorly conserved between yeast and metazoans. Notably, some human replication factors are 2-3x larger than their yeast counterparts and containing novel domains with unknown functions. My laboratory recently developed a single-molecule imaging platform to visualize replication initiation in real time. We will use this powerful approach to (i) provide a quantitative description of replication initiation; (ii) understand how this process changed from yeast to humans; (iii) delineate the role of key replication proteins and their domains that are unique to metazoans; and (iv) understand why mutations in some replication initiation factors cause disease. Our work will also provide valuable mechanistic insights into how cancer cells sustain rapid proliferation by over-activating replication initiation and inform how replication initiation factors may be used as biomarkers or targets for novel therapies.