Project Summary The survival of living organisms depends on the timely and accurate duplication of chromosomal DNA. In all domains of life, the onset of DNA replication (or initiation) relies on dedicated initiator proteins that bind genomic sites, termed replication origins, and help load replicative helicases onto DNA. In eukaryotes, the initiator is the origin recognition complex (ORC), which recruits and deposits the Mcm2-7 helicase motor module onto DNA to ‘license’ origins. Although core replication initiation factors are conserved across most eukaryotes and are well-studied in budding yeast, important differences exist with respect to origin recognition and the regulation of origin licensing between S. cerevisiae and higher eukaryotes. Numerous outstanding questions therefore remain in the metazoan system regarding origin recruitment and function of core and accessory DNA replication initiation factors, and the specific contributions of chromatin-associated proteins to these events. In this proposal, we aim to define at a mechanistic level in metazoan systems how ORC- dependent DNA remodeling contributes to Mcm2-7 loading, how disease-linked mutations alter initiator activities, and how ORC-partner proteins promote chromosomal recruitment and function of the initiator, by integrating biochemical, structural, and cell-based approaches. The findings from this work will have broad implications for multiple scientific fields, as they will not only help generate models for origin specification, origin processing, and origin licensing in metazoans, but also contribute to our understanding of DNA- and chromatin-dependent macromolecular machines. Our studies are also of significant biomedical relevance since the failure to precisely replicate chromosomal DNA causes genome instability, which in turn underpins many human diseases, including cancer. In the long-term, the outcomes of our efforts thus have the potential to provide important starting points for the development of novel therapeutic strategies.