Molecular and structural mechanisms of metazoan origin licensing - Abstract The survival of living organisms depends on the timely and accurate duplication of chromosomal DNA. Defects in DNA replication jeopardize genome integrity and organismal viability and are linked to numerous diseases ranging from developmental syndromes to cancer. Research in my laboratory is broadly focused on elucidating the mechanisms that control the onset of DNA replication. In eukaryotes, DNA replication begins with the binding of initiator proteins, the origin recognition complex (ORC), to DNA at replication origins. ORC subsequently recruits and deposits the Mcm2-7 helicase motor onto DNA as an MCM double hexamer to ‘license’ these origins for DNA replication. Much of our knowledge about origin licensing and DNA replication initiation is derived from studies using budding yeast as a model system, but how these steps are accomplished in metazoan systems remains ill-defined mechanistically. In the past, we have employed biochemical, biophysical, and structural approaches to uncover new, fundamental principles for how eukaryotic ORC operates to license replication origins. For example, we have determined high-resolution structures of ORC-containing assemblies in various functional states to define how ORC binds DNA and cofactors, and how these activities are regulated. More recently, we have reconstituted human origin licensing in vitro, which revealed that human Mcm2-7 can be loaded onto DNA through multiple pathways, suggesting that not all human origins may be licensed through the same mechanism. Despite these advancements, numerous questions persist with respect to the mechanisms that regulate origin recognition and origin licensing in multicellular eukaryotes, which will provide the basis for our research program in the next 5 years. Building on our strengths in biochemical reconstitution and in structural biology, we plan to resolve how metazoan MCM loading intermediates mature into MCM double hexamers through the various licensing pathways, how these pathways have evolved across multicellular eukaryotes, and how origin licensing occurs in different types of chromatin contexts. In the long-term, our studies will help explain disease mechanisms and aid in the development of new treatment modalities for diseases linked to dysregulated DNA replication initiation such as cancer.
