Mechanisms of replication origin licensing studied by real-time single-molecule fluorescence

Project Summary DNA replication is essential to maintain the genome of all organisms. During each round of cell division, eukaryotic cells must establish hundreds to thousands of replication forks that coordinately replicate each chromosome. These events begin during G1, when two copies of the replicative helicase, the Mcm2-7 complex, are loaded at all potential origins of DNA replication. Once loaded, the two ring-shaped, heterohexameric Mcm2-7 complexes encircle the DNA and interact tightly via their N-terminal domains. Although inactive, the resulting head-to-head Mcm2-7 double hexamer licenses each origin for subsequent bidirectional initiation upon entry into S phase. Consistent with their importance, mutations in or misregulation of the proteins mediating helicase loading lead to cancer and developmental abnormalities. Thus, understanding the mechanism of these processes will provide critical information concerning the maintenance of genome integrity and potential targets for therapeutics. The biochemical reconstitution of helicase loading using budding yeast proteins has been a powerful tool to understand these events, however, bulk biochemical assays are poorly suited to study the complex dynamics involved in helicase loading due to their frequently incomplete and asynchronous nature. Single- molecule fluorescence microscopy experiments bypass these problems by monitoring events on individual DNA molecules in real time, defining the sequence of biochemical events, detecting short-lived intermediates and specific protein-protein interactions, and defining quantitative kinetic mechanisms. We propose single- molecule experiments on helicase loading using reconstituted yeast proteins in vitro, supplemented with molecular genetics experiments on live cells. Together, these studies will provide critical insights into the dynamic mechanisms of helicase loading and will complement and aid in the interpretation of the static structures revealed in recent cryoelectron microscopy studies. The proposed research primarily focuses on events of helicase loading that are conserved across all eukaryotic organisms. Both yeast and metazoan ORC induce a strong bend in the DNA upon binding. In Specific Aim one, we investigate the role of this activity in origin selection and determine which steps in helicase loading require this function. The MO complex is a key helicase-loading intermediate that ensures the second recruited Mcm2-7 forms head-to-head interactions with the first. In Aim two, we will determine the role of this complex in closing of the Mcm2-7 ring around DNA and define the pathways by which ORC and Mcm2- 7 form this complex. In the final Aim, we will determine how nucleosomes and sequence-nonspecific ORC DNA binding change helicase loading, both key elements of origin selection in metazoan species.