The timely complete and accurate replication of the genome is essential to the normal proliferation of all eukaryotic cells. Either too much or too little replication can have lethal consequences for the cell. Accordingly, the initiation of DNA replication is tightly coordinated with progression through the cell division cycle. The overall goal of this proposal is to define the molecular mechanisms that direct the initiation of eukaryotic DNA replication and determine how they are integrated into and regulated by the cell cycle machinery. To ensure that replication does not initiate more than once per cell cycle, eukaryotic cells divide the replication initiation into two temporally distinct events: origin selection and origin activation. During G1-phase, all potential origins of replication are selected through the assembly of pre-replication complexes (pre-RCs). During S-phase, these sites are activated to initiate replication by the formation of two higher order complexes: the pre-initiation complex (pre-IC) and the replisome. During pre-RC formation the six-protein, replicative DNA helicase (the Mcm2-7 complex) is loaded onto origin DNA in an inactive state. Formation of the pre-IC activates the Mcm2-7 helicase and the helicase-dependent unwinding of origin DNA that is required for replisome assembly. Using biochemical assays that recapitulate pre-RC formation and mutant and modified proteins involved in these events Dr. Bell will: 1. Determine the mechanism of Mcm2-7 origin loading. 2. Determine the impact of nucleosomes on pre-RC formation. 3. Reconstitute and dissect pre-IC formation. These studies will be performed using the yeast S. cerevisiae rather than human cells. The availability of highly defined sequences that act as origins of DNA replication and powerful cell cycle tools make studies of DNA replication in this organism particularly advantageous. Nevertheless, the highly conserved nature of eukaryotic DNA replication indicates that progress made in this yeast will be translated rapidly to human cells. New understanding of the events of replication initiation will lead to candidate targets for anti-fungal compounds. Moreover, the understanding eukaryotic replication provided by these yeast studies will direct studies to identify inhibitors of the same events in humans, which represent important candidates for chemotherapeutic agents. Project Narrative In this proposal Dr. Bell will study the fundamental events that control the duplication of animal cell chromosomes. In particular, he will investigate how the hundreds, if not thousands, of sites at which this process is initiated are selected and activated to start chromosome duplication at the appropriate times. The findings will provide fundamentally important information that will be relevant to the proliferation of all animal cells including humans.
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