MCM PROTEIN FUNCTION DURING EUKARYOTIC DNA REPLICATION

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The timely and accurate replication of the genome is essential to the normal proliferation of all eukaryotic cells. Accordingly, the initiation of DNA replication is carefully coordinated with the progress of the cell cycle. The long term objective of this proposal is to determine the events that direct the initiation of replication and how these factors then contribute to the elongation phases of DNA replication. The MCM proteins are a family of six related proteins that play a central role in the replication process in the yeast S. cerevisiae. Recent studies indicate that these proteins are among the earliest factors assembled at origins of replication (prior to the initiation of DNA replication) but also participate in the elongation phase of the replication process. These and other findings suggest that the MCM proteins act as the replicative DNA helicase in eukaryotic cells. This hypothesis is tested in this proposal by addressing the genetic and biochemical properties of the MCM proteins. Specifically Dr. Bell will: Identify conditional mutants in MCM genes that distinguish between the initiation and elongation functions of these proteins. The resulting mutants will be characterized for their effects on the association of the MCM and other proteins with origin and non-origin DNA sequences. Determine the requirement for origin DNA unwinding to assemble MCM proteins at the origin and identify similar elements in other origins of replication. Determine the biochemical properties of purified MCM proteins with particular attention on their ability to bind and hydrolyze ATP and to act as a DNA helicase. Based on previous studies, any new understanding of the fundamental mechanisms of eukaryotic DNA replication in yeast will be readily translated to our understanding of the same process in human cells. The rapid progress that can be made in S. cerevisiae make it a ideal organism to perform these studies. New understanding of these proteins will lead to strong candidate targets for novel anti-fungal compounds. In addition, the understanding of the yeast MCM proteins gained in these studies will direct studies to identify inhibitors of the human analogs of these proteins, which are strong candidate targets for novel chemotherapeutic compounds based on the success of previous DNA replication inhibitors in chemotherapeutic regimens.