Initiation of DNA replication signals commitment of a cell to enter S phase and to complete the next cell cycle. This commitment made at the GI/S boundary is the control point at which cell growth and cell division are coordinated. Cancer cells are characterized by their uncoordinated cell divisions. Our current understanding of the initiation of DNA replication in eukaryotes is derived mostly from viral model systems which provide molecular detail of the initiation event but lack information on the regulation of these events. Our laboratory has identified a family of genes, called MCM, whose products participate in the initiation of DNA synthesis at replication origins in yeast. MCM1 is a transcription factor which also acts as a replication initiation factor when bound to multiple sites at yeast replication origins. MCM2, MCM3 and MCM5 are a family of structurally and functionally related proteins which has a cell cycle dependent nuclear localization. The most conserved region of this family of proteins contains a potential DNA helicase motif. Mammalian homologs for each of these proteins have been identified suggesting that the functions of these MCM proteins are likely to be conserved in all eukaryotes. In this proposal, the functional relationship between the MCM proteins and their roles in the initiation of DNA synthesis at replication origins will be investigated further. The structure and function of MCM1 will be examined by biochemical and mutational analyses. The functional relationship between the MCM1 and MCM2-3-5 protein family will be examined by DNA binding studies, two-hybrids and suppressor analyses. The binding sites of the MCM proteins on replication origins will be analyzed by DNase I and chemical footprinting analyses. The interactions between members of the MCM2-3-5 protein family will be investigated by genetic and biochemical analyses. Purified MCM proteins will be analyzed individually and as a reconstituted complex for ATPase and DNA helicase activities. Other gene products that interact with the MCM proteins will be identified from the two hybrids library and by suppressor analyses. Our long term objective is to reconstitute a replication initiation complex that directs origin- specific initiation of DNA synthesis in vitro.
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