The goal of the research sponsored by this grant is to understand the mechanisms involved in eukaryotic DNA replication and its control. Our studies began with the development and characterization of the cell-free SV40 DNA replication system, which made possible the identification of proteins involved in the initiation and elongation of DNA chains in mammalian cells. More recently our work has evolved toward understanding how cellular DNA replication is initiated at chromosomal origins of replication. During the last grant period we identified the human origin recognition complex (HsORC) in extracts of HeLa cells, reconstituted the complex from recombinant subunits, characterized its physical organization, studied its DNA binding properties, and analyzed its function in a cell-free replication system. During the next grant period we plan to continue our biochemical studies of HsORC with the ultimate goal of defining the mechanisms involved in the assembly of functional initiation complexes on origin DNA. Our immediate objectives are (1) to characterize the structural organization of human ORC, (2) to define the DNA binding properties of the complex, (3) to identify cellular proteins that modulate the interaction of HsORC with origins, and (4) to reconstitute the human initiation complex in vitro and define the biochemical functions of its components. This work is focused on a fundamental problem of modern biology and has direct relevance for the more general problem of uderstanding the mechanisms that control cell proliferation.