The overall goal of this project is to elucidate the organizational, functional and regulatory properties of eucaryotic DNA replication in relation to nuclear structure. This area of research is of paramount importance to furthering the understanding of growth and proliferation in normal cells and how these properties are altered in disease states. The proposed research will focus on three basic questions. (1) What are the cell cycle relationships of different replicational components associated with the nuclear matrix? Various properties of DNA polymerase alpha, DNA primase, diadenosine tetraphosphate (AP4A) binding sites, DNA topoisomerase II and other replicative components will be studied at different stages in the cell cycle of synchronized HeLa S3 cells. Particular emphasis will be placed on the possible pre-replicative assembly of these replicative components into multicomponent complexes. (2) What is the structural topography of nuclear matrix-bound replicational components and how does this be performed in mammalian cells grown in monolayers versus nuclear matrix monolayer preparations using antibodies to DNA polymerase alpha, DNA topoisomerase II, 5-bromodeoxyuridine (following in vivo incorporation of BUdR) and fluorescent conjugated avidin following in vitro incorporation of biotin-dUTP. These studies will be performed at different stages in the cell cycle as well as in G1- arrested cells and will then be extended by immunogold labeling to electron microscopic thin sectioning, reinless thick sectioning and whole mount three-dimensional analysis. (3) What are the properties of matrix-bound replicational complexes? Various organizational and functional properties of multi-component replicational complexes solubilized from the nuclear matrix will be studied. For example, we will (1) further purify the complexes by immunoaffinity separation; (2) study the purified complexes by electron microscopy including immunogold localization; (3) determine the polypeptide composition as well as identify specific polypeptides (e.g., DNA polymerase alpha, primase and topoisomerase II) in the released complexes and use this reconstitution system to identify components involved in ATP stimulation of processive DNA synthesis by matrix-bound polymerase alpha.
NIH ExPorter
NIH ExPorter