NUCLEAR MATRIX STRUCTURE AND DNA REPLICATION

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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 four basic questions. 1. Is the nuclear matrix involved in the functioning of the matrix-bound DNA polymerase alpha? This question will be addressed with experiments aimed at determining whether the nuclear matrix-bound DNA polymerase alpha enzyme has a greater degree of processivity compared to soluble enzyme preparations. Further experiments will attempt to determine whether increased processivity is a property of the matrix-bound state of the enzyme complex or a special property of the alpha polymerase bound to the matrix. 2. Is the matrix-bound DNA polymerase alpha organized into a replicational complex on the nuclear matrix? As a first step in addressing this question we are developing procedures to release DNA polymerase alpha activity from the nuclear matrix structure. We will then determine whether in vivo replicating DNA is bound to solubilized complexes containing the polymerase activity. 3. What are the properties of the matrix-released complexes? Organizational and functional properties of the released complexes containing DNA polymerase alpha will be studied. For example, we will determine: (1) whether the alpha polymerase in the released complexes continues the synthesis of DNA in vitro along in vivo replicating DNA fragments; (2) whether the alpha polymerase in the released complex has a high degree of processivity; (3) the polypeptide composition as well as identify specific polypeptides of DNA polymerase in the released complex; (4) whether other replicative factors are associated with the released complex; and (5) whether the complexes are dynamically assembled on the matrix pre-replicatively or during in vivo replication. 4. What is the relationship of these matrix-bound replicational complexes to chromatin structure? As a first step in addressing this question we will determine whether DNA is attached to the nuclear matrix in a nucleosomal repeat structure. We will then attempt to isolate replicational complexes containing matrix-attached chromatin fragments and study the structural organization and functional properties of these putative replicational complexes.