The overall goals of this project are to elucidate the structure, function and regulation of the mammalian replication complex and the mechanisms by which replication fidelity is maintained in mammalian cells. This proposal focuses on two components of the replication complex: DNA polymerase delta (pol delta), a replicative DNA polymerase which has been postulated to be the leading strand replicase, and delta helicase, a putative replicative DNA helicase. We have recently cloned and sequenced the cDNAs for the 125 and 50 kDa subunits of pol delta from both bovine and human sources and are currently attempting to express the polypeptides both in vitro and in heterologous systems. We plan to express the polypeptides individually and in combination to determine the function of the 50 kDa subunit. Deletion mutagenesis will be used to delineate the domains on the subunits which interact in the heterodimer as well as the domains which are required for interaction with other replication proteins. We plan to identify residues at or near the active sites of the polymerase and 3' -5' exonuclease activities of pol delta by photoaffinity labeling of the heterodimer with nucleotide analogs containing azido groups, and to use site-directed mutagenesis to examine the functions of residues identified by affinity labeling. We plan to elucidate the mechanisms which regulate the expression of pol delta in vivo by determining the level of expression of pol delta at the mRNA and protein levels as a function of cell cycle position and following serum stimulation of quiescent cells, and by mapping the promoter region of the genes encoding the 125 and 50 kDa subunits of pol delta. We have recently purified a 56 kDa DNA helicase from calf thymus, delta helicase, which has several properties in common with prokaryotic replicative DNA helicases. We plan to clone and sequence the cDNA for delta helicase, to analyze the structure and function of the enzyme, and to develop antibody and oligonucleotide reagents to elucidate the physiological role of this helicase.
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