The goals of this proposal are to further our understanding of the molecular enzymology of the human placental DNA polymerases. The major focus will be on a new type of mammalian DNA polymerase, DNA polymerase Delta, which possesses an associated Delta to 5 ft. exonuclease activity, and is therefore unlike the classical DNA polymerase Alpha. The existence and properties of DNA polymerase Delta in human tissue will be established, and its relationship to other high molecular weight DNA polymerases will be investigated.
The specific aims of the proposal are as follows: The human DNA polymerases Delta and Alpha will be isolated to homogeneity from placental tissue. The enzymes of interest are the DNA polymerase Delta types, of which there are at least two forms, Delta 1 and Delta 2, and polymerase Alpha. The goal is the development of methods for the reproducible isolation of these enzymes from human placenta in their native forms, so as to provide sufficient amounts in the pure state for rigorous characterization. The placental DNA polymerases will be characterized. The subunit structures and physicochemical properties of the DNA polymerases (Delta 1, Delta 2 and Alpha) will be determined and compared. Possible interrelationships will be explored by peptide mapping, and by the use of immunological methods using monoclonal antibodies. The goal is the rigorous description of polymerases Delta 1 and Delta 2, and the determination of their relationships, as well as those, if any, with polymerase Alpha. Attempts will be made to localize the active sites of the exonuclease and polymerase activities of DNA polymerase Delta 1 and Delta 2. The exonuclease and polymerase sites will be labeled by photoaffinity methods using 8-azido-adenosine mono and triphosphates. The goal is to obtain unequivocal evidence that DNA polymerase Delta 1 and Delta 2 each possess two active sites. Monoclonal antibodies against polymerases Delta 1, Delta 2 and Alpha will be made. These antibodies will be used as tools to further the goals above, to prepare affinity columns for the rapid and large scale isolation of these proteins, and to provide tools for the radioimmunoassay and subcellular localization of these enzymes. A long term aim is to study changes of DNA polymerase activity during the cell cycle and also their subcellular localization.
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