The focus of these studies is the molecular enzymology of human DNA polymerase Delta, which is novel in possessing an associated 3' to 5' exonuclease activity, and is therefore unlike the classical form of mammalian polymerase Alpha. Two forms of polymerase Delta have been separated during purification. These, and DNA polymerase Alpha, will be purified in forms with well-defined polypeptide compositions, by exploitation of HPLC methods and by immunoaffinity chromatography. The integrity of the catalytic subunits will be monitored by Western blotting and activity gel staining during purification. The enzymatic and physicochemical properties of DNA polymerase Delta will be studied, with a major emphasis on the identification of the subunit structures of the native enzymes. The identity of the subunits of the native enzymes will be established by crosslinking with crosslinking reagents and by 2-D PAGE. The polypeptide(s) containing the catalytic sites of the 3' to 5' exonuclease and DNA polymerase activities, respectively, will be identified by several approaches, including activity staining after SDS PAGE, the use of photoaffinity labeling procedures using 8-azido-analogs of AMP and dATP. The relationships of the two forms of polymerase Delta with each other and with purified human DNA polymerase Alpha will be studied by biochemical and immunological methods, including peptide mapping, Western blot analysis and cross-reactivity of specific antibodies to the enzymes. A panel of mouse hybridoma cell cultures secreting monoclonal antibodies to both forms of polymerase Delta has been established. These will be screened for useful characteristics, and the monoclonal antibodies will be isolated and characterized. The subcellular localization of DNA polymerase Delta will be studied by optical and electron immunomicroscopy using specific monoclonal antibodies. The role of DNA polymerase Delta in replication will be examined by effects of monoclonal antibodies on DNA synthesis in permeabilized cells. Fundamental knowledge of this DNA polymerase which possesses an associated 3' to 5' exonuclease capable of a proof-reading function is of significance to our understanding of mechanisms of DNA replication and mutagenesis.
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