My research has two long range goals. The first is to develop inhibitor/probes that will be useful for study of the active site of replication-specific DNA polymerases, especially DNA polymerase alpha (pol alpha) from eukaryotic cells, and the less well characterized DNA polymerase delta. The second is to exploit the selectivity of such inhibitor probes to design cytotoxic compounds that may be developed into antitumor drugs.
The specific aims of the present application approach these long-term goals on three fronts. 1. We first will employ a labelled form of the potent DNA polymerase alpha inhibitor N2-(p-n-butylphenyl)-2'- deoxyguanosine 5'-triphosphate, (3H)BupdGTP, to determine the polypeptide(s) of holoenzyme forms of pol alpha from calf thymus and Chinese hamster ovary cells that bind the inhibitor. Chemical synthesis of the target nucleotide and related inhibitor/probes, equilibrium dialysis, and activity gel analyses will be used to accomplish these experiments. If, as expected, the inhibitor binds to the core pol alpha protein of calf thymus, we will pursue questions about the relationship of that site to the pol active, catalytic site. 2. Synthesis and structure activity relationships of selective inhibitors of DNA polymerase delta, a recently described DNA polymerase that may be important in nuclear DNA replication, will be pursued. Based on observations that the potent pol alpha inhibitors BuPdGTP and its adenine counterpart BuAdATP are weakly inhibitory to pol delta, and that several 3,4- dichlorophenyl-substituted purine analogs appear to inhibit the latter enzyme, similar base and deoxyribonucleotides bearing the dichlorophenyl group will be synthesized. Assays of compounds against both pol alpha and pol delta from calf thymus will be used to identify a pol delta-selective inhibitor class, and to study the mechanism and site of inhibitor binding. 3. The effects of inhibitors of purified pol alpha and pol delta on DNA synthesis in cells in culture will be studied. we will use the Chinese hamster ovary cell line and measure incorporation of labelled macromolecule presursors, net macromulecule synthesis, and DNA synthesis in permeabilized cells to help identify the target(s) for cytotoxicity, and to provide reagents that may serve to study the role(s) of pol alpha and/or pol delta in eukaryotic DNA replication.
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