INHIBITION OF HUMAN DNA POLYMERASES BY ANTIVIRAL DRUGS

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The proposal is directed toward examining the effects of antiviral drugs on the two human DNA polymerases (alpha and delta) which. are involved in human chromosomal DNA replication. The effects of antiviral drugs on the isolated enzymes and in the presence of other replication factors will be examined and compared to their effects on recombinant HIV reverse transcriptase. A knowledge of the effects of such drugs on the human DNA polymerases may provide important inforrnation in relation to the assessment of potential drug cytotoxicity and for the future design of antiviral drugs. 1. The kinetics of inhibition of DNA polymerase a and DNA polymerase 8 by antiviral drugs will be studied. Parallel studies with HIV reverse transcriptase will be performed. 2. The mechanisms of the inhibition of the DNA polymerases will be studied in detail. DNA sequencing gel methods and model template-primers in the form of oligonucleotide primed ss-circular DNA will be used to analyze the mechanisms involved. Parallel studies using HIV reverse- transcriptase will be performed using model oligonucleotide-primed RNA templates in combination with sequencing methodology. 3. The effects of nucleotide analogs on processivity and on the strand displacement reactions will be studied by the use of model defined substrates, including one carrying a topologically stable replication fork. 4. The particular role of the 3' to 5' exonuclease in the inhibition processes will be studied, particularly in relation to chain-terminating inhibitors; the effects of mononucleotide analogs on this activity will also be determined.