Many anticancer and antiviral agents are analogs of purines or pyrimidines that are thought to act by being incorporated into DNA. Most of the studies performed to date concerning the mechanism by which these drugs act have used DNA into which the analog has been randomly incorporated. For one such analog, 6-thioguanine (TG), we have performed experiments that suggest that the action of this drug may depend on its being incorporated into a specific DNA sequence. The overall objective of the proposed work is to determine if such """"""""sequence-specific incorporation"""""""" is necessary for the action of Tg, and also of three other analogs which are believed to act via incorporation into DNA. This objective will be met by achieving the following specific goals: 1. Construction of specifically substituted viral DNA molecules. The eukaryotic virus SV40 will be the model system for these studies. SV40 DNA contains a sequence at its origin of replication whose integrity has been shown to be critical for initiation of viral DNA synthesis. Analog substitutions will be performed either collectively within this sequence or in other regions of the viral genome known to be insensitive to mutation. In addition to TG, substitutions will be performed using ara-C, ara-A or bromodeoxyuridine. 2. Testing the biological effects of sequence-specific substitution. Once the contructed molecules are in hand their ability to function will be tested in two systems. First, infectivity of these species will be tested by DEAE-dextran mediated transfection. Second, the ability of the substituted molecules to function as templates in a recently reported cell-free system will be assayed. 3. Testing the protein binding properties of substituted DNA. The virally-coded """"""""T-antigen"""""""" (T-ag), whose function is critical for initiation of replication, binds to the site within the origin where specific substitutions will be made. The affinity of analog-substituted monlecules for T-ag will be measured by means of an immunoprecipitation procedure using an anti-T-ag monoclonal antibody.
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