02-Alkyl(methylhethyl) thymidine, 04-alkyl thymidine and-02-alkyl cytidine are formed in vivo by a variety of N-nitroso carcinogens. In all organs and eukaryotic cells examined, these derivatives persist for long periods. The promutagenic nature of each of these pyrimidines has been demonstrated in vitro and in vivo for 04-alkyl T. 04-Methyl and -ethyl thymidines are also directly implicated in initiation of hepato-carcinogenesis. This project is directed toward understanding how the presence of 0-alkyl pyrimidines in DNA or model oligomers affects secondary structure and polymerase recognition in replication. 02- and O4-alkyl thymine will be site-specifically incorporated in template-primer complexes by DNA polymerase of varying fidelity. They will be placed opposite A or G, the known partners, or T, a mismatch. The kinetics of insertion and elongation (km Vmax) will be determined by gel electrophoresis or radiolabeled nucleotide incorporation. Structures of complete duplexes, e.g., a 22-mer, containing 0 to 3 alkyl thymines, will be examined by thermal denaturation, enzyme sensitivity and antibody recognition. A hexamer capable of the B to Z transition will be synthesized with a single alkyl thymine and the result of the substitution studied by circular dichroism. Duplex oligomers with 2-aminopurine opposite 04-alkyl thymine will be used to measure fidelity and structure in the absence of steric hindrance. Fluorescence changes are a sensitive indicator of the degree of stacking and bonding. Sequence effects on kinetics and structure will be determined by varying the template oligomers. Sequences with stability may be less likely to be repaired, so mutation can occur. Preparation of 02-alkyl CDP and CTP will use mild chemical or enzymatic methods to attempt prevention of dealkylation and depyrimidination. If the desired products are obtained, polymers can be made for mispairing studies. Finally, a pair of cell lines, isolated from human brain tumors, will be used to study the time course and extent of 0-alkyl pyrimidine repair. These cells differ greatly in their ability to repair the initial O6-alkyl G adduct formed by chemotherapeutic halonitrosoureas. Either cell one may be suitable for further study of pyrimidine repair mechanisms.
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