The research is aimed at understanding basic mechanisms by which alkylating carcinogens, such as dimethylnitrosamine, lead to initiation of tumors, and the physiological processes by virtue of which cells are resistant to this initiation. More specifically, the studies cover two separate factors which play key roles in tumor initiation; the interaction of alkylating agents with DNA and the subsequent repair of these alkylation lesions. Experiments will be carried out to investigate the sequence specificity and mechanism of DNA alkylation with particular reference to the formation of O6-alkylguanine, O4-methylthymine, 7- methylguanine and the ring opened form of 7-methylguanine. Preliminary studies with oligodeoxynucleotides of defined sequence suggest that there may be a major effect of DNA sequence on the extent of methylation of guanines which has so far been overlooked. More detailed investigation of the extent of specificity and the reasons for it will be carried out. DNA repair systems for the alkylation products described above will be characterized and the factors regulating their activity will be studied in detail. It is intended to obtain specific antibodies to these proteins and to obtain cDNA and genomic clones for them in order to facilitate this work. Continued investigations of the mammalian alkyltransferase protein responsible for the repair of O6-alkylguanine in DNA will be a major portion of the proposed studies. These will include: (a) investigation of its sequence specificity by synthesizing oligodeoxynucleotides of defined sequence containing O6-alkylguanine and measuring their rate of repair; (b) studying the mechanism of the reaction and designing inhibitors of it; (c) purifying the protein from human liver and raising antibodies to it; (d) isolating the gene and cDNA clones for the protein by transfection procedures and by screening of human cDNA libraries in lambda gt 11; and e) using these tools to study the regulation of the protein in normal and neoplastic cells. The studies of the regulation of the alkyltransferase will be combined with studies of the concomitant changes in the amounts of proteins responsible for the repair of other alkylation products. Dimethylnitrosamine and related compounds containing the N-nitroso-group form a large class of chemical carcinogens which have considerable potential environmental significance since they are so widespread that it may not be possible to avoid exposure completely. The processes underlying DNA alkylation and repair which will be studied in this work may greatly influence the sensitivity of response to nitrosamines. The understanding of these factors at the molecular level will permit more detailed response to nitrosamines. The understanding of these factors at the molecular level will permit more detailed analysis of the risks associated with exposure to N-nitroso compounds and may enable strategies to be designed to increase resistance to the hazards associated with exposure to them.
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