ERROR PRONE DNA SYNTHESIS AND ONCOGENE MUTAGENESIS

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Activation of the c-Ha-ras oncogene can occur by point mutation at a limited number of sites. c-Ha-ras-containing plasmids treated with chemical carcinogens yield transformants on introduction into recipient human cells. These observations suggest the following questions: a) Are the mutations the result of mispairing at a targeted site or does carcinogen treatment induce an error-prone condition in the cells?; b) Are segments of the c-Ha-ras oncogene particularly susceptible to mutation because of their sequence or does selection alone account for the frequency of change to a transformed state. We plan to study these questions utilizing an in vitro DNA system obtained from human cells and with plasmids containing c-Ha-ras. We propose: 1) to replicate such plasmids in single and double stranded form in vitro using purified prokaryotic and eukaryotic enzymes; 2) to develop mammalian cell extracts and partially purified systems which will replicate c-Ha-ras; 3) to apply previously developed methodology to the replication of carcinogen-reacted c-Ha-ras templates; 4) to utilize extracts prepared from carcinogen-reacted cells to see whether they promote adduct bypass more readily, as would be expected if an inducible system had developed; 5) to assay the reaction products for mutation by transfection of rodent cells to the transformed state; and 6) to study the role of deoxynucleoside triphosphate pool bias, as affected by treatment with carcinogen, to see whether it is possible to account for increases in mutability on the basis of pool effects. These in vitro studies should suggest answers to the questions asked and should also provide information useful in the design of plasmids for in vivo tests of mutability. The long term goal of these studies is the determination of how nucleotide sequence and the physiological state of the cell interact in the production of precarcinogenic mutagenic events.