The long-range objectives of this project are to clarify the mechanisms by which human cells maintain the genetic integrity of their DNA in the face of cytotoxic, mutagenic or carcinogenic insults. These goals are being approached through biochemical studies to identify and characterize the enzymes and reactions involved in repair of alkylation or radiation damage of DNA. The experiments are designed to answer questions about the critical initial steps of repair that involve recognition of a lesion or class of lesions by endonucleases, N-glycosylase or alkyltransferases. The endonuclease specific for UV- or -irradiated or oxidized DNA will be purified further from human lymphoblasts and attempts will be made to establish unequivocally that it comprises a combination of N-glycosylase and AP-endonuclease activities. The identity of the damaged bases recognized by this activity will be determined rigorously by derivitization of reaction products, HPLC and DNA-sequencing methods. The highly purified human DNA glycosylase specific for N-alkylpurines in DNA will be investigated for activity against O-alkylated pyrimidines, while the O6-alkylguanine-DNA alkyltransferase, purified 300-fold from human lymphoblasts, will be purified further and tested for activity against O-alkylated pyrimidines. A new endeavor concerns enzymes for repair of DNA interstrand cross-links induced by antineoplastic bifunctional alkylating agents. The mechanism(s) for O6-alkyltransferase-mediated repair of cross-link presursors induced by chloroethylnitrosoureas will be investigated. Preliminary studies indicating that the sensitivity of human rhabdomyosarcoma xenografts to the antineoplastic nitrosoureas relates to levels of transferase will be extended to other human tumor xenografts. Knowledge gained from the proposed research should contribute to an understanding of certain pathologic changes such as cancer and aging, or help explain variations in chemotherapeutic and radiotherapeutic responses of different cells. It may ultimately suggest approaches for predicting cellular sensitivity or interfering with the agents' biological effects.
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