Journal article

Synthesis and applications of chemical probes for human O6-alkylguanine-DNA alkyltransferase

The design and synthesis of chem. probes to study and to elucidate complex biol. problems is becoming an increasingly important field in chem. We are interested in the repair of O6-alkylated guanines in DNA, a DNA lesion that results from alkylation by S-adenosylmethionine or exogenous toxins and which has been shown to be highly mutagenic and carcinogenic. The DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT; EC reverses this alkylation by transferring the alkyl group to a reactive cysteine residue in the protein, leading to repaired DNA and an irreversibly alkylated protein. The expression level of human AGT (hAGT) in tumor cells is also crucial for their sensitivity to chemotherapeutic agents that alkylate DNA, such as 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and temozolomide. Consequently, hAGT has become a target in cancer chemotherapy, as its inhibition would increase the efficiency of currently used DNA-alkylating drugs. A simple and reliable assay to measure the activity of hAGT in cell exts. would be of great importance for research on the role of hAGT in the chemotherapy of tumors, as currently used assays rely on radioactively labeled substrates and a subsequent HPLC sepn. We describe here the synthesis of oligonucleotides contg. O6-alkylated guanine derivs. of the type 1 and 2 that serve as affinity labels for hAGT and their use in a highly specific assay for this alkyltransferase. In addn., we introduce a novel system for the directed mol. evolution of hAGT, which relies on the display of active hAGT on phage l and on oligonucleotides contg. O6-alkylated guanine derivs. of the type 1 and 2. [on SciFinder (R)]


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