DNA-BINDING MECHANISM OF O-6-ALKYLGUANINE-DNA ALKYLTRANSFERASE - STOICHIOMETRY AND EFFECTS OF DNA-BASE COMPOSITION AND SECONDARY STRUCTURE ON COMPLEX STABILITY

O-6-Alkylguanine-DNA alkyltransferase (AGT) is an important cellular d efense against the mutagenic effects of DNA alkylating agents. In huma ns this defense can contribute to the ability of some tumors to resist the effects of chemotherapeutic agents that act through DNA alljlatio n. We report here studies that characterize the interaction of AGT wit h DNA. We show that although AGT sediments as a monomer in the absence of DNA, it binds cooperatively to single stranded deoxyribonucleotide s. The stoichiometries of complexes formed with 16-, 30-, and 80-base oligodeoxyribonucleotides are 3.8 +/- 0.3, 5.3 +/- 0.2, and 8.9 +/- 0. 2, respectively; the binding density decreasing from similar to 4 nt/m onomer to similar to 9 nt/monomer as DNA length increases over this ra nge. Binding competition assays show that DNA affinities depend only w eakly on base composition or secondary structure, although in general G+C-rich sequences are bound with greater affinity than are A+T-rich o nes and single stranded DNA is bound with greater affinity than duplex forms. These results suggest mechanisms by which AGT may search for a lkylated sites and interact with them to effect DNA repair.