The mutagenic and cytotoxic effects of many endogenous and exogenous alkylating agents are mitigated by the actions of O⁶-alkylguanine-DNA alkyltransferase (AGT). In humans this protein protects the integrity of the genome, but it also contributes to the resistance of tumors to DNA-alkylating chemotherapeutic agents. Here, we report properties of the interaction between AGT and short DNAs and between AGT and covalently closed circular plasmids. We show that while AGT sediments as a monomer in the absence of DNA, it binds with high cooperativity to both single-stranded and double-stranded DNAs. This result is surprising in view of the 1:1 binding mechanism found in crystalline AGT-DNA complexes. Crosslinking analysis allows identification of protein residues juxtaposed in the cooperative complex. The simplest models consistent with the data place the protein in a two-start helical structure with a net pitch similar to that of B-form DNA. Topoisomerase assays test this notion and reveal that AGT unwinds DNA by only ∼7.5 deg/protein molecule. These results have significant implications for the mechanisms by which AGT locates and interacts with O⁶-alkylguanine lesions to effect DNA repair. Supported by NIH grant GM070622.


Above: Topoisomerase analysis of the binding of AGT to closed-circular pUC19 DNA. Reactions were carried out at 20 °C in 50 mM potassium acetate, 20 mM Tris acetate, 10 mM magnesium acetate, 1 mM DTT; pH 7.9. Samples were resolved by electrophoresis in 3% agarose gels at 2 V/cm. Relaxed (R) and supercoiled (S) bands are indicated. Sample a contained supercoiled DNA only, sample b contained DNA and E. coli topoisomerase I. Samples c-j contained DNA, topoisomerase and AGT protein, with [AGT] increasing in regular steps from 4μM to 36μM.