Book of Abstracts: Albany 2007
June 19-23 2007
Cooperativity, Crosslinking, and Supercoiling in the Interaction of AGT with DNA
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.
Department of Molecular and Cellular Biochemistry