Topologies of Complexes Containing O6-Alkylguanine–DNA Alkyltransferase and DNA

The mutagenic and cytotoxic effects of many alkylating agents are reduced by O6-alkylguanine–DNA alkyltransferase (AGT). In humans, this protein not only protects the integrity of the genome, but also contributes to the resistance of tumors to DNA-alkylating chemotherapeutic agents. Here we describe and test models for cooperative multiprotein complexes of AGT with single-stranded and duplex DNAs that are based on in vitro binding data and the crystal structure of a 1:1 AGT–DNA complex. These models predict that cooperative assemblies contain a three-start helical array of proteins with dominant protein–protein interactions between the amino-terminal face of protein n and the carboxy-terminal face of protein n + 3, and they predict that binding duplex DNA does not require large changes in B-form DNA geometry. Experimental tests using protein cross-linking analyzed by mass spectrometry, electrophoretic and analytical ultracentrifugation binding assays, and topological analyses with closed circular DNA show that the properties of multiprotein AGT–DNA complexes are consistent with these predictions.