title: Resolving the contributions of two cooperative mechanisms to the DNA binding of AGT.
abstract: The O(6)-alkylguanine DNA alkyltransferase (AGT) is a DNA repair enzyme that binds DNA with moderate cooperativity. This cooperativity is important for its search for alkylated bases. A structural model of the cooperative complex of AGT with DNA predicts short-range interactions between nearest protein neighbors and long-range interactions between proteins separated in the array. DNA substrates ranging from 11bp to 30bp allowed us to use differences in binding stoichiometry to resolve short- and long-range protein contributions to the stability of AGT complexes. We found that the short-range component of ΔG°(coop) was nearly independent of DNA length and protein packing density. In contrast the long-range component oscillated with DNA length, with a period equal to the occluded binding site size (4bp). The amplitude of the long-range component decayed from ∼-4 kcal/mole of interaction to ∼-1.2 kcal/mol of interaction as the size of cooperative unit increased from 4 to 7 proteins, suggesting a mechanism to limit the size of cooperative clusters. These features allow us to make testable predictions about AGT distributions and interactions with chromatin structures in vivo.
authors.affiliation: Department of Molecular and Cellular Biochemistry, Center for Structural Biology, University of Kentucky, Lexington, KY, 40536.
authors.forename: Manana
authors.initials: M
authors.lastname: Melikishvili
authors.affiliation: Department of Molecular and Cellular Biochemistry, Center for Structural Biology, University of Kentucky, Lexington, KY, 40536.
authors.forename: Michael G
authors.initials: MG
authors.lastname: Fried
mesh_terms: D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004260:DNA Repair / Q000235:genetics / Q000502:physiology; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D004247:DNA; D019853:O(6)-Methylguanine-DNA Methyltransferase
keywords: DNA repair; O6-alkylguanine; binding cooperativity; protein-DNA interaction