Resolving the contributions of two cooperative mechanisms to the DNA binding of AGT.

Abstract

ABSTRACTThe O6‐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. © 2015 The Authors Biopolymers Published by Wiley Periodicals, Inc. Biopolymers 103: 509–516, 2015.