Chapter Fifteen - Characterization of Homogeneous, Cooperative Protein–DNA Clusters by Sedimentation Equilibrium Analytical Ultracentrifugation and Atomic Force Microscopy

Strong, positively cooperative binding can lead to the clustering of proteins on DNA. Here, we describe one approach to the analysis of such clusters. Our example is based on recent studies of the interactions of O-6-alkylguanine DNA alkyltransferase (AGT) with high-molecular- weight DNAs (Adams et al., 2009; Tessmer, Melikishvili, & Fried, 2012). Cooperative cluster size distributions are predicted using the simplest homogeneous binding and cooperativity (HBC) model, together with data obtained by sedimentation equilibrium analysis. These predictions are tested using atomic force microscopy imaging; for AGT, measured cluster sizes are found to be significantly smaller than those predicted by the HBC model. A mechanism that may account for cluster size limitation is briefly discussed.