Topological Probes of a Cooperative, Nonspecific Protein–DNA Complex
Many proteins bind DNA with moderate cooperativity and low sequence discrimination. Important among these are bacterial and eukaryotic chromosome-structuring proteins [2, 22] and single-stranded DNA-binding proteins . In addition, many sequence-specific DNA-binding proteins interact with nontarget sequences cooperatively and with low sequence discrimination as part of their target-search mechanisms. Examples can be found among the transcription-regulatory proteins (human glucocorticoid receptor  and NFkB ; E. coli CAP [27, 30] and lambda repressor ), among the bacterial restriction endonucleases , and among the DNA-repair enzymes . The ubiquity of such complexes calls for a better understanding of the interactions that stabilize them and the structures that result. Here we describe DNA-directed cross-linking and topoisomer analyses that are useful for characterizing topologies of protein–DNA complexes. Each is a variation on a classical approach, adapting it to features that are present in cooperative, nonspecific assemblies.
KeywordsSedimentation Equilibrium Binding Stoichiometry Partial Specific Volume Human Glucocorticoid Receptor Bind Site Size
Mass spectrometric analyses were performed at the University of Kentucky Center for Structural Biology Protein Core Facility. This facility is supported, in part, by funds from NIH National Center for Research Resources (NCRR) grant P20 RR020171. We gratefully acknowledge the help of Dr. Carol Beach in acquiring these data. This research was supported by NIH grant GM-070662 (to M.G.F.).
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