title: Direct observation of enhanced translocation of a homeodomain between DNA cognate sites by NMR exchange spectroscopy.
abstract: A novel approach is presented for studying the kinetics of specific protein-DNA interactions by NMR exchange spectroscopy. The experimental design involves the direct observation of translocation of a homeodomain between cognate sites on two oligonucleotide duplexes, differing by only a single base pair at the edge of the DNA recognition sequence. The single base-pair change perturbs the 1H-15N correlation spectrum of a number of residues, while leaving the affinity for the DNA unchanged. The exchange process has apparent rate constants in the 5-20 s-1 range which are linearly dependent upon the concentration of free DNA. These rates are about 3 orders of magnitude larger than the dissociation rate constant determined by gel shift assays at nanomolar DNA concentrations. The complete NMR exchange data set, comprising auto- and cross-peak intensities as a function of mixing time at five concentrations of free DNA, can be fit simultaneously to a simple model in which protein translocation between DNA duplexes occurs via a second-order process (with rate constants of approximately 6 x 104 M-1 s-1) involving direct collision of a protein-DNA complex with free DNA. This is akin to intersegmental transfer, and a physical model for the process is discussed. Rapid translocation at high concentrations of free DNA observed directly by NMR exchange spectroscopy reconciles the long half-lives of protein-DNA complexes measured by biochemical analysis in vitro with the highly dynamic behavior of such complexes observed in vivo. The relevance of this mechanism to the kinetics of protein-DNA interactions within the cell is discussed.
authors.affiliation: Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0520, USA.
authors.forename: Junji
authors.initials: J
authors.lastname: Iwahara
authors.forename: G Marius
authors.initials: GM
authors.lastname: Clore
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D005801:Genes, Homeobox; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D013379:Substrate Specificity
chemical_list: D018398:Homeodomain Proteins; D004247:DNA