Direct observation of enhanced translocation of a homeodomain between DNA cognate sites by NMR exchange spectroscopy

J Am Chem Soc. 2006 Jan 18;128(2):404-5. doi: 10.1021/ja056786o.


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.

Publication types

  • Research Support, N.I.H., Intramural
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Base Sequence
  • DNA / chemistry*
  • DNA / metabolism
  • Genes, Homeobox
  • Homeodomain Proteins / chemistry*
  • Homeodomain Proteins / metabolism
  • Kinetics
  • Nuclear Magnetic Resonance, Biomolecular / methods*
  • Substrate Specificity


  • Homeodomain Proteins
  • DNA