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

Junji Iwahara, G. Marius Clore

Research output: Contribution to journalArticlepeer-review

81 Scopus citations


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 ∼6 × 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.

Original languageEnglish (US)
Pages (from-to)404-405
Number of pages2
JournalJournal of the American Chemical Society
Issue number2
StatePublished - Jan 18 2006
Externally publishedYes

ASJC Scopus subject areas

  • Catalysis
  • General Chemistry
  • Biochemistry
  • Colloid and Surface Chemistry


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