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 journalArticle

70 Citations (Scopus)

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 ∼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
Volume128
Issue number2
DOIs
StatePublished - Jan 18 2006
Externally publishedYes

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DNA
Magnetic Resonance Spectroscopy
Nuclear magnetic resonance
Observation
Spectroscopy
Proteins
Rate constants
Base Pairing
Physical Phenomena
Kinetics
Protein Transport
Oligonucleotides
Electronic data interchange
Cell Communication
Design of experiments
Research Design
Assays
Gels

ASJC Scopus subject areas

  • Chemistry(all)

Cite this

Direct observation of enhanced translocation of a homeodomain between DNA cognate sites by NMR exchange spectroscopy. / Iwahara, Junji; Clore, G. Marius.

In: Journal of the American Chemical Society, Vol. 128, No. 2, 18.01.2006, p. 404-405.

Research output: Contribution to journalArticle

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