Concerted binding and bending of DNA by Eschericia coli integration host factor

Gauri M. Dhavan, Donald M. Crothers, Mark R. Chance, Michael Brenowitz

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Abstract

Integration host factor (IHF) is a heterodimeric Eschericia coli protein that plays essential roles in a variety of cellular processes including site-specific recombination, transcription, and DNA replication. The IHF-DNA interface extends over three helical turns and includes sequential minor groove contacts that present strong, sequence specific protection patterns against hydroxyl radical cleavage. Synchrotron X-ray footprinting has been used to follow the kinetics of formation of DNA-protein contacts in the IHF-DNA complex with single base-pair spatial, and millisecond time, resolution. The three sites of IHF protection on the DNA develop with similar time-dependence, indicating that sequence specific binding and bending occur concertedly. Two distinct phases are observed in the association process. The first "burst" phase is characterized by a rate that is greater than diffusion limited (>1010 s-1 M-1) and the second phase is on the order of diffusion controlled (∼108 M-1 s-1). The overall kinetics of association become faster with increasing IHF concentration showing that complex formation is second-order with protein. The rate of association is maximal between 100 and 200 mM KCl decreasing at higher and lower concentrations. The rate of IHF dissociation from site-specifically bound DNA increases monotonically as KCl concentration is increased. The dissociation progress curves are biphasic with the amplitude of the first phase dependent upon competitor DNA concentration. These results are the first analysis by synchrotron footprinting of the fast kinetics of a protein-DNA interaction and suggest that IHF binds its specific site through a multiple-step mechanism in which the first step is facilitated diffusion along the length of the duplex followed by subsequent binding and bending of the DNA in a concerted manner. 2002 Elsevier Science Ltd.

Original languageEnglish (US)
Pages (from-to)1027-1037
Number of pages11
JournalJournal of Molecular Biology
Volume315
Issue number5
DOIs
Publication statusPublished - Jan 1 2002

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Keywords

  • Footprinting
  • IHF
  • Integration host factor
  • Kinetics
  • Synchrotron

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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