Micromechanical analysis of the binding of DNA-bending proteins HMGB1, NHP6A, and HU reveals their ability to form highly stable DNA-protein complexes

Dunja Skoko, Ben Wong, Reid C. Johnson, John F. Marko*

*Corresponding author for this work

Research output: Contribution to journalArticle

109 Scopus citations

Abstract

The mechanical response generated by binding of the nonspecific DNA-bending proteins HMGB1, NHP6A, and HU to single tethered 48.5 kb λ-DNA molecules is investigated using DNA micromanipulation. As protein concentration is increased, the force needed to extend the DNA molecule increases, due to its compaction by protein-generated bending. Most significantly, we find that for each of HMGB1, NHP6A, and HU there is a well-defined protein concentration, not far above the binding threshold, above which the proteins do not spontaneously dissociate. In this regime, the amount of protein bound to the DNA, as assayed by the degree to which the DNA is compacted, is unperturbed either by replacing the surrounding protein solution with protein-free buffer or by straightening of the molecule by applied force. Thus, the stability of the protein-DNA complexes formed is dependent on the protein concentration during the binding. HU is distinguished by a switch to a DNA-stiffening function at the protein concentration where the formation of highly stable complexes occurs. Finally, introduction of competitor DNA fragments into the surrounding solution disassembles the stable DNA complexes with HMGB1, NHP6A, and HU within seconds. Since spontaneous dissociation of protein does not occur on a time scale of hours, we conclude that this rapid protein exchange in the presence of competitor DNA must occur only via "direct" DNA-DNA contact. We therefore observe that protein transport along DNA by direct transfers occurs even for proteins such as NHP6A and HU that have only one DNA-binding domain.

Original languageEnglish (US)
Pages (from-to)13867-13874
Number of pages8
JournalBiochemistry
Volume43
Issue number43
DOIs
StatePublished - Nov 2 2004

ASJC Scopus subject areas

  • Biochemistry

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