Micromechanical Analysis of the Binding of DNA-Bending Proteins HMGB1, NHP6A, and HU Reveals Their Ability To Form Highly Stable DNA−Protein Complexes

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.Non-histone DNA-bending proteins are associated with chromosomes of prokaryotic and eukaryotic cells. Prominent among these in eukaryotes are the nonspecifically binding class of HMGB 1 proteins, present in the nucleus at micromolar concentrations (1-3). HMGB proteins possess a ∼75 amino acid residue DNA-minor-groove-binding domain, which induces a bend of about 90°. Charged residues within extended peptide segments at the N-or C-termini of the HMGB domain can strongly modulate DNA binding affinities of these proteins. Saccharomyces cereVisiae NHP6A contains one HMGB domain but binds DNA with considerably higher affinity than mammalian HMGB1, which contains two domains (4). HMGB proteins function in a variety of localized DNA transactions including assembly of transcription and recombination complexes and chromatin remodeling (1-3). A general role for HMGB proteins in modulating global DNA organization in eukaryotes is not yet established, but yeast HMGB proteins have been shown to condense chromosomal DNA in prokaryotic models (4,5).

Membrane protein expression triggers chromosomal locus repositioning in bacteria

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Topoisomerase V relaxes supercoiled DNA by a constrained swiveling mechanism

“…All single-molecule micromanipulation experiments were performed by using the magnetic tweezers setup described (23,24). Flow-cell preparation, force measurements, and calibration were performed as described (23,24).…”

Micromanipulation Studies of Chromatin Fibers in Xenopus Egg Extracts Reveal ATP-dependent Chromatin Assembly Dynamics

“…Controlled forces were applied to the bead by using permanent magnets (Smith et al, 1992;Strick et al, 1996Strick et al, , 1998Strick et al, , 2000, and bead positions were measured using digital videomicroscopy and bead-tracking computer software. We used two setups: in the first "vertical MT," the bead is pulled perpendicular to the focal plane, so DNA extension is determined by autofocusing software ( Figure 2a); details are described in Skoko et al (2004). In the second "transverse MT" system the DNA is extended in the focal plane ( Figure 2b); for details, see Yan et al (2004).…”