Nonspecifically bound proteins spin while diffusing along DNA

Abstract

It is known that DNA-binding proteins can slide along the DNA helix while searching for specific binding sites, but their path of motion remains obscure. Do these proteins undergo simple onedimensional (1D) translational diffusion, or do they rotate to maintain a specific orientation with respect to the DNA helix? We measured 1D diffusion constants as a function of protein size while maintaining the DNA-protein interface. Using bootstrap analysis of single-molecule diffusion data, we compared the results to theoretical predictions for pure translational motion and rotation-coupled sliding along the DNA. The data indicate that DNA-binding proteins undergo rotation-coupled sliding along the DNA helix and can be described by a model of diffusion along the DNA helix on a rugged free-energy landscape. A similar analysis including the 1 D diffusion constants of eight proteins of varying size shows that rotation-coupled sliding is a general phenomenon. The average free-energy barrier for sliding along the DNA was 1.1 ± 0.2 k B T. Such small barriers facilitate rapid search for binding sites.Many nucleic acid enzymes and proteins that act on DNA quickly locate target sites by diffusing along nonspecific DNA. It has been shown that proteins can both hop and slide along doublestranded DNA [1][2][3] , although the microscopic mechanism of protein motion along DNA molecules is still not understood in molecular detail. In particular, the path traced by a sliding protein molecule along the surface of DNA has not been established. Both linear paths, parallel to the DNA axis, and helical paths, following a strand or groove of the DNA around the DNA axis, have been taken as assumptions in biophysical and biochemical models. Although rotation of sliding proteins around the DNA helix was implicitly 4 and explicitly 5,6 anticipated, such rotation was not shown to occur during diffusive sliding. The concept of rotational coupling has also arisen among structural biologists based on concepts of molecular recognition and observations of detailed structural complementarity between proteins and DNA 7-9 . Despite © 2009 Nature America, Inc. All rights reserved.Correspondence to: Biman Bagchi; X Sunney Xie.Correspondence should be addressed to B.B. (bagchibiman@yahoo.com) the persistent high profile of this question in the literature, it remains unknown whether sliding proteins track the DNA helix. Such tracking would have major biophysical and biochemical implications: for example, only a limited set of enzyme-helix juxtapositions would need to be considered in questions of protein-DNA interaction. In this work, we examine the dependence on protein size of the diffusion constant for sliding along DNA in order to distinguish pure translational diffusion (Fig. 1a) along DNA from rotation-coupled (or -slaved) diffusion (Fig. 1b). The result offers insights into the mechanism of target search and recognition of all DNAbinding proteins.As a protein moves along DNA, it experiences three different frictional forces arising from rando...

Protein–DNA Electrostatics

“…Indeed, as it was imagined by Schurr [51], some DNA-binding proteins slide with an helical motion along DNA [34,35]. The resulting effective diffusion coefficient then depends on the DNA-protein distance in the bound state [91,34,35] and we have seen that the latter depends on the salt concentration; the sliding diffusion coefficient therefore depends on the salt concentration. This In the diffusing mode, the partially denatured protein is much less sensitive to the sequence and its mobility is therefore increased [23].…”

“…The last hypothesis has the advantage to keep the protein in closest and constant contact with the DNA base-pairs, allowing the protein to maintain a specific orientation with respect to the DNA helix. An helical trajectory has been then indirectly proved for the case of some DNAbinding proteins [34,35], but the question remains open in general [36].…”

Two glycosylase families diffusively scan DNA using a wedge residue to probe for and identify oxidatively damaged bases

“…Given that glycosylases are conjugated to ∼20-nm Qdots, the viscous drag of this label will reduce the diffusive motion of the enzyme on DNA. In fact, Blainey et al (19) demonstrated that glycosylases conjugated to fluorescent labels with increasing Stokes radii diffused along DNA molecules with diffusion constants that were consistent with the glycosylase tracking the DNA's helical contour (i.e., rotating along the DNA), most probably in the minor groove where they bind (7). By this analysis, our Qdot labeling strategy gives an apparent onedimensional diffusion constant of ∼0.05 μm 2 /s (18) as the glycosylase diffuses along the helical path of the DNA.…”

“…In E. coli Nth, Ile79, Leu81, and Gln41 have been shown to play a similar role in lesion extrusion (15). Although crystal structures have shed light on the glycosylase-DNA interactions upon lesion binding, single-molecule biophysical studies may define the mechanism by which glycosylases locate damaged bases while rapidly diffusing along the DNA (17)(18)(19).…”

Protein−DNA binding in the absence of specific base-pair recognition

“…The second mechanism is largely DNA sequence independent, and it involves electrostatic binding modulated by the overall DNA geometry (6). Despite significant experimental progress, molecular mechanisms responsible for these two types of nonspecific binding remain poorly understood, and the free energy of nonspecific protein−DNA binding has not been systematically characterized (11)(12)(13)(14). The interplay between consensus and nonconsensus DNA sequence elements emerges as a dominant factor that governs protein−DNA binding preferences.…”