DNA repair proteins slide in contact with DNA to find defect bases

(see Blainey et al. "A base-excision DNA-repair protein finds intrahelical lesion bases by fast sliding in contact with DNA," Proc. Natl. Aca. Sci., 103, 5752-5757, 2006).

We have combined the DNA flow-stretching assay developed in our group with single-molecule fluorescence detection to monitor single fluorescently labeled DNA-binding proteins interact with stretched DNA molecules in real time. For these measurements, we do not add a bead to the free end of the DNA. Using wide-field total internal reflection fluorescence microscopy, we can image the DNA-bound protein molecules while minimizing the fluorescence background from protein molecules in solution.

Many classes of DNA-binding proteins need to locate target sites in DNA very quickly. These proteins face a difficult needle-in-a-haystack problem: the rare target sites are hidden among billions of nonspecific sites in a large DNA genome. These proteins are thought to solve the search problem through a combination of “hopping” among different DNA segments and “sliding” along DNA segments. While there exists evidence for diffusion of proteins along DNA, it has not been clear if this took place exclusively by hopping or whether the proteins can slide in contact with the DNA.

Oxoguanine (OxoG) is a highly mutagenic DNA lesion formed in cells when free radicals react with DNA. With colleagues from the Verdine Group, we investigated the search behavior of human oxoguanine DNA glycosylase 1 (hOgg1), the DNA repair protein that initiates repair of OxoG (structure* shown below). To prevent harmful genetic changes from occurring in our bodies, hOgg1 must find and initiate repair of OxoG quickly.

We label hOgg1 with the fluorescent dye Cy3B at the protein’s C-terminous. When a small amount of the labeled protein is added to the flow solution, we observe the DNA repair enzymes attach to and move along the flow-stretched DNA strands. Over the course of a few minutes we see hundreds to thousands of protein molecules attach to, move along, and dissociate from each DNA strand in the field of view. By determining the positions of the molecules in each frame of a recorded movie, we can trace out the path each protein molecule took as it moved along DNA searching for OxoG.

Using many such molecular trajectories, we can construct population-averaged mean-square displacement plots to accurately measure the protein’s diffusion constant under a given buffer condition.

hOgg1’s diffusion constant is independent of salt concentration. Since a protein hopping along DNA would appear to diffuse much more quickly as its binding to DNA is weakened by increasing salt, we conclude the hOgg1 diffuses rapidly along DNA while maintaining persistent contact, literally sliding along the DNA.

hOgg1’s sliding activity is also pH-dependent. The fast one-dimensional sliding (D > 5,000,000 bp²/s) observed at higher pH requires the presence of a certain amino acid (histidine 270) on the part of the protein that contacts DNA. Arrhenius analysis using Schurr's** hydrodynamic limit rate allows calculation of mean free energy barrier for sliding, which is about 1 kBT at physiological pH. These single-molecule tracking measurements in combination with ongoing structural work in the Verdine Lab shed new light on how hOgg1 recognizes OxoG. The high sliding rate excludes possibilities that hOgg1 extrudes each base for inspection in the extra-helical active site and that hOgg1 captures spontaneously extruded oxoG bases. The redundancy of sliding indicates that hOgg1 need not identify oxoG on every encounter, but must avoid time-consuming sampling of native bases. These points, together with an increasing body of structural evidence, suggest that oxoG glycosylases identify intrahelical lesion bases and selectively accelerate their extrusion from DNA into the enzyme active site.

* Bruner, S. D., Norman, D. P. G. & Verdine, G. L. (2000) Nature 403, 859–866.
** Schurr, J. M. (1979) Biophys. Chem. 9, 413–414.