A base-excision DNA-repair protein finds intrahelical lesion bases by fast sliding in contact with DNA

Abstract

A central mystery in the function of site-specific DNA-binding proteins is the detailed mechanism for rapid location and binding of target sites in DNA. Human oxoguanine DNA glycosylase 1 (hOgg1), for example, must search out rare 8-oxoguanine lesions to prevent transversion mutations arising from oxidative stress. Here we report high-speed imaging of single hOgg1 enzyme molecules diffusing along DNA stretched by shear flow. Salt-concentration-dependent measurements reveal that such diffusion occurs as hOgg1 slides in persistent contact with DNA. At near-physiologic pH and salt concentration, hOgg1 has a subsecond DNA-binding time and slides with a diffusion constant as high as 5 x 10(6) bp(2)/s. Such a value approaches the theoretical upper limit for one-dimensional diffusion and indicates an activation barrier for sliding of only 0.5 kcal/mol (1 kcal = 4.2 kJ). This nearly barrierless Brownian sliding indicates that DNA glycosylases locate lesion bases by a massively redundant search in which the enzyme selectively binds 8-oxoguanine under kinetic control.

Fig. 1.

hOgg1 and its cognate lesion oxoG. (A) hOgg1 cocrystal structure with oxoG:C-containing DNA (5). The location of His-270 is indicated by an asterisk and arrow. The C terminus is indicated by a double asterisk. (B) Scheme depicting the formation of the oxoG lesion by attack of reactive oxygen species (ROS) on guanine.

Fig. 2.

Single-molecule assay for protein translocation along DNA. (A and B) Inverted microscope fitted for total internal reflection fluorescence imaging (A) with mounted flow cell (B). (C) Schematic of flow-stretched DNA molecule (not to scale). Buffer solution flows over the glass coverslip to which double-stranded λ DNA, 16 μm in length, is attached by one end. (D) Image of SYTOX orange-stained λ DNA molecule stretched by flow (integration time, 0.040 s). (E) Image of 10 single hOgg1 enzymes bound to an undamaged DNA molecule (integration time, 0.050 s). We selected an image from an experiment with a higher than usual protein concentration (0.2 nM) for the given buffer condition (0.01 M NaCl, pH 7.5) to obtain the larger-than-usual number of DNA-bound enzyme molecules. Noncollinear signals arise from surface-adsorbed enzymes. (F) Trajectories of 700 enzyme molecules diffusing on an undamaged λ DNA molecule over the course of 150 s (note broad distribution of transverse positions at the free end due to fluctuations of the DNA position). Buffer flows toward the right in A and C–F. The DNA extension in D is greater because of the intercalating dye. (Scale bars, 1.0 μm.)

Fig. 3.

Diffusion of single hOgg1 enzyme molecules along DNA. (A) Trajectories of two hOgg1 molecules diffusing along an undamaged DNA molecule at pH 7.5 with 0.01 M NaCl (molecule no. 1, black solid line; molecule no. 2, gray solid line) in the direction of DNA extension/flow (toward positive x-axis direction). Starting times and positions are defined as zero (starting position indicated by gray dashed line). (B) Histogram of net displacement (= x_final − x_initial) for 723 trajectories collected in one experiment on an undamaged DNA molecule. The histogram mean is 0.013 μm.

Fig. 4.

pH and salt concentration-dependence of hOgg1’s sliding activity. (A) Mean-square displacement of hOgg1 K249Q (blue points) and hOgg1 H270A (red points) along undamaged DNA at various indicated pH values. Data for each measurement represent an average over >100 binding events (except for hOgg1 H270A at pH 7.5, for which the average is over eight events). Solid lines are least-squares fits to the data. The case for free sliding is shown, indicated by the dashed line with slope 2.46 μm²/s. The salt concentration for all plots in A is 0.010 M. (B) Mean binding lifetime (□, left axis as indicated) and diffusion constant (■, right axis as indicated) of the K249Q mutant at pH 7.0 as a function of salt concentration along undamaged DNA. (C) Diffusion constant versus pH for the two mutants along undamaged DNA with salt concentration 0.010 M. Error bars represent SD. Where no error bar appears in B and C, the uncertainty is comparable to the dimension of the plotted symbol.

Fig. 5.

Mean activation energy for hOgg1 sliding on undamaged DNA. Shown is the mean activation energy for sliding versus pH. The shaded area indicates activation energies at which fast searching is possible (29). Also indicated are the range of intranuclear pH values measured in mammalian cells (7.55–7.79) (33) and the pH of the mitochondrial matrix (mito) (pH 8). Error bars represent SD.