Electrostatic Properties of Complexes along a DNA Glycosylase Damage Search Pathway

Human uracil DNA glycosylase (hUNG) follows an extended reaction coordinate for locating rare uracil bases in genomic DNA. This process begins with diffusion-controlled engagement of undamaged DNA, followed by a damage search step in which the enzyme remains loosely associated with the DNA chain (translocation), and finally, a recognition step that allows the enzyme to efficiently bind and excise uracil when it is encountered. At each step along this coordinate, the enzyme must form DNA interactions that are highly specialized for either rapid damage searching or catalysis. Here we make extensive measurements of hUNG activity as a function of salt concentration to dissect the thermodynamic, kinetic, and electrostatic properties of key enzyme states along this reaction coordinate. We find that the interaction of hUNG with undamaged DNA is electrostatically driven at a physiological concentration of potassium ions (Δ<i>G</i>_elect = −3.5 ± 0.5 kcal mol^–1), with only a small nonelectrostatic contribution (Δ<i>G</i>_non = −2.0 ± 0.2 kcal mol^–1). In contrast, the interaction with damaged DNA is dominated by the nonelectrostatic free energy term (Δ<i>G</i>_non = −7.2 ± 0.1 kcal mol^–1), yet retains the nonspecific electrostatic contribution (Δ<i>G</i>_elect = −2.3 ± 0.2 kcal mol^–1). Stopped-flow kinetic experiments established that the salt sensitivity of damaged DNA binding originates from a reduction of <i>k</i>_on, while <i>k</i>_off is weakly dependent on salt. Similar findings were obtained from the salt dependences of the steady-state kinetic parameters, where the diffusion-controlled <i>k</i>_cat/<i>K</i>_m showed a salt dependence similar to <i>k</i>_on, while <i>k</i>_cat (limited by product release) was weakly dependent on salt. Finally, the salt dependence of translocation between two uracil sites separated by 20 bp in the same DNA chain was indistinguishable from that of <i>k</i>_on. This result suggests that the transition-state for translocation over this spacing resembles that for DNA association from bulk solution and that hUNG escapes the DNA ion cloud during translocation. These findings provide key insights into how the ionic environment in cells influences the DNA damage search pathway

AP-Endonuclease 1 Accelerates Turnover of Human 8‑Oxoguanine DNA Glycosylase by Preventing Retrograde Binding to the Abasic-Site Product

A major product of oxidative DNA damage is 8-oxoguanine. In humans, 8-oxoguanine DNA glycosylase (hOGG1) facilitates removal of these lesions, producing an abasic (AP) site in the DNA that is subsequently incised by AP-endonuclease 1 (APE1). APE1 stimulates turnover of several glycosylases by accelerating rate-limiting product release. However, there have been conflicting accounts of whether hOGG1 follows a similar mechanism. In pre-steady-state kinetic measurements, we found that addition of APE1 had no effect on the rapid burst phase of 8-oxoguanine excision by hOGG1 but accelerated steady-state turnover (<i>k</i>_cat) by ∼10-fold. The stimulation by APE1 required divalent cations, could be detected under multiple-turnover conditions using limiting concentrations of APE1, did not require flanking DNA surrounding the hOGG1 lesion site, and occurred efficiently even when the first 49 residues of APE1’s N-terminus had been deleted. Stimulation by APE1 does not involve relief from product inhibition because thymine DNA glycosylase, an enzyme that binds more tightly to AP sites than hOGG1 does, could not effectively substitute for APE1. A stimulation mechanism involving stable protein–protein interactions between free APE1 and hOGG1, or the DNA-bound forms, was excluded using protein cross-linking assays. The combined results indicate a mechanism whereby dynamic excursions of hOGG1 from the AP site allow APE1 to invade the site and rapidly incise the phosphate backbone. This mechanism, which allows APE1 to access the AP site without forming specific interactions with the glycosylase, is a simple and elegant solution to passing along unstable intermediates in base excision repair