Electrostatic Properties of Complexes along a DNA Glycosylase Damage Search Pathway

ABSTRACT: 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, fol-lowed 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 (ΔGelect = −3.5 ± 0.5 kcal mol−1), with only a small nonelectrostati