BASE EXCISION-REPAIR INITIATION REVEALED BY CRYSTAL-STRUCTURES AND BINDING-KINETICS OF HUMAN URACIL-DNA GLYCOSYLASE WITH DNA

Three high-resolution crystal structures of DNA complexes with wild-ty pe and mutant human uracil-DNA glycosylase (UDG), coupled kinetic char acterizations and comparisons with the refined unbound UDG structure h elp resolve fundamental issues in the initiation of DNA base excision repair (BER): damage detection, nucleotide flipping versus extrahelica l nucleotide capture, avoidance of apurinic/apyrimidinic (AP) site tox icity and coupling of damage-specific and damage-general BER steps. St ructural and kinetic results suggest that UDG binds, kinks and compres ses the DNA backbone with a 'Ser-Pro pinch' and scans the minor groove for damage. Concerted shifts in UDG simultaneously form the catalytic ally competent active site and induce further compression and kinking of the double-stranded DNA backbone only at uracil and AP sites, where these nucleotides can flip at the phosphate-sugar junction into a com plementary specificity pocket. Unexpectedly, UDG binds to AP sites mor e tightly and more rapidly than to uracil-containing DNA, and thus may protect cells sterically from AP site toxicity. Furthermore, AP-endon uclease, which catalyzes the first damage-general step of BER, enhance s UDG activity, most likely by inducing UDG release via shared minor g roove contacts and flipped AP site binding. Thus, AP site binding may couple damage-specific and damage-general steps of BER without requiri ng direct protein-protein interactions.