Sammendrag
Three high-resolution crystal structures of DNA complexes with wild-t ype and mutant human uracil-DNA glycosylase (UDG), coupled kinetic ch aracteriz and comparisons with the refined unbound UDG structure help resolve fundamental issues in the initiation of DNA base excision re pair (BER): damage detection, nucleotide flipping versus extrahelical nucleotide capture, avoidance of apurinic/apyrimidinic (AP) site tox icity and coupling of damage-specific and damage-general BER steps. S tructural and kinetic results suggest that UDG binds, kinks and compr esses the DNA backbone with a 'Ser-Pro pinch' and scans the minor gro ove for damage. Concerted shifts in UDG simultaneously form the catal ytically competent active site and induce further compression and kin king of the double-stranded DNA backbone only at uracil and AP sites, where these nucleotides can flip at the phosphate-sugar junction int o a complementary specificity pocket. Unexpectedly, UDG binds to AP s ites more tightly and more rapidly than to uracil-containing DNA, and thus may protect cells sterically from AP site toxicity. Furthermore , AP-endonuclease, which catalyzes the first damage-general step of B ER, enhances UDG activity, most likely by inducing UDG release via sh ared minor groove contacts and flipped AP site binding. Thus, AP site binding may couple damage-specific and damage-general steps of BER w ithout requiring direct protein-protein interactions.
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