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Three high-resolution crystal structures of DNA complexes with wild-type and
mutant human uracil-DNA glycosylase (UDG), coupled kinetic characterizations and
comparisons with the refined unbound UDG structure help resolve fundamental
issues in the initiation of DNA base excision repair (BER): damage detection,
nucleotide flipping versus extrahelical nucleotide capture, avoidance of
apurinic/apyrimidinic (AP) site toxicity and coupling of damage-specific and
damage-general BER steps. Structural and kinetic results suggest that UDG binds,
kinks and compresses the DNA backbone with a 'Ser-Pro pinch' and scans the minor
groove for damage. Concerted shifts in UDG simultaneously form the catalytically
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 complementary
specificity pocket. Unexpectedly, UDG binds to AP sites 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 BER, enhances UDG activity, most likely by inducing UDG
release via shared minor groove contacts and flipped AP site binding. Thus, AP
site binding may couple damage-specific and damage-general steps of BER without
requiring direct protein-protein interactions.
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