The structural and chemical biology of DNA glycosylases for base-excision repair

DNA glycosylases initiate DNA base-excision repair (BER) by removing damaged bases from DNA by cleaving the glycosylic bond. Human uracil-DNA glycosylase (UDG), an archetypical BER enzyme excises the damaged base uracil. Crystal structures of DNA complexes with wild-type and mutant human UDG and coupled biochemical characterizations help resolve fundamental issues in the initiation of DNA BER: damage detection, catalytic mechanism, and avoidance of apurinic/apyrimidinic (AP) site toxicity. Fluorescence experiments indicate that UDG does not flip every nucleotide out of the DNA base stack when scanning for damage. UDG likely binds, bends and compresses the DNA backbone with a 'pinch-progression' to scan the minor groove for damage. Analysis of a human-cytomegalovirus chimeric UDG highlights structural features of the damage-scanning mechanism. Closure of UDG forms the catalytically-competent active center and induces further contortions in DNA only at uracils and AP sites, where these nucleotides can flip into the active site and glycosylic bond cleavage occurs. UDG remains active even when active-site functional groups are deleted by mutagenesis. Enzyme-DNA substrate binding energy from the macromolecular interface is funneled into catalytic power at the active site. The architecturally-stablized closing of UDG distorts the flipped-out uridine, which is relieved by glycosylic bond cleavage in the product complex. The substrate stereochemistry implies coupling of the anomeric effect to delocalization of the glycosylic bond electrons into the uracil aromatic system. This mechanism resolves paradoxes concerning the transpositions of electrons among orthogonal orbitals and the retention of catalytic efficiency despite deletion of active-site functional groups. UDG binds to AP sites tighter than to uracil-containing DNA, and thus may sterically protect cells from AP site toxicity. Furthermore, AP endonuclease, which catalyzes the next step of BER, enhances UDG activity. AP site binding may couple damage-specific and damage-general steps of BER without requiring direct protein-protein interactions. The DNA glycosylase MutY, which is a member of the Helix-hairpin-Helix DNA glycosylase superfamily, excises mispaired adenines from dsDNA. Crystal structures of the MutY catalytic core, mutants, and a MutY-adenine complex reveal the basis for adenine specificity and glycosylic bond cleavage. Common themes from both the UDG and MutY systems are identified and discussed.