Base excision repair initiation revealed by crystal structures and binding kinetics of human uracil-DNA glycosylase with DNA

Abstract

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 damagegeneral 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 phosphatesugar 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.

DNA-binding mechanism of the Escherichia coli Ada O6-alkylguanine-DNA alkyltransferase

“…Various DNA glycosylases also only contact the phosphate backbone of a single DNA strand when bound to duplex DNA (49)(50)(51). This contrasts to the majority of DNA-binding proteins, which form contacts to both DNA strands.…”

Uracil in DNA – occurrence, consequences and repair

“…This established a 'push -pull' hypothesis in which the L272 side chain penetrates into the minor groove expelling uracil (push), and complementary interactions from the specificity pocket facilitate productive binding (pull). The mechanistics of this was further refined by high resolution co-crystal structures of wild type and mutant UNG bound to U : A and U : G oligonucleotides (Parikh et al, 1998). …”

“…This implied that the extrahelical conformation could be achieved even in the absence of the insertion of a hydrophobic side chain (push). A mechanism was proposed in which serine-proline rich loops compress the phosphates flanking uracil by a 'pinching' mechanism and thereby stabilize the extrahelical conformation (Parikh et al, 1998) without contributing significant energy to the cleavage reaction itself . Such a 'pinch -push -pull' mechanism was also hypothesised for E. coli Ung .…”

Targeting base excision repair suggests a new therapeutic strategy of fludarabine for the treatment of chronic lymphocytic leukemia

“…UDG protein was significantly upregulated after drug treatments, particularly in cells treated with the combination treatment of fludarabine and MX. On the basis of previous observation that UDG has a higher affinity for the products at the AP-site than the actual substrate, 8 it has been proposed that subsequent to a base release, UDG remains bound to the AP site to protect the cell until the AP site is transferred to the downstream BER-pathway enzymes, AP endonuclease and polymerase b. Although the upregulation of UDG in response to fludarabine and MX occurs at the transcriptional level (data not shown), it is also possible that the persistence of MX-bound AP sites results in the accumulation of UDG at un-repairable MX-bound AP sites.…”