Structural Basis for the Inhibition of Human Alkyladenine DNA Glycosylase (AAG) by 3,N4-Ethenocytosine-containing DNA

Gondichatnahalli M Lingaraju,C Ainsley Davis,Jeremy W Setser,Leona D Samson,Catherine L Drennan

doi:10.1074/jbc.m110.192435

Abstract

Reactive oxygen and nitrogen species, generated by neutrophils and macrophages in chronically inflamed tissues, readily damage DNA, producing a variety of potentially genotoxic etheno base lesions; such inflammation-related DNA damage is now known to contribute to carcinogenesis. Although the human alkyladenine DNA glycosylase (AAG) can specifically bind DNA containing either 1,N6-ethenoadenine (ϵA) lesions or 3,N4-ethenocytosine (ϵC) lesions, it can only excise ϵA lesions. AAG binds very tightly to DNA containing ϵC lesions, forming an abortive protein-DNA complex; such binding not only shields ϵC from repair by other enzymes but also inhibits AAG from acting on other DNA lesions. To understand the structural basis for inhibition, we have characterized the binding of AAG to DNA containing ϵC lesions and have solved a crystal structure of AAG bound to a DNA duplex containing the ϵC lesion. This study provides the first structure of a DNA glycosylase in complex with an inhibitory base lesion that is induced endogenously and that is also induced upon exposure to environmental agents such as vinyl chloride. We identify the primary cause of inhibition as a failure to activate the nucleotide base as an efficient leaving group and demonstrate that the higher binding affinity of AAG for ϵC versus ϵA is achieved through formation of an additional hydrogen bond between Asn-169 in the active site pocket and the O2 of ϵC. This structure provides the basis for the design of AAG inhibitors currently being sought as an adjuvant for cancer chemotherapy.

Highlights

alkyladenine DNA glycosylase (AAG) has been previously characterized by crystallography
The crystal structures of an N-terminally truncated, but catalytically active, construct of AAG (⌬79AAG), both bound to a pyrrolidine abasic site mimic (Pyr) and bound to an ⑀A-containing piece of DNA, suggest a mode by which AAG recognizes a wide range of lesions while still discriminating against undamaged bases [9, 10]
It was surprising that AAG has the ability to recognize and bind a number of DNA base lesions that it is incapable of excising, in particular the ⑀C lesion

Summary

EXPERIMENTAL PROCEDURES

⌬79AAG Plasmid Construction, Creation of Mutants, and Protein Preparation—Constructs of full-length AAG and a truncated form of AAG (with 84 residues at the N terminus deleted) were cloned into pET19b-PPS vectors for protein expression (UniprotID: P29372). Binding reactions were set up as 10-␮l solutions containing 1ϫ binding buffer (50 mM Hepes-NaOH, pH 7.5, 100 mM NaCl, 1 mM EDTA, 9.5% v/v glycerol, 50 ␮g/ml BSA, and 5 mM DTT), 2 nM 32P-labeled oligonucleotide, and 0 –1000 nM of the purified ⌬79AAG protein. The reactions were set up as 20-␮l solutions containing 1ϫ glycosylase assay buffer, 1 nM 32P-labeled ⑀A:T (T paired opposite ⑀A) 25-mer oligonucleotide duplex DNA (5Ј-GCA ATC TAG CCA ⑀AGT CGA TGT ATG C-3Ј), 5 nM of the purified ⌬79AAG enzyme, and increasing concentrations of competitor DNA (0 –3000 nM). Dissociation constant (Kd) values measured using gel shift assays and 50% inhibitory concentration (IC50) for the inhibition of ⌬79AAG activity on ⑀A:T 25-mer, measured using competition DNA glycosylase assay at 37 °C, in the presence of increasing concentration of cold competitor 13-mer duplexes

20 Ϯ 2 13 Ϯ 2 46 Ϯ 6 21 Ϯ 3 31 Ϯ 4 47 Ϯ 6

RESULTS

DISCUSSION