An unprecedented nucleic acid capture mechanism for excision of DNA damage

Abstract

DNA glycosylases that remove alkylated and deaminated purine nucleobases are essential DNA repair enzymes that protect the genome, and at the same time confound cancer alkylation therapy, by excising cytotoxic N3-methyladenine bases formed by DNA-targeting anticancer compounds. The basis for glycosylase specificity towards N3- and N7-alkylpurines is believed to result from intrinsic instability of the modified bases and not from direct enzyme functional group chemistry. Here we present crystal structures of the recently discovered Bacillus cereus AlkD glycosylase in complex with DNAs containing alkylated, mismatched and abasic nucleotides. Unlike other glycosylases, AlkD captures the extrahelical lesion in a solvent-exposed orientation, providing an illustration for how hydrolysis of N3- and N7-alkylated bases may be facilitated by increased lifetime out of the DNA helix. The structures and supporting biochemical analysis of base flipping and catalysis reveal how the HEAT repeats of AlkD distort the DNA backbone to detect non-Watson-Crick base pairs without duplex intercalation.

Figure 1

Base excision repair of alkylated DNA by AlkD. a, AlkD catalyzes the hydrolysis of the N-glycosidic bond to liberate an abasic site and free nucleobase. The enzyme is specific for positively charged N3-methyladenine (a) and N7-methylguanine (b). c,d, Structures of 3-deaza-3-methyladenosine (c) and tetrahydrofuran (d) used to trap AlkD in complex with alkylated and abasic DNA. e, Crystal structure of AlkD bound to 3d3mA-DNA. Each of the 6 HEAT-repeats are colored red-to-violet. The DNA is colored silver with the 3d3mA nucleotide colored magenta.

Figure 2

Crystal structures of AlkD in complex with 3d3mA-DNA (a) and THF-DNA (b). The top of each panel shows orthogonal views of the AlkD protein (green) wrapping around the DNA duplex (gold). The modified 3d3mA and THF nucleotides are colored blue, and the opposing thymine is magenta. At the bottom, a side view of the atomic model and corresponding schematic illustrates the interactions between the modified base pairs and the protein. Dashed lines represent hydrogen bonds and wavy lines represent van der Waals interactions.

Figure 3

Recognition of DNA damage by AlkD. a, 3d3mA-DNA (substrate) complex; b, THF-DNA (product) complex. Composite omit electron density (contoured to 1σ) for the modified base pairs is superimposed against the crystallographic models. Dashed arrows denote displacement of THF and opposing thymine from their positions in B-DNA. Hydrogen bonds are shown as dashed lines. Views are down the DNA helix axis.

Figure 4

Excision of N7- and O²- pyridyloxobutyl (POB) base adducts by AlkD. a, Chemical structures of N7-POB-deoxyguanine and O²-POB-deoxycytosine. b,c, Time courses for the release of N7-POB-dG (black squares) and O²-POB-dC (red circles) in the presence (closed symbols, solid lines) and absence (open symbols, dashed lines) of Bacillus cereus AlkD (b) or human AAG (c). Error bars represent the standard deviation from three independent measurements.

Figure 5

Remodeling of a G•T wobble base pair by AlkD. a, AlkD/G•T-DNA complex viewed down the helical axis. b, The structure of a G•T wobble base pair in DNA alone (PDB 113D) is superimposed onto the AlkD/G•T complex. Steric clashes between the protein and DNA are highlighted by yellow stars, and disrupted hydrogen bonds (dashed lines) are shown by a red X. c, Relative single-turnover rates (k_st) of 7mG excision from a 25mer oligonucleotide duplex by wild-type and mutants of AlkD. Wild-type, D113N, and R148A data from ref. . Error bars represent the standard deviation from three independent measurements.