Structural insight into repair of alkylated DNA by a new superfamily of DNA glycosylases comprising HEAT-like repeats

3-methyladenine DNA glycosylases initiate repair of cytotoxic and promutagenic alkylated bases in DNA. We demonstrate by comparative modelling that Bacillus cereus AlkD belongs to a new, fifth, structural superfamily of DNA glycosylases with an alpha–alpha superhelix fold comprising six HEAT-like repeats. The structure reveals a wide, positively charged groove, including a putative base recognition pocket. This groove appears to be suitable for the accommodation of double-stranded DNA with a flipped-out alkylated base. Site-specific mutagenesis within the recognition pocket identified several residues essential for enzyme activity. The results suggest that the aromatic side chain of a tryptophan residue recognizes electron-deficient alkylated bases through stacking interactions, while an interacting aspartate–arginine pair is essential for removal of the damaged base. A structural model of AlkD bound to DNA with a flipped-out purine moiety gives insight into the catalytic machinery for this new class of DNA glycosylases

Separation-of-Function Mutants Unravel the Dual-Reaction Mode of Human 8-Oxoguanine DNA Glycosylase

Summary7,8-Dihydro-8-oxoguanine (8oxoG) is a major mutagenic base lesion formed when reactive oxygen species react with guanine in DNA. The human 8oxoG DNA glycosylase (hOgg1) recognizes and initiates repair of 8oxoG. hOgg1 is acknowledged as a bifunctional DNA glycosylase catalyzing removal of the damaged base followed by cleavage of the backbone of the intermediate abasic DNA (AP lyase/β-elimination). When acting on 8oxoG-containing DNA, these two steps in the hOgg1 catalysis are considered coupled, with Lys249 implicated as a key residue. However, several lines of evidence point to a concurrent and independent monofunctional hydrolysis of the N-glycosylic bond being the in vivo relevant reaction mode of hOgg1. Here, we present biochemical and structural evidence for the monofunctional mode of hOgg1 by design of separation-of-function mutants. Asp268 is identified as the catalytic residue, while Lys249 appears critical for the specific recognition and final alignment of 8oxoG during the hydrolysis reaction

Structural model with proposed active site and lesion recognition pocket of AlkD

<p><b>Copyright information:</b></p><p>Taken from "Structural insight into repair of alkylated DNA by a new superfamily of DNA glycosylases comprising HEAT-like repeats"</p><p></p><p>Nucleic Acids Research 2007;35(7):2451-2459.</p><p>Published online 29 Mar 2007</p><p>PMCID:PMC1874660.</p><p>© 2007 The Author(s)</p> The model contains residues 11 through to 226 and is lacking α-helix α1 and the 11 C-terminal residues (predicted to be disordered). () Cartoon rendering of the protein which comprises 13 α-helices contributing to the six repeats in . () APBS () calculated electrostatic potential mapped onto the protein surface (red = negative, white = neutral and blue = positive) showing the 20–25 Å wide, positively charged, putative DNA binding groove. () Amino acid residue conservation in 43 AlkD homologs mapped onto the space filling representation of the model generated with ConSurf (). The scale extends from magenta (highly conserved), through white to cyan (highly variable). There is a nest of conserved residues in the putative DNA binding groove, and several conserved basic amino acid residues (Arg and Lys) are sited along the upper and lower edge of the groove. () Stereo view of a close-up of the highly conserved nest shows the eight conserved residues that were mutated by site-directed mutagenesis. The catalytic activity and MMS sensitivity of the resulting mutants were determined (). The eight conserved residues have identical geometry in the experimental structure 2B6C. The orientation of the protein in space is identical in all panels. The model is also available as Supplementary Video 1 online