No abstract

DNA glycosylase MutY plays a critical role in suppression of mutations resulted from oxidative damage, as highlighted by cancer-association of the human enzyme. MutY requires a highly conserved catalytic Asp residue for excision of adenines misinserted opposite 8-oxo-7,8-dihydroguanine (OG). A nearby Asn residue hydrogen bonds to the catalytic Asp in structures of MutY and its mutation to Ser is an inherited variant in human MUTYH associated with colorectal cancer. We captured structural snapshots of N146S Geobacillus stearothermophilus MutY bound to DNA containing a substrate, a transition state analog and enzyme-catalyzed abasic site products to provide insight into the base excision mechanism of MutY and the role of Asn. Surprisingly, despite the ability of N146S to excise adenine and purine (P) in vitro, albeit at slow rates, N146S-OG:P complex showed a calcium coordinated to the purine base altering its conformation to inhibit hydrolysis. We obtained crystal structures of N146S Gs MutY bound to its abasic site product by removing the calcium from crystals of N146S-OG:P complex to initiate catalysis in crystallo or by crystallization in the absence of calcium. The product structures of N146S feature enzyme-generated β-anomer abasic sites that support a retaining mechanism for MutY-catalyzed base excision.

DNA glycosylases preserve genome integrity and define the specificity of the base excision repair pathway for discreet, detrimental modifications, and thus, the mechanisms by which glycosylases locate DNA damage are of particular interest. Bacterial AlkC and AlkD are specific for cationic alkylated nucleobases and have a distinctive HEAT-like repeat (HLR) fold. AlkD uses a unique non-base-flipping mechanism that enables excision of bulky lesions more commonly associated with nucleotide excision repair. In contrast, AlkC has a much narrower specificity for small lesions, principally N3-methyladenine (3mA). Here, we describe how AlkC selects for and excises 3mA using a non-base-flipping strategy distinct from that of AlkD. A crystal structure resembling a catalytic intermediate complex shows how AlkC uses unique HLR and immunoglobulin-like domains to induce a sharp kink in the DNA, exposing the damaged nucleobase to active site residues that project into the DNA This active site can accommodate and excise N3-methylcytosine (3mC) and N1-methyladenine (1mA), which are also repaired by AlkB-catalyzed oxidative demethylation, providing a potential alternative mechanism for repair of these lesions in bacteria.

DNA glycosylases of the Base Excision Repair (BER) pathway are front-line defenders in removing compromising modifications of the DNA nucleobases. Aberrantly modified nucleobases mediate genomic mutations and inhibit DNA replication leading to adverse health consequences such as cancer, neurological diseases and aging. In an effort towards developing high-affinity transition state (TS) analogs as chemical biology probes for DNA glycosylases, oligonucleotides containing a propargyl-modified pyrrolidine TS mimic nucleotide were synthesized. A small library of TS-mimic containing oligonucleotides was generated using a structurally diverse set of five azides via copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) "click" chemistry. The relative affinity (K d) was evaluated for BER glycosylases E. coli MutY (Ec MutY), bacterial formamidopyrimidine glycosylase (Fpg), and human OG glycosylase 1 (hOGG1) with the library of TS-mimic DNA duplexes. All of the BER glycosylases were found to exhibit extremely high affinity (K d ~ pM) for the TS mimics. However, binding preferences, distinct for each glycosylase, for the TS mimic library members were observed, suggesting different modes of binding and transition state stabilization among the three glycosylases. Fpg bound all of the TS mimics with exceptionally high affinity, while MutY binding affinity correlated inversely with size of the appended moiety. Of note, we identified one member of the small TS mimic library that exhibited particularly high affinity for hOGG1. These results strongly support use of the propargyl-TS mimic oligonucleotides and elaboration via click chemistry in screening and identification of highaffinity ligands for a BER glycosylases of interest.