Mechanism-Based DNA−Protein Cross-Linking of MutY via Oxidation of 8-Oxoguanosine

Introduction of a covalent bond cross-linking a protein to its nucleic acid target can be used to stabilize a protein-DNA complex or to investigate contacts between the two biopolymers. Many cross-linking methods rely on photochemical triggers to generate reactive intermediates of suitably modified nucleotides or amino acid residues, while other DNA-protein cross- links are a natural result of oxidative damage to chromatin arising from endogenous metal ions, oxidants, or ionizing radiation. Of the four bases, guanine is the most susceptible to oxidative damage, but one of its common oxidation products, 8-oxoG, is dramatically more reactive due to its low redox potential ( similar to 0.6 V compared to 1.29 V vs NHE for guanosine). Thus, placement of an 8-oxoG:C pair into a DNA duplex yields little perturbation of DNA structure but significantly enhances DNA reactivity. Even though the one-electron oxidation of a DNA base may initially occur at a distant site, rapid electron transfer in the duplex will result in exclusive formation of [8-oxoG] super(+ times ). The ultimate fate of [8-oxoG] super(+ times ) appears to mimic the urate oxidation pathway leading, via 5-hydroxy-8-oxoG, to a guanidi nohydantoin moeity. This mechanism likely involves trapping of the initially formed radical cation by a solvent water molecule; in the presence of bound protein, we reasoned that an active site nucleophile might participate instead, leading to a covalent DNA-protein cross-link analogous to 5-hydroxy-8-oxoguanosine. The E. coli DNA repair enzyme MutY provides an ideal system in which to test this cross-linking hypothesis. MutY binds to 8-oxoG:A and G:A mispairs in duplex DNA and catalyzes deglycosylation of the 2'-deoxyadenosine. Importantly, release of the product is slow, providing a relatively long-lived E times P complex. The high mutagenicity of 8-oxoG stems from the misincorporation of A to form 8- oxoG:A mispairs, and the activity of MutY is therefore critical for prevention of deleterious DNA mutations. Thus, specific 8-oxoG cross linking will also provide insight into the amino acid residues involved in the recognition of damaged and mismatched DNA by MutY.