The adenine glycosylase MutY selectively initiates repair of OG:A lesions and, by comparison, avoids G:A mispairs. The ability to distinguish these closely related substrates relies on the Cterminal domain of MutY which structurally resembles MutT. To understand the mechanism for substrate specificity, we crystallized MutY in complex with DNA containing G across from the high-affinity azaribose transition state analog. Our structure shows that G is accommodated by the OG site and highlights the role of a serine residue in OG versus G discrimination. The functional significance of Ser308 and its neighboring residues was evaluated by mutational analysis, revealing the critical importance of a beta-loop in the C-terminal domain for mutation suppression in cells, and biochemical performance in vitro. This loop comprising residues Phe307, Ser308, and His309 (Geobacillus stearothermophilus sequence positions) is conserved in MutY but absent in MutT and other DNA repair enzymes, and may therefore serve as a MutY-specific target exploitable by chemical biological probes.Aberrant DNA modifications that arise from chemical reactions with exogenous and endogenous agents are considered "DNA damage" since these modifications put biological systems at risk. DNA repair enzymes mitigate this risk by counteracting chemical damage that otherwise would erode information content of DNA. 1 Guanine is particularly prone to oxidative damage due to its low redox potential. 2 Oxidation of G results in 8-oxo-7,8dihydroguanine (OG) which differs from G by only two atoms (Figure 1). The OG lesion is especially problematic because the syn conformer mispairs with adenine during DNA replication. The guanine oxidation (GO) repair pathway prevents mutations that otherwise would arise from OG template ambiguity (Figure 2). The GO repair pathway features ‡
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.
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