Unique Hydrogen Bonding of Adenine with the Oxidatively Damaged Base 8-Oxoguanine Enables Specific Recognition and Repair by DNA Glycosylase MutY

Abstract

The DNA glycosylase MutY prevents deleterious mutations resulting from guanine oxidation by recognition and removal of adenine (A) misincorporated opposite 8-oxo-7,8-dihydroguanine (OG). Correct identification of OG:A is crucial to prevent improper and detrimental MutY-mediatedadenine excision from G:A or T:A base pairs. Here we present a structure–activity relationship (SAR) study using analogues of A to probe the basis for OG:A specificity of MutY. We correlate observed in vitro MutY activity on A analogue substrates with their experimental and calculated acidities to provide mechanistic insight into the factors influencing MutY base excision efficiency. These data show that H-bonding and electrostatic interactions of the base within the MutY active site modulate the lability of the N-glycosidic bond. A analogues that were not excised from duplex DNA as efficiently as predicted by calculations provided insight into other required structural features, such as steric fit and H-bonding within the active site for proper alignment with MutY catalytic residues. We also determined MutY-mediated repair of A analogues paired with OG within the context of a DNA plasmid in bacteria. Remarkably, the magnitudes of decreased in vitro MutY excision rates with different A analogue duplexes do not correlate with the impact on overall MutY-mediated repair. The feature that most strongly correlated with facile cellular repair was the ability of the A analogues to H-bond with the Hoogsteen face of OG. Notably, base pairing of A with OG uniquely positions the 2-amino group of OG in the major groove and provides a means to indirectly select only these inappropriately placed adenines for excision. This highlights the importance of OG lesion detection for efficient MutY-mediated cellular repair. The A analogue SARs also highlight the types of modifications tolerated by MutY and will guide the development of specific probes and inhibitors of MutY.

Structural snapshots of base excision by the cancer-associated variant MutY N146S reveal a retaining mechanism

“…Indeed, Glu188 adopts a different rotamer away from the base that is not completely engaged in the LRC structure ( 39 ) as well as in the N146S-OG:P structure (Figure 4F ). The disengaged base position seen in the LRC and the N146S-OG:P structure described here suggests subtle changes in active site residues alter the proper alignment of the base, which is likely a key quality control mechanism by MutY, and is consistent with previous work that showed reduced activity with structural changes in A ( 35 ).…”

“…The purine substrate is structurally similar to adenine except for the lack of exocyclic amino group that alters base-pairing with OG, as well as potential contacts within the MutY active site. Purine has been previously reported to show a higher acid lability than adenine and is excised only ∼2-fold slower than adenine by WT Ec MutY ( 33 , 35 ) with a product release rate of 0.003 ± 0.001 min −1 ( 33 ). In contrast, the rate constants k 2 for purine removal by N146S Gs MutY and N140S Ec MutY revealed a much more dramatic reduction in the activities by ∼92-fold and ∼200-fold in comparison to the respective WT enzymes (Table 1 , Figure 2A&B ).…”

Functionalization of Mesoporous Silica with a G-A-Mismatched dsDNA Chain for Efficient Identification and Selective Capturing of the MutY Protein

“…In the dsDNA chain with a G-A mismatch, the mismatched adenine (A) base is extruded from the double-helix structure of the DNA chain and is bound firmly in the catalytic center pocket of the MutY protein via H-bond interactions with the amino acid residues of MutY, e.g., the N1 of adenine in the DNA chain, the Gln25 residue of MutY, the N7 of adenine, and the carboxyl group of the Glu37 residue/hydroxyl group of the Ser120 residue. At the same time, as for guanine (G), H-bonds are formed between the N1/N2 of guanine in the DNA chain and the Gln42/Thr43 residues, and the Leu80 main chain of the MutY protein. ,,− These multiple H-bonds form a strong and stable hydrogen-bond network, enabling the effective binding of the dsDNA chain with the MutY protein.…”

Gas-Phase Studies of Hypoxanthine-Guanine-(Xanthine) Phosphoribosyltransferase (HG(X)PRT) Substrates

“…The Pf HG­(X)­PRT purine binding site is formed by hydrophobic residues (Tyr116, Ile146, Phe197, Val198, and Leu203) . Prior studies have shown that gas-phase data lend insight into the reactivity in the nonpolar environment of enzyme active sites. − Specifically, it has been found that if the role of the enzyme is to provide a hydrophobic environment, then ease of reaction depends on the intrinsic lability of the substrate, and the gas-phase acidities and/or proton affinities (depending on mechanism) will track, trendwise, with enzyme rate. We therefore aimed to ascertain whether our gas-phase data could provide insight into the Pf HG­(X)­PRT mechanism.…”

“…Herein, we focus on the properties of HG­(X)­PRT substrates. Prior studies have shown that the examination of properties in the gas phase, which provides the “ultimate” nonpolar environment, uncovers intrinsic, inherent reactivity that correlates to activity in other nonpolar media, including hydrophobic enzyme active sites. − In this paper, we calculate and measure the gas-phase acidities and proton affinities (PA) of a series of Pf HG­(X)­PRT purine substrates not heretofore studied in vacuo . We also conduct energetics and kinetic isotope effect (KIE) calculations; all of these results are discussed in the context of the HG­(X)­PRT mechanism.…”