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B. stearothermophilus Adenine DNA Glycosylase Protein MutY

Elizabeth Bailey '12 and Jonathan Weil '11


I. Introduction

The mutated base 8-oxoguanine (oxoG) has the potential to induce errors in DNA replication. DNA polymerase misreads the oxoG and pairs it with an adenine residue instead of the necessary cytosine residue (oxoG vs thymine). If left uncorrected, this mispair can lead to permanent mutations following the next round of replication. The adenine DNA glycosylase protein, MutY, has the vital function of removing the mispaired A of the oxoG:A base pair. A sequence specific glycosylase, MutY recognizes oxoG and cleaves the mispaired adenine. MutY uses base flipping to twist the mispaired adenine out of the DNA helix and into MutY’s active site. Such adenine DNA glycosylases are found in both bacteria and humans (called MutY and nMYH, respectivly). This tutorial presents the structure of MutY from Bacillus stearothermophilus bound to DNA and inorganic pyrophosphate.

II. General Structure

MutY is composed of 2 distinct domains: the C-terminal domain and the catalytic domain . The C-terminal domain of MutY contacts the oxoG containing DNA strand and the catalytic domain. The catalytic domain is composed of the 6-helix barrel module and the 4Fe-4S module . The C-terminal and catalytic domains are connected by an extended linker that transverses the DNA major groove near the lesion. The linker has beneficial electrostatic interactions with the phosphate backbone because it is rich in positively-charged lysines (Lys 228, 230, 231, 235). The contacts between the C-terminal domain and the catalytic region allow MutY to encircle the DNA. The adenine is completely extruded from the DNA helix, while the modified guanine (oxoG) remains within the normal DNA structure. The adenine is held within a pocket in the catalytic domain of the protein. As with other members of the structural superfamily base excision and repair proteins, the catalytic domain in MutY contains a signature helix-hairpin-helix element, followed by a Gly/Pro-rich loop and a catalyticly essential Asn 144.  

III. Interactions with 8-Oxoguanine

The oxoG is found in the syn conformation when bound to adenine prior to association with MutY. OxoG’s glycosidic bond rotates 180° into the anti conformation in allow MutY to bind (OxoG syn vs. anti conformation). This rotation is thought to force the adenine to flip out of the helix since the anti oxoG would clash sterically with the adenine. Tyr 88 is intercalated into the DNA on the 5' side of OxoG, which nearly doubles the distance between OxoG and its 5' neighbor. The side chain of Gln 48 is inserted into the space vacated by the extruded adenine and forms pi-stacking interactions with the 3'-neighboring base and a hydrogen bond to the phosphate 3' to the substrate adenine. The R-subunit of Gln 48 forms multiple direct and indirect stabilizing bonds to Tyr 88, Ser 308, and the oxoG purine ring. OxoG is also contacted by the main chain amide carbonyl of Gln 48. These contacts are further stabilized by interactions with the side chain hydroxyl of Thr 49. A final pairs of contacts are contributed by the main chain carbonyl of Leu 86 and the main chain amide of Ser 308. The Ser 308 residue is responsible for MutY’s discrimination between oxoguanine and guanine. If MutY bound to a guanine, lost contacts would include the hydrogen bonding of Ser 308’s N-H to oxoG’s O8, as well as the extensive network between Ser 308, Tyr 88, and Gln 48.

IV. Adenine Binding and Excision Reaction

MutY removes the mispaired adenine by a dissociative SN1-like reaction mechanism . The reaction has two distinct steps: breaking of the gylcosidic bond and nucleophilic attack by water. MutY is able to catalyze this reaction by stabilizing the reaction intermediates. Asn 144 is located near of the O4' of the adenine sugar and stabilizes the charged ion intermediate in the base-excision step. The water below the C1' of the adenine sugar functions as the nucleophile which reacts with the charged intermediate at C1' to form the final product. The second water molecule hydrogen bonds to the adenine N7, stabilizing the base leaving group. Both water molecules have hydrogen bond contacts with the Glu 43 side chain. Glu 43 functions as a catalyst by accepting a proton then transferring it to N7 via the water-bound intermediate, effectively lowering the transition state energy for the base excision.

V. References

Fromme, J. C.; Banerjee, A.; Huang, S. J.; and G. L Verdine. 2004. Structural basis for removal of adenine mispaired with 8-oxoguanine by MutY adenine DNA glycosylase. Nature 427:652-656.

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