B.
stearothermophilus Adenine DNA
Glycosylase Protein MutY
Elizabeth Bailey '12 and Jonathan Weil '11
Contents:
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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