Trapping and structural elucidation of a very advanced intermediate in the lesion-extrusion pathway of hOGG1

Article abstract of DOI:10.1021/ja800821t

Here we present the first structure of a very advanced intermediate in the lesion-extrusion pathway of a DNA glycosylase, human 8-oxoguanine DNA glycosylase (hOGG1), and a substrate DNA containing a mutagenic lesion, 8-oxoguanine (oxoG). The structure was

Full text of DOI:10.1021/ja800821t

Published on Web 05/29/2008
Trapping and Structural Elucidation of a Very Advanced Intermediate in the
Lesion-Extrusion Pathway of hOGG1
Seongmin Lee, Christopher T. Radom, and Gregory L. Verdine*
Department of Chemistry and Chemical Biology, HarVard UniVersity, Cambridge, Massachusetts 02138
Received February 1, 2008; E-mail: verdine@chemistry.harvard.edu
DNA glycosylases, key components of the base-excision DNA
repair pathway,1,2 recognize damaged nucleobases in DNA and
catalyze their excision. Structural studies have revealed that
structurally diverse DNA glycosylases share the common feature
of extruding their target lesion from DNA and inserting it into an
extrahelical active site pocket on the enzyme; only after this does
catalysis ensue.3 We have shown that this extrusion process
proceeds via a multistep pathway characterized by discrete pre-
and postextrusion intermediates.4a In a series of structural studies,4
we have captured human 8-oxoguanine DNA glycosylase (hOGG1)
bound to DNA at different stages along the extrusion pathway for
its cognate lesion, the mutagen 8-oxoguanine (oxoG).5 None of
these studies, however, revealed a catalytically active hOGG1
presenting oxoG to the active site. Here we report the first synthesis
of a photocaged analogue of oxoG, and the use of this derivative
in combination with flash-cryotrapping to capture and structurally
Figure 1. Overview of the experimental strategy
characterize the most advanced intermediate yet observed along
Scheme 1. Preparation of DNA Containing a Photocaged oxoG a
any base-extrusion pathway. The structure reveals that insertion of
the lesion nucleobase into the enzyme active site occurs much faster
than subsequent rearrangements in active site side-chains that
produce a catalytically active state.
In previous work,4a we used intermolecular disulfide cross-linking
(DXL) technology6 to capture hOGG1 attempting to present the
normal base G to its active site pocket. The structure of this
G-interrogation complex revealed that the target G, though fully
extrahelical, failed to insert into the active site pocket, and instead
resided in a nearby ‘exosite’. We reasoned that an oxoG base affixed
with a photocleavable group at the O6-position might similarly be
denied entry in the lesion-recognition pocket, owing to a steric clash
between the bulky photocleavable group and active site residues
(Figure 1). If so, then photodeprotection would liberate the oxoG
and thereby trigger the final sequence of the base-extrusion pathway,
namely transit of the oxoG from the exosite to the active site. We
hoped that irradiation of the caged complex followed by flash-
freezing might lead to the cryogenic trapping of an intermediate in
either base-extrusion or in base-excision. To stabilize the otherwise
unstable complex formed between hOGG1 and DNA containing
the photocaged-oxoG DNA complex, we employed DXL technol-
ogy, just as with the G-complex and other extrusion intermediates
(Figure 1).4
a Reagents and conditions: (a) Ac2O, DMAP, MeCN, 25 °C, 3 h; (b)
PacCl, pyridine, 0 °C; (c) NBS, pyridine (2 equiv), MeCN, 25 °C; (d) PPh3,
DEAD, 2-(2-nitrophenyl)propanol, THF, 25 °C; (e) NaOAc, AcOH, 80 °C;
(f) 1 N NaOH, EtOH, 0 °C; (g) DMTrCl, pyridine, 25 °C; (h) 4,5-
dicyanoimidazole, 2-cyanoethyltetraisopropyl phosphordiamidite, CH2Cl2,
25 °C; (i) solid phase DNA synthesis.
O6-photocaged oxoG). The oligonucleotide was deprotected with
methanolic potassium carbonate, purified by urea-PAGE gel elec-
trophoresis, and its structure was verified by MALDI-TOF analysis.
The photocaged nucleotide was annealed to a complementary strand,
5′-TGGTAGACCTGGACGG containing a site-specific thiol-tether
(indicated by C), and the duplex was DXLed to hOGG1 containing
an engineered Cys at position-149.4 Following purification and
concentration, the DXLed complex containing O6-photocaged oxoG
(designated photocaged complex or PCC) was crystallized by the
hanging droplet vapor diffusion method. A base-excision assay
performed on these crystals unambiguously revealed the presence
of an intact O6-photocaged oxoG, with no evidence of processing
by hOGG1 (see Supporting Information).
The synthesis of O6-photocaged oxoG DNA began with O-
acetylation of 2′-deoxyguanosine 1 (Scheme 1). Protection of the
N2-amine followed by bromination provided 8-bromo-2′-deoxy
guanosine 2. Mitsonobu coupling gave the O6-o-nitrophenisopropyl
ether 3 (as a mixture of stereoisomers); this was converted to 8-oxo-
2′-deoxyguanosine 4 by treatment with sodium acetate. Following
removal of the 3′ and 5′-acetyl groups, the free nucleoside (not
shown) was elaborated into the corresponding O6-photocaged oxoG
phosphoramidite 5, which was then employed in the solid phase
synthesis of 6, a 16-mer oligodeoxynucleotide bearing a single O6-
photocaged 8-oxoG residue (5′-AGCGTCCAXGTCTACC-3′; X )
The structure of the PCC is similar overall to that of the
G-interrogation complex,4a having an extrahelical target nucleobase
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10.1021/ja800821t CCC: $40.75
2008 American Chemical Society

C O M M U N I C A T I O N S
the side chain of the catalytic nucleophile Lys249 is disordered at
its distal end in the FC and does not appear to be engaged in the
key salt bridge with Cys253 [Lys249(NH3+)/Cys253(S-)] impli-
cated computationally4a in oxoG recognition. These differences
between the FC and LRC are not limited to side-chain motions.
For example, the R-O helix, which bears three active site residues,
Gln315, Phe319, and Asp322, is retracted from the active site in
the FC (Figure 2D).
The DNA conformation in the FC is also perturbed relative to
the LRC. On the 5′-side of the oxoG lesion, the DNA backbone in
that region would require a crankshaft conformational shift about
the oxoG C4′-C5′-O5′-P5′ to move into position for a productive
contact with His270 (denoted by curved arrow in Figure 2C). The
ribose ring of oxoG is shifted slightly in position, as is the
catalytically essential residue Asp268.
The simplest explanation for the structural features of the FC is
that it represents a very advanced intermediate in the lesion-
extrusion pathway, indeed the most advanced thus far observed
for any DNA glycosylase, with the oxoG nucleobase having
undergone nearly complete insertion into the active site, but the
protein and to some extent the DNA having not yet adopted their
final, catalytically productive conformations.8 These observations
thus suggest that the transit of the lesion from the exosite to the
active site occurs faster than the subsequent round of protein
conformational adjustments required to produce a catalytically
competent active site.
Figure 2. (A) Active site view of the hOgg1/oxoG lesion-recognition
complex (LRC), with the protein backbone in light gray, side chains in
green, oxoG in pink, and DNA in pale yellow. An ordered water is shown
as a pink sphere. Noteworthy hydrogen bonding interactions are indicated
by a dashed line. (B) Active site view of the FC (this work), with the protein
backbone in dark gray; side chains, dark green; oxoG, red; DNA, gold;
water, red sphere. (C) Heavy atom superposition of the FC with the LRC
(color scheme as in B and A, respectively). Curved arrows denote bonds
that have undergone substantial rotations. (D) Superposition as in C, but
from a perspective that emphasizes the different positions of the R-O helix.
Dotted line and distances illustrate the positional shifts at the CR carbons
at the residues indicated in red.
Acknowledgment. This work was supported by the NIH (CA
100742). X-ray data were collected at the 19-ID beamline at the
Structural Biology Center of the Advanced Photon Source of
Argonne National Laboratory. We are grateful to Brian Bowman
for helpful advice with crystallography and Paul Blainey for
assistance with the laser apparatus construction.
not bound in the active site, except that in the PCC the density for
the caged oxoG is weak, suggesting the caged oxoG is conforma-
tionally mobile in the exosite (Supporting Information). To obtain
the structure of this complex following photodeprotection (desig-
nated flashed complex or FC), crystals of the PCC were irradiated
with 373 nm laser light for 30 s at 4 °C and immediately
cryotrapped by being plunged into liquid nitrogen. The structure
of the FC, solved to 2.8 Å resolution, reveals clear electron density
for the oxoG nucleobase, but none corresponding to the photocaging
group, consistent with the results of base-excision assays showing
that 30 s irradiation caused >90% removal of the photocaging group
(see Supporting Information). Remarkably, the oxoG lesion in the
FC is inserted into the active site pocket in roughly the same
position as in the hOgg1/oxoG lesion-recognition complex (LRC),
which is believed to represent a state poised for catalysis (compare
Figure 2 panels A and B, overlays in C and D). Indeed, the hallmark
specificity-determining contact of oxoG recognitionsthe hydrogen
bonding contact between N7-H of oxoG and the main-chain
carbonyl oxygen of Gly42sis evidently present in the FC, though
it is significantly longer than that observed in the LRC (3.4 vs 2.8
Å, respectively). Apart from this one signature contact, however,
none of the other active site contacts to the oxoG always observed
in LRC structures is formed in the FC. Three key residues known
structurally and biochemically7 to play an important role in
contacting oxoG, namely Phe319, Cys253, Gln315, are all dislodged
in the FC from their positions in the LRC. In addition, the contact
between His270 and the oxoG 5′-phosphate is disrupted in the FC,
and instead His270 stacks with Phe319. The hydrogen bonding
interaction between His270 and Asp322 in the FC is not observed
in the LRC, but is seen in earlier extrusion intermediates. Finally,
Supporting Information Available: Difference electron density
map, photodeprotection assay, synthetic procedures. This material is
available free of charge via the Internet at http://pubs.acs.org.
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