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PDBsum entry 2nol
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Hydrolase, lyase/DNA
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PDB id
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2nol
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References listed in PDB file
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Key reference
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Title
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Structural characterization of human 8-Oxoguanine DNA glycosylase variants bearing active site mutations.
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Authors
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C.T.Radom,
A.Banerjee,
G.L.Verdine.
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Ref.
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J Biol Chem, 2007,
282,
9182-9194.
[DOI no: ]
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PubMed id
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Abstract
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The human 8-oxoguanine DNA glycosylase (hOGG1) protein is responsible for
initiating base excision DNA repair of the endogenous mutagen 8-oxoguanine. Like
nearly all DNA glycosylases, hOGG1 extrudes its substrate from the DNA helix and
inserts it into an extrahelical enzyme active site pocket lined with residues
that participate in lesion recognition and catalysis. Structural analysis has
been performed on mutant versions of hOGG1 having changes in catalytic residues
but not on variants having altered 7,8-dihydro-8-oxoguanine (oxoG) contact
residues. Here we report high resolution structural analysis of such recognition
variants. We found that Ala substitution at residues that contact the phosphate
5' to the lesion (H270A mutation) and its Watson-Crick face (Q315A mutation)
simply removed key functionality from the contact interface but otherwise had no
effect on structure. Ala substitution at the only residue making an
oxoG-specific contact (G42A mutation) introduced torsional stress into the DNA
contact surface of hOGG1, but this was overcome by local interactions within the
folded protein, indicating that this oxoG recognition motif is
"hardwired." Introduction of a side chain intended to sterically
obstruct the active site pocket (Q315F mutation) led to two different
structures, one of which (Q315F(*149)) has the oxoG lesion in an exosite
flanking the active site and the other of which (Q315F(*292)) has the oxoG
inserted nearly completely into the lesion recognition pocket. The latter
structure offers a view of the latest stage in the base extrusion pathway yet
observed, and its lack of catalytic activity demonstrates that the transition
state for displacement of the lesion base is geometrically demanding.
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Figure 2.
FIGURE 2. Effect of the H270A mutation. A, view of the
active site region of the H270A structure (side chains in teal
and DNA backbone in gold) showing four water molecules (green
spheres) that coordinate the 5'-phosphate of oxoG to the protein
with dashed lines denoting hydrogen bonds. B, least squares
superposition showing the active site region of the H270A
structure (colored as in A) with that of the proximally
cross-linked recognition complex (LRC^*149; Protein Data Bank
code 1YQR (28)) (white side chains and DNA backbone).
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Figure 3.
FIGURE 3. Role of Gly-42 and consequences of the G42A
mutation. A, stereoview of the G42A complex (protein chain in
teal except for Ala-42, which is shown in magenta; DNA backbone
in gold; and waters as green spheres) showing extensive hydrogen
bonding interactions among residues of the Gly-42 loop and
surrounding residues. B, schematic detailing the interactions
from A. C, superposition of the G42A complex (colored as in A)
with the LRC (protein and DNA in white and waters in gray).
Inset, close-up view of the G42A complex showing the near
eclipsing interaction of Ala-42 and the hydrogen bond formed
between N7-H of oxoG and the carbonyl oxygen of Ala-42.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2007,
282,
9182-9194)
copyright 2007.
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