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PDBsum entry 1tk0
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Transferase/electron transport/DNA
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PDB id
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1tk0
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Contents |
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* Residue conservation analysis
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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 basis for the dual coding potential of 8-Oxoguanosine by a high-Fidelity DNA polymerase.
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Authors
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L.G.Brieba,
B.F.Eichman,
R.J.Kokoska,
S.Doublié,
T.A.Kunkel,
T.Ellenberger.
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Ref.
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EMBO J, 2004,
23,
3452-3461.
[DOI no: ]
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PubMed id
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Abstract
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Accurate DNA replication involves polymerases with high nucleotide selectivity
and proofreading activity. We show here why both fidelity mechanisms fail when
normally accurate T7 DNA polymerase bypasses the common oxidative lesion
8-oxo-7, 8-dihydro-2'-deoxyguanosine (8oG). The crystal structure of the
polymerase with 8oG templating dC insertion shows that the O8 oxygen is
tolerated by strong kinking of the DNA template. A model of a corresponding
structure with dATP predicts steric and electrostatic clashes that would reduce
but not eliminate insertion of dA. The structure of a postinsertional complex
shows 8oG(syn).dA (anti) in a Hoogsteen-like base pair at the 3' terminus, and
polymerase interactions with the minor groove surface of the mismatch that mimic
those with undamaged, matched base pairs. This explains why translesion
synthesis is permitted without proofreading of an 8oG.dA mismatch, thus
providing insight into the high mutagenic potential of 8oG.
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Figure 2.
Figure 2 T7 DNA polymerase bypass of an 8oG lesion. Primer
extension reactions were performed with exo- (left) and
wild-type (right) T7 DNA polymerase with undamaged guanine (G)
and 8-oxoguanine (8oG) in comparison to controls containing no
enzyme. The images shown are for 3 min incubations of reaction
mixtures containing 200- to 400-fold excess of DNA over
polymerase. The most intense band in each lane is unreacted
primer, at least 80% of which remains unextended for all
efficiency reactions performed in this study. The location of
8oG within the template strand is as indicated and enhanced
images of products using 8oG are shown to the right of the boxed
images. The probability of insertion at each template site,
listed in percent to the right of each lane, is an average of 7
-16 determinations and is calculated as described previously
(Kokoska et al, 2003).
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Figure 4.
Figure 4 Comparison of an open 8oG complex and a closed T  ddATP
insertion complex. The open 8oG complex (red) and a dT dATP
insertion complex (gray) were superimposed using C[  ]atoms.
The proteins are depicted as cylinders and the DNA as sticks.
Both structures are largely similar but they specifically differ
in the orientation adopted by their fingers subdomains. In the
closed structure,  -helices
O and O1 pack against the incoming ddATP (blue) and the template
thymine, respectively. In the open structure, the fingers move
outwards from the palm subdomain, as shown by the  45°
rotation of the O and O1 helices relative to the closed
conformation. Residue Tyr530, which moves to the position that
would correspond to the templating base of the closed complex,
has been omitted for clarity. The templating 8oG, the 5'
template strand, and residues 532 -536 located at the junction
between helices
O and O1 are disordered in the open complex. No interpretable
electron density is observed for the metal ions or incoming
nucleotide.
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The above figures are
reprinted
from an Open Access publication published by Macmillan Publishers Ltd:
EMBO J
(2004,
23,
3452-3461)
copyright 2004.
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