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PDBsum entry 2c2d
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References listed in PDB file
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Key reference
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Title
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Efficient and high fidelity incorporation of dctp opposite 7,8-Dihydro-8-Oxodeoxyguanosine by sulfolobus solfataricus DNA polymerase dpo4.
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Authors
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H.Zang,
A.Irimia,
J.Y.Choi,
K.C.Angel,
L.V.Loukachevitch,
M.Egli,
F.P.Guengerich.
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Ref.
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J Biol Chem, 2006,
281,
2358-2372.
[DOI no: ]
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PubMed id
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Note In the PDB file this reference is
annotated as "TO BE PUBLISHED".
The citation details given above were identified by an automated
search of PubMed on title and author
names, giving a
perfect match.
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Abstract
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DNA polymerases insert dATP opposite the oxidative damage product
7,8-dihydro-8-oxodeoxyguanosine (8-oxoG) instead of dCTP, to the extent of >90%
with some polymerases. Steady-state kinetics with the Y-family Sulfolobus
solfataricus DNA polymerase IV (Dpo4) showed 90-fold higher incorporation
efficiency of dCTP > dATP opposite 8-oxoG and 4-fold higher efficiency of
extension beyond an 8-oxoG:C pair than an 8-oxoG:A pair. The catalytic
efficiency for these events (with dCTP or C) was similar for G and 8-oxoG
templates. Mass spectral analysis of extended DNA primers showed >/=95%
incorporation of dCTP > dATP opposite 8-oxoG. Pre-steady-state kinetics showed
faster rates of dCTP incorporation opposite 8-oxoG than G. The measured
K(d)(,dCTP) was 15-fold lower for an oligonucleotide containing 8-oxoG than with
G. Extension beyond an 8-oxoG:C pair was similar to G:C and faster than for an
8-oxoG:A pair, in contrast to other polymerases. The E(a) for dCTP insertion
opposite 8-oxoG was lower than for opposite G. Crystal structures of Dpo4
complexes with oligonucleotides were solved with C, A, and G nucleoside
triphosphates placed opposite 8-oxoG. With ddCTP, dCTP, and dATP the
phosphodiester bonds were formed even in the presence of Ca(2+). The 8-oxoG:C
pair showed classic Watson-Crick geometry; the 8-oxoG:A pair was in the syn:anti
configuration, with the A hybridized in a Hoogsteen pair with 8-oxoG. With dGTP
placed opposite 8-oxoG, pairing was not to the 8-oxoG but to the 5' C (and in
classic Watson-Crick geometry), consistent with the low frequency of this
frameshift event observed in the catalytic assays.
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Figure 1.
Extension of primers by Dpo4. Extension of a ^32P-labeled
primer (13-mer, oligomer 1 of Scheme 1) opposite G or 8-oxoG
(oligomer 2 of Scheme 1) was analyzed with increasing reaction
times, as indicated by the gradient bars (0, 10, 30, 60, 90,
120, and 180 min, respectively).
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Figure 8.
Quality of the electron density and DNA duplex conformations
at the active sites in the ternary complexes. A, Dpo4-dG; B,
Dpo4-dA; C, Dpo4-dC; D, Dpo4-ddG; E, Dpo4-ddC. The views are
into the major groove, Fourier (3F[o] - 2F[c]) sum electron
density is drawn at the 1σ level, Ca^2+ ions are yellow
spheres, and phosphorus atoms of the α-, β-, and γ-phosphate
groups of (d)dNTPs are highlighted in cyan, gray, and white,
respectively.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2006,
281,
2358-2372)
copyright 2006.
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