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PDBsum entry 2bdp
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Transferase/DNA
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PDB id
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2bdp
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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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Visualizing DNA replication in a catalytically active bacillus DNA polymerase crystal.
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Authors
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J.R.Kiefer,
C.Mao,
J.C.Braman,
L.S.Beese.
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Ref.
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Nature, 1998,
391,
304-307.
[DOI no: ]
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PubMed id
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Abstract
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DNA polymerases copy DNA templates with remarkably high fidelity, checking for
correct base-pair formation both at nucleotide insertion and at subsequent DNA
extension steps. Despite extensive biochemical, genetic and structural studies,
the mechanism by which nucleotides are correctly incorporated is not known. Here
we present high-resolution crystal structures of a thermostable bacterial
(Bacillus stearothermophilus) DNA polymerase I large fragments with DNA primer
templates bound productively at the polymerase active site. The active site
retains catalytic activity, allowing direct observation of the products of
several rounds of nucleotide incorporation. The polymerase also retains its
ability to discriminate between correct and incorrectly paired nucleotides in
the crystal. Comparison of the structures of successively translocated complexes
allows the structural features for the sequence-independent molecular
recognition of correctly formed base pairs to be deduced unambiguously. These
include extensive interactions with the first four to five base pairs in the
minor groove, location of the terminal base pair in a pocket of excellent steric
complementarity favouring correct base-pair formation, and a conformational
switch from B-form to underwound A-form DNA at the polymerase active site.
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Figure 1.
Figure 1 Structure of the Bacillus fragment with duplex DNA
bound at the polymerase active site. The Bacillus fragment
molecular surface is coloured according to its proximity to the
DNA, with all points less than 3.5 ? coloured magenta, between
3.5 and 5.0 ? yellow, and greater than 5 ? blue. Bound water
molecules were not included in this calculation.
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Figure 4.
Figure 4 Polymerase active site with observed DNA and
modelled dTTP. The position of dTTP (violet) was based on the
 -polymerase
complex18, adjusted such that the base ring stacks with the
primer and one oxygen from each phosphate group was within 3 ?
of the observed metal ion (gold). The sugar pucker of the primer
terminus was made C3'-endo, which shifted its 3'-OH to within
1.7 ? of the modelled  -phosphate
of the dTTP. A second metal ion (violet) was modelled to be
within 3 ? of the 3'-OH of the primer, the -phosphate
group, and residues Asp 830 and Glu 831. The observed 5'
template overhang cannot accept an incoming dNTP without a
conformational change of the O helix.
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The above figures are
reprinted
by permission from Macmillan Publishers Ltd:
Nature
(1998,
391,
304-307)
copyright 1998.
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Secondary reference #1
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Title
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Crystal structure of a thermostable bacillus DNA polymerase i large fragment at 2.1 a resolution.
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Authors
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J.R.Kiefer,
C.Mao,
C.J.Hansen,
S.L.Basehore,
H.H.Hogrefe,
J.C.Braman,
L.S.Beese.
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Ref.
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Structure, 1997,
5,
95.
[DOI no: ]
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PubMed id
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Figure 3.
Figure 3. Comparison of 3'-5' exonuclease active sites.
Stereo diagram of the BF polymerase vestigial exonuclease active
site (red) with the position of a portion of the structure of
the KF active site (gold) [4] superimposed. The KF Ca backbone
schematic is accompanied by is two bound zinc atoms (green), and
three nucleotides (black) from the KF editing complex [11]. The
KF residues shown (yellow) are the four residues that bind the
two metal ions essential for catalysis. These essential KF
sidechains Asp355, Glu357, Asp424, and Asp501 correspond to BF
residues Val319, Glu321, Ala376, and Lys450, respectively (shown
in blue). Also shown in blue are two BF proline residues (438
and 441) that may be responsible for the collapse of a loop
between helices E[1] and F (dotted line) into the exonuclease
cleft not observed in KF. (Drawn with RIBBONS [71].)
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The above figure is
reproduced from the cited reference
with permission from Cell Press
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