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PDBsum entry 1xwl
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DNA replication
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PDB id
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1xwl
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References listed in PDB file
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Key reference
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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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Abstract
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BACKGROUND: The study of DNA polymerases in the Pol l family is central to the
understanding of DNA replication and repair. DNA polymerases are used in many
molecular biology techniques, including PCR, which require a thermostable
polymerase. In order to learn about Pol I function and the basis of
thermostability, we undertook structural studies of a new thermostable DNA
polymerase. RESULTS: A DNA polymerase large, Klenow-like, fragment from a
recently identified thermostable strain of Bacillus stearothermophilus (BF) was
cloned, sequenced, overexpressed and characterized. Its crystal structure was
determined to 2.1 A resolution by the method of multiple isomorphous
replacement. CONCLUSIONS: This structure represents the highest resolution view
of a Pol I enzyme obtained to date. Comparison of the three Pol I structures
reveals no compelling evidence for many of the specific interactions that have
been proposed to induce thermostability, but suggests that thermostability
arises from innumerable small changes distributed throughout the protein
structure. The polymerase domain is highly conserved in all three proteins. The
N-terminal domains are highly divergent in sequence, but retain a common fold.
When present, the 3'-5' proofreading exonuclease activity is associated with
this domain. Its absence is associated with changes in catalytic residues that
coordinate the divalent ions required for activity and in loops connecting
homologous secondary structural elements. In BF, these changes result in a
blockage of the DNA-binding cleft.
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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
reprinted
by permission from Cell Press:
Structure
(1997,
5,
95-0)
copyright 1997.
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Secondary reference #1
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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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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
reproduced from the cited reference
with permission from Macmillan Publishers Ltd
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