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PDBsum entry 1kfs
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Transferase/DNA
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PDB id
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1kfs
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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 principles for the inhibition of the 3'-5' Exonuclease activity of escherichia coli DNA polymerase i by phosphorothioates.
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Authors
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C.A.Brautigam,
T.A.Steitz.
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Ref.
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J Mol Biol, 1998,
277,
363-377.
[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
percentage match of
95%.
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Abstract
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A two-metal-ion catalytic mechanism has previously been proposed for several
phosphoryl-transfer enzymes. In order to extend the structural basis of this
mechanism, crystal structures of three single-stranded DNA substrates bound to
the 3'-5' exonucleolytic active site of the large fragment of DNA polymerase I
from Escherichia coli have been elucidated. The first is a 2.1 A resolution
structure of a Michaelis complex between the large fragment (or Klenow fragment,
KF) and a single-stranded DNA substrate, stabilized by low pH and
flash-freezing. The positions and identities of the catalytic metal ions, a Zn2+
at site A and a Mg2+ at site B, have been clearly established. The structural
and kinetic consequences of sulfur substitutions in the scissile phosphate have
been explored. A complex with the Rp isomer of phosphorothioate DNA, refined at
2.2 A resolution, shows Zn2+ bound to both metal sites and a mispositioning of
the substrate and attacking nucleophile. The complex with the Sp
phosphorothioate at 2. 3 A resolution reveals that metal ions do not bind in the
active site, having been displaced by a bulky sulfur atom. Steady-state kinetic
experiments show that catalyzed hydrolysis of the Rp isomer was reduced only
about 15-fold, while no enzyme activity could be detected with the Sp
phosphorothioate, consistent with the structural observations. Furthermore, Mn2+
could not rescue the activity of the exonuclease on the Sp phosphorothioate.
Taken together, these studies confirm and extend the proposed two-metal-ion
exonuclease mechanism and provide a structural context to explain the effects of
sulfur substitutions on this and other phosphoryl-transfer enzymes. These
experiments also suggest that the possibility of metal-ion exclusion be taken
into account when interpreting the results of Mn2+ rescue experiments.
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Figure 2.
Figure 2. the configurations of the oxygen (or sulfur)
atoms about the scissile phosphate of normal or phos-
phorothioate DNA. The phosphates are shown in the
orientation that will occur in all other Figures. (a) Nor-
mal, or all-oxygen phosphate. The pro-R and pro-S pos-
itions are marked. The negative charge is distributed
between the non-bridging oxygens. (b) Rp phosphor-
othioate phosphate. Note that the sulfur atom has only
a single covalent bond to the phosphorus atom and is
negatively charged. The pro-S oxygen features a double
bond to phosphorus. (c) The Sp phosphorothioate.
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Figure 5.
Figure 5. Schematic drawing of the
R isomer structure. The same color-
ing scheme as in Figure 4 is used,
with the pro-R sulfur and ``attack''
water highlighted in yellow and
purple, respectively. The two Zn
ions are about 4.0 Å apart.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(1998,
277,
363-377)
copyright 1998.
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Secondary reference #1
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Title
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Structural basis for the 3'-5' Exonuclease activity of escherichia coli DNA polymerase i: a two metal ion mechanism.
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Authors
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L.S.Beese,
T.A.Steitz.
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Ref.
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Embo J, 1991,
10,
25-33.
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PubMed id
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