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PDBsum entry 2kzm
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Transferase/DNA
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PDB id
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2kzm
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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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Structures of normal single-Stranded DNA and deoxyribo-3'-S-Phosphorothiolates bound to the 3'-5' Exonucleolytic active site of DNA polymerase i from escherichia coli.
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Authors
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C.A.Brautigam,
S.Sun,
J.A.Piccirilli,
T.A.Steitz.
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Ref.
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Biochemistry, 1999,
38,
696-704.
[DOI no: ]
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PubMed id
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Abstract
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The interaction of a divalent metal ion with a leaving 3' oxygen is a central
component of several proposed mechanisms of phosphoryl transfer. In support of
this are recent kinetic studies showing that thiophilic metal ions (e.g., Mn2+)
stimulate the hydrolysis of compounds in which sulfur takes the place of the
leaving oxygen. To examine the structural basis of this phenomenon, we have
solved four crystal structures of single-stranded DNA's containing either oxygen
or sulfur at a 3'-bridging position bound in conjunction with various metal ions
at the 3'-5' exonucleolytic active site of the Klenow fragment (KF) of DNA
polymerase I from Escherichia coli. Two structures of normal ssDNA bound to KF
in the presence of Zn2+ and Mn2+ or Zn2+ alone were refined at 2.6- and 2.25-A
resolution, respectively. They serve as standards for comparison with other
Mn2+- and Zn2+-containing structures. In these cases, Mn2+ and Zn2+ bind at
metal ion site B in a nearly identical position to Mg2+ (Brautigam and Steitz
(1998) J. Mol. Biol. 277, 363-377). Two structures of KF bound to a
deoxyoligonucleotide that contained a 3'-bridging sulfur at the scissile
phosphate were refined at 2.03-A resolution. Although the bridging sulfur
compounds bind in a manner very similar to that of the normal oligonucleotides,
the presence of the sulfur changes the metal ion binding properties of the
active site such that Mn2+ and Zn2+ are observed at metal ion site B, but Mg2+
is not. It therefore appears that the ability of the bridging sulfur compounds
to exclude nonthiophilic metal ions from metal ion site B explains the low
activity of KF exonuclease on these substrates in the presence of Mg2+ (Curley
et al. (1997) J. Am. Chem. Soc. 119, 12691-12692) and that the 3'-bridging atom
of the substrate is influencing the binding of metal ion B prior to catalysis.
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Secondary reference #1
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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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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
reproduced from the cited reference
with permission from Elsevier
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