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The refined crystal structures of the large proteolytic fragment (Klenow
fragment) of Escherichia coli DNA polymerase I and its complexes with a
deoxynucleoside monophosphate product and a single-stranded DNA substrate offer
a detailed picture of an editing 3'-5' exonuclease active site. The structures
of these complexes have been refined to R-factors of 0.18 and 0.19 at 2.6 and
3.1 A resolution respectively. The complex with a thymidine tetranucleotide
complex shows numerous hydrophobic and hydrogen-bonding interactions between the
protein and an extended tetranucleotide that account for the ability of this
enzyme to denature four nucleotides at the 3' end of duplex DNA. The structures
of these complexes provide details that support and extend a proposed two metal
ion mechanism for the 3'-5' editing exonuclease reaction that may be general for
a large family of phosphoryltransfer enzymes. A nucleophilic attack on the
phosphorous atom of the terminal nucleotide is postulated to be carried out by a
hydroxide ion that is activated by one divalent metal, while the expected
pentacoordinate transition state and the leaving oxyanion are stabilized by a
second divalent metal ion that is 3.9 A from the first. Virtually all aspects of
the pretransition state substrate complex are directly seen in the structures,
and only very small changes in the positions of phosphate atoms are required to
form the transition state.
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