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J. Biol. Chem., Vol. 276, Issue 7, 5044-5051, February 16, 2001
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From the Joseph Gottstein Memorial Cancer Laboratory,
Departments of Pathology and Biological Structure, University of
Washington School of Medicine, Seattle, Washington 98195
DNA polymerases contain active sites that are
structurally superimposable and conserved in amino acid sequence. To
probe the biochemical and structure-function relationship of DNA
polymerases, a large library (200,000 members) of mutant Thermus
aquaticus DNA polymerase I (Taq pol I) was created
containing random substitutions within a portion of the dNTP binding
site (Motif A; amino acids 605-617), and a fraction of all selected
active Taq pol I (291 out of 8000) was tested for base
pairing fidelity; seven unique mutants that efficiently misincorporate
bases and/or extend mismatched bases were identified and sequenced.
These mutants all contain substitutions of one specific amino acid,
Ile-614, which forms part of the hydrophobic pocket that binds the base
and ribose portions of the incoming nucleotide. Mutant Taq
pol Is containing hydrophilic substitution I614K exhibit 10-fold lower
base misincorporation fidelity, as well as a high propensity to extend
mispairs. In addition, these low fidelity mutants containing
hydrophilic substitution for Ile-614 can bypass damaged templates that
include an abasic site and vinyl chloride adduct ethenoA. During
polymerase chain reaction, Taq pol I mutant I614K exhibits
an error rate that is >20-fold higher relative to the wild-type enzyme
and efficiently catalyzes both transition and transversion errors.
These studies have generated polymerase chain reaction-proficient
mutant polymerases containing substitutions within the active site that
confers low base pairing fidelity and a high error rate.
Considering the structural and sequence conservation of Motif A,
it is likely that a similar substitution will yield active low fidelity
DNA polymerases that are mutagenic.
A Single Highly Mutable Catalytic Site Amino Acid Is Critical for
DNA Polymerase Fidelity*
*
This work was supported by Medical Scientist Training
Program Grant NIH NIGMS5T3207266 and Molecular Training Program in
Cancer Research Grant CA09437 (to P. H. P.) and by National Cancer
Institute Grants R35 CA39903 and CA78885 (to L. A. L.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 206-543-6015;
Fax: 206-543-3967; E-mail: laloeb@u.washington.edu.
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