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J. Biol. Chem., Vol. 275, Issue 51, 40266-40272, December 22, 2000
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From the Joseph Gottstein Memorial Cancer Laboratory, Department of
Pathology, University of Washington School of Medicine,
Seattle, Washington 98195-7705
DNA and RNA polymerase exhibit similarities in
structures and catalytic mechanisms, suggesting that both classes of
enzymes are evolutionarily related. To probe the biochemical and
structure-function relationship between the two classes of 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 of 8000) was tested for the ability
to incorporate successive ribonucleotides; 23 unique mutants that added
rNTPs into a growing polynucleotide chain were identified and
sequenced. These mutants, each containing one to four substitutions,
incorporate ribonucleotides at a efficiency approaching
103-fold greater than that of wild type Taq pol
I. Several mutants added successive ribonucleotides and thus can
catalyze the synthesis of RNA. Sequence analysis of these mutants
demonstrates that at least two amino acid residues are involved in
excluding ribonucleotides from the active site. Interestingly, wild
type DNA polymerases from several distinct families selectively
discriminate against rUTP. This study suggests that current DNA and RNA
polymerases could have evolved by divergent evolution from an ancestor
that shared a common mechanism for polynucleotide synthesis.
Multiple Amino Acid Substitutions Allow DNA Polymerases to
Synthesize RNA*
*
This work was supported by Medical Scientist Training
Program Grant 5T3207266 from the National Institutes of Health,
National Institute of General Medical Sciences (to P. H. P.)
and 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. Fax:
206-543-3967; E-mail: laloeb@u.washington.edu.
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