Random mutagenesis of Thermus aquaticus DNA polymerase I: Concordance of immutable sites in vivo with the crystal structure

Motoshi Suzuki, Dale Baskin, Leroy Hood, Lawrence A. Loeb

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Abstract

Expression of Thermus aquaticus (Taq) DNA polymerase I (pol I) in Escherichia coli complements the growth defect caused by a temperature- sensitive mutation in the host pol I. We replaced the nucleotide sequence encoding amino acids 659-671 of the O-helix of Taq DNA pol I, corresponding to the substrate binding site, with an oligonucleotide containing random nucleotides. Functional Taq pol I mutants were selected based on colony formation at the nonpermissive temperature. By using a library with 9% random substitutions at each of 39 positions, we identified 61 active Taq pol I mutants, each of which contained from one to four amino acid substitutions. Some amino acids, such as alanine-661 and threonine-664, were tolerant of several or even many diverse replacements. In contrast, no replacements or only conservative replacements were identified at arginine-659, lysine-663, and tyrosine-671. By using a library with totally random nucleotides at five different codons (arginine-659, arginine-660, lysine-663, phenylalanine-667, and glycine 668), we confirmed that arginine-659 and lysine-663 were immutable, and observed that only tyrosine substituted for phenylalanine- 667. The two immutable residues and the two residues that tolerate only highly conservative replacements lie on the side of O-helix facing the incoming deoxynucleoside triphosphate, as determined by x-ray analysis. Thus, we offer a new approach to assess concordance of the active conformation of an enzyme, as interpreted from the crystal structure, with the active conformation inferred from in vivo function.

Original languageEnglish
Pages (from-to)9670-9675
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume93
Issue number18
DOIs
Publication statusPublished - 03-09-1996

All Science Journal Classification (ASJC) codes

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