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

研究成果: Article査読

64 被引用数 (Scopus)

抄録

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.

本文言語English
ページ(範囲)9670-9675
ページ数6
ジャーナルProceedings of the National Academy of Sciences of the United States of America
93
18
DOI
出版ステータスPublished - 03-09-1996
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All Science Journal Classification (ASJC) codes

  • 一般

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