| Proc Natl Acad Sci U S A. 1996 September 3; 93(18): 9670–9675. | PMCID: PMC38487 |
Random mutagenesis of Thermus aquaticus DNA polymerase I: concordance of immutable sites in vivo with the crystal structure. M Suzuki, D Baskin, L Hood, and L A Loeb Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology, Seattle, WA, USA. 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. Full text Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.4M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References. Images in this article Click on the image to see a larger version. These references are in PubMed. This may not be the complete list of references from this article. - Joyce, CM; Steitz, TA. Function and structure relationships in DNA polymerases. Annu Rev Biochem. 1994;63:777–822. [PubMed]
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