RANDOM MUTAGENESIS OF THERMUS-AQUATICUS DNA-POLYMERASE-I - CONCORDANCE OF IMMUTABLE SITES IN-VIVO WITH THE CRYSTAL-STRUCTURE

Expression of Thermus aquaticus (Tag) DNA polymerase I (pol I) in Esch erichia coli complements the growth defect caused by a temperature-sen sitive mutation in the host pol I. We replaced the nucleotide sequence encoding amino acids 659-671 of the O-helix of Tag DNA pol I, corresp onding to the substrate binding site, with an oligonucleotide containi ng random nucleotides. Functional Tag pol I mutants were selected base d on colony formation at the nonpermissive temperature, By using a lib rary with 9% random substitutions at each of 39 positions, we identifi ed 61 active Taq pol I mutants, each of which contained from one to fo ur amino acid substitutions, Some amino acids, such as alanine-661 and threonine-664, were tolerant of several or even many diverse replacem ents, In contrast, no replacements or only conservative replacements m ere identified at arginine-659, lysine-663, and tyrosine-671. By using a library with totally random nucleotides at five different codons (a rginine-659, arginine-660, lysine-663, phenylalanine-667, and glycine- 668), we confirmed that arginine-659 and lysine-663 were immutable, an d observed that only tyrosine substituted for phenylalanine-667. The t wo immutable residues and the two residues that tolerate only highly c onservative replacements lie on the side of O-helix facing the incomin g deoxynucleoside triphosphate, as determined by x-ray analysis. Thus, we offer a new approach to assess concordance of the active conformat ion of an enzyme, as interpreted from the crystal structure, with the active conformation inferred from in vivo function.