Low Fidelity Mutants in the O-Helix of Thermus aquaticus DNA Polymerase I

Abstract

We screened 67 mutants in the O-helix of Thermus aquaticus (Taq) DNA polymerase I (pol I) for altered fidelity of DNA synthesis. These mutants were obtained (Suzuki, M., Baskin, D., Hood, L., and Loeb, L. A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 9670 -9675) by substituting an oligonucleotide containing random sequences for codons 659 -671, and selecting for complementation of a growth defect in Escherichia coli caused by temperature-sensitive host pol I. Thirteen mutants decreased fidelity in a screen that employed primer extension reactions lacking one of four complementary deoxynucleoside triphosphates (dNTPs). Three mutants were purified and exhibited 29 -68% of wild-type specific activity. Homogeneous polymerases A661E, A661P, and T664R extended primers further than the wild-type, synthesizing past template nucleotides for which the complementary dNTP was absent. The data indicate that both misinsertion of incorrect nucleotides and extension of mispaired primer termini were increased. In a lacZ␣ forward mutation assay, A661E and T664R yielded mutation frequencies at least 7-and 25-fold greater, respectively, than that of the wild-type polymerase. These findings emphasize the importance of the O-helix in substrate recognition and are compatible with a role for pyrophosphate release in enhancing fidelity of DNA synthesis. Thermus aquaticus (Taq) DNA polymerase I (pol I)1 is used extensively in the amplification of DNA by the polymerase chain reaction. Based on its primary sequence, crystal structure, and catalytic mechanism, Taq pol I is classified in the same family as Escherichia coli pol I (1-3). The amino acid sequences of Taq pol I and E. coli pol I are 38% homologous, both enzymes have 5Ј-3Ј exonuclease domains (4, 5), and the crystal structures of the polymerase domains are nearly identical (5, 6). The structure of the vestigial 3Ј-5Ј exonuclease domain of Taq pol I is very different, however; unlike E. coli pol I, purified Taq pol I exhibits no proofreading activity (7).The O-helix in E. coli pol I is constituted of 15 amino acids which form part of the DNA binding cleft (8). Crystallographic, biochemical, and mutagenesis studies indicate that four conserved residues in the O-helix which face toward the cleft interact with either the incoming dNTP or the templateprimer. Considerable evidence indicates that in E. coli pol I, Arg-754 and Lys-758 interact with the phosphate groups in the incoming dNTP, that Phe-762 interacts with the deoxyribose moiety in the dNTP, and that Tyr-766 binds to the templateprimer (9 -11). The nearly identical structures of the polymerase domains of Taq pol I and E. coli pol I (6, 7) suggest that the corresponding amino acids in Taq pol I have analogous functions. By using random sequence mutagenesis together with genetic complementation, we recently observed that the Ohelix of Taq pol I contains four amino acids which are immutable (or nearly immutable) and are apparently essential for catalysis in vivo (12). These residues, Arg-659, Lys-663, Phe-667, and Tyr-671, corres...

Directed evolution of novel polymerase activities: Mutation of a DNA polymerase into an efficient RNA polymerase

“…Loeb and coworkers (11,23) have used a combination of selection and screening to identify interesting members of polymerase libraries. Taq polymerase libraries consisting of 10 4 -10 5 mutants were generated and preselected for wild-type activity by genetic complementation of E. coli with a temperature-sensitive host polymerase I. Mutants that supported bacterial growth at the nonpermissive temperature were isolated and then screened for interesting activity, including altered fidelity and RNAP activity.…”

“…Many techniques have been reported that can produce large libraries of mutant proteins, such as cassette mutagenesis (1, 2), error-prone PCR (3,4), staggered extension process PCR (5), and gene shuffling (6,7). Methods to identify proteins with desired activities include phage display (8,9), ribosomal display (10), and complementation (11). Phage display has emerged as a particularly powerful technique to select desired mutants from large libraries based on affinity for a given target molecule.…”

Amino Acid Substitutions at Conserved Tyrosine 52 Alter Fidelity and Bypass Efficiency of Human DNA Polymerase η

“…In contrast, the finger and thumb domains are smaller and differ significantly from those of the high fidelity enzymes (17), resulting in a reduction of the surface area in the active site that interacts with DNA. Interestingly, the O-helix present in high fidelity DNA polymerases is absent in the Y-family; the O-helix region is believed to enhance fidelity by tightening the surface formed between the incoming nucleotide and template DNA (18). The more open active site of the Y-family DNA polymerases can accommodate 2 nucleotides (13,15), allowing bypass of bulky lesions such as cis-syn thymine dimers and resulting in a greater frequency of misincorporation in copying natural DNA templates (19).…”

One‐step purification of Taq DNA polymerase using nucleotide‐mimetic affinity chromatography

“…Heat-treatment of the cell lysate, as the only means of effecting Taq Pol purification, has been reported to lead to Taq Pol with a specific activity of 342 612 U/mg [32]. Finally, another possibility is to combine heat-treatment and ammonium sulfate-Polymin P precipitation, followed by phenyl-Sepharose and heparinSepharose chromatography [11]. The present strategy (Table 2) leads, in a single chromatography step, to Taq Pol with a specific activity of 61 538 U/mg, employing a low-cost elution agent (80 mM sodium phosphate buffer, pH 7.0).…”

“…Taq Pol belongs to the family of DNA polymerases I (DNA Pol I), as does the E. coli DNA polymerase I [9]. Because of its high turnover number [10], lack of proofreading activity (3'-5' exonuclease activity) [11], high temperature optimum (72-74°C), and ability to incorporate 7-deaza-3-deoxyquanosine, Taq Pol has been used extensively in DNA sequencing and other molecular biology techniques [12,13]. This enzyme holds academic interest and commercial importance and was, therefore, chosen for further evaluation of the ligand library.…”