Thermus aquaticus DNA Polymerase I Mutants with Altered Fidelity

Phe667 in the conserved O-helix of Thermus aquaticus (Taq) DNA polymerase I (pol I) is known to be important for discrimination against dideoxy-NTPs. We show here that Phe667 is also important for base selection fidelity. In a forward mutation assay at high polymerase concentration, wild type pol I catalyzed frequent A → T and G → T transversions and −1 frameshifts at nonreiterated sites involving loss of a purine immediately downstream of a pyrimidine. The mutants F667L and A661E,I665T,F667L exhibited large decreases in A → T and G → T transversions, and the triple mutant displayed reduction in the aforementioned −1 frameshifts as well. Kinetic analysis showed that the F667L and A661E,I665T,F667L polymerases discriminated against synthesis of A:A mispairs more effectively and catalyzed less extension of A:A mispairs than the wild type enzyme. These data indicate that Phe667 functions in maintaining the error frequency and spectrum, and the catalytic efficiency, of wild type pol I. We also found that the strong general mutator activity conferred by the single A661E substitution was entirely suppressed in the A661E, I665T,F667L polymerase, exemplifying how interactions among O-helix residues can contribute to fidelity. We discuss the mutator and anti-mutator mutations in light of recently obtained three-dimensional structures of T. aquaticus pol I.

[1]  T. Steitz,et al.  Structure of large fragment of Escherichia coli DNA polymerase I complexed with dTMP , 2020, Nature.

[2]  G. Waksman,et al.  Structure-based design of Taq DNA polymerases with improved properties of dideoxynucleotide incorporation. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[3]  T. Kunkel,et al.  Side Chains That Influence Fidelity at the Polymerase Active Site of Escherichia coli DNA Polymerase I (Klenow Fragment)* , 1999, The Journal of Biological Chemistry.

[4]  Gabriel Waksman,et al.  Crystal structures of open and closed forms of binary and ternary complexes of the large fragment of Thermus aquaticus DNA polymerase I: structural basis for nucleotide incorporation , 1998, The EMBO journal.

[5]  C. M. Joyce,et al.  How E. coli DNA polymerase I (Klenow fragment) distinguishes between deoxy- and dideoxynucleotides. , 1998, Journal of molecular biology.

[6]  S. Doublié,et al.  Crystal structure of a bacteriophage T7 DNA replication complex at 2.2 Å resolution , 1998, Nature.

[7]  James R. Kiefer,et al.  Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal , 1998, Nature.

[8]  L. Loeb,et al.  Low Fidelity Mutants in the O-Helix of Thermus aquaticus DNA Polymerase I* , 1997, The Journal of Biological Chemistry.

[9]  T. Kunkel,et al.  Base Miscoding and Strand Misalignment Errors by Mutator Klenow Polymerases with Amino Acid Substitutions at Tyrosine 766 in the O Helix of the Fingers Subdomain* , 1997, The Journal of Biological Chemistry.

[10]  L. Hood,et al.  Random mutagenesis of Thermus aquaticus DNA polymerase I: concordance of immutable sites in vivo with the crystal structure. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[11]  M. Nayal,et al.  Crystal structure of the large fragment of Thermus aquaticus DNA polymerase I at 2.5-A resolution: structural basis for thermostability. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[12]  Dae-Sil Lee,et al.  Crystal structure of Thermus aquaticus DNA polymerase , 1995, Nature.

[13]  C. Richardson,et al.  A single residue in DNA polymerases of the Escherichia coli DNA polymerase I family is critical for distinguishing between deoxy- and dideoxyribonucleotides. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[14]  C. M. Joyce,et al.  Deoxynucleoside Triphosphate and Pyrophosphate Binding Sites in the Catalytically Competent Ternary Complex for the Polymerase Reaction Catalyzed by DNA Polymerase I (Klenow Fragment) (*) , 1995, The Journal of Biological Chemistry.

[15]  E A Merritt,et al.  Raster3D Version 2.0. A program for photorealistic molecular graphics. , 1994, Acta crystallographica. Section D, Biological crystallography.

[16]  L. Loeb,et al.  Reverse chemical mutagenesis: identification of the mutagenic lesions resulting from reactive oxygen species-mediated damage to DNA. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[17]  J. Ito,et al.  Compilation, alignment, and phylogenetic relationships of DNA polymerases. , 1993, Nucleic acids research.

[18]  C. M. Joyce,et al.  Reactions at the polymerase active site that contribute to the fidelity of Escherichia coli DNA polymerase I (Klenow fragment). , 1992, The Journal of biological chemistry.

[19]  N. Arnheim,et al.  Extension of base mispairs by Taq DNA polymerase: implications for single nucleotide discrimination in PCR. , 1992, Nucleic acids research.

[20]  D. Lilley,et al.  DNA replication, 2nd edn , 1992 .

[21]  P. Kraulis.  A program to produce both detailed and schematic plots of protein structures , 1991 .

[22]  J. Zou,et al.  Improved methods for building protein models in electron density maps and the location of errors in these models. , 1991, Acta crystallographica. Section A, Foundations of crystallography.

[23]  S. Benkovic,et al.  A mutant of DNA polymerase I (Klenow fragment) with reduced fidelity. , 1991, Biochemistry.

[24]  Smita S. Patel,et al.  Pre-steady-state kinetic analysis of processive DNA replication including complete characterization of an exonuclease-deficient mutant. , 1991, Biochemistry.

[25]  T. Kunkel,et al.  Frameshift errors initiated by nucleotide misincorporation. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[26]  T. Kunkel,et al.  Fidelity of DNA synthesis by the Thermus aquaticus DNA polymerase. , 1988, Biochemistry.

[27]  S. Benkovic,et al.  Elementary steps in the DNA polymerase I reaction pathway. , 1983, Biochemistry.

[28]  G J Williams,et al.  The Protein Data Bank: a computer-based archival file for macromolecular structures. , 1978, Archives of biochemistry and biophysics.

[29]  T. Kunkel,et al.  Analyzing fidelity of DNA polymerases. , 1995, Methods in enzymology.

[30]  T. Steitz,et al.  Function and structure relationships in DNA polymerases. , 1994, Annual review of biochemistry.

[31]  S. Creighton,et al.  Biochemical basis of DNA replication fidelity. , 1993, Critical reviews in biochemistry and molecular biology.

[32]  H. Echols,et al.  Fidelity mechanisms in DNA replication. , 1991, Annual review of biochemistry.

[33]  S. Benkovic,et al.  Mechanism of the idling-turnover reaction of the large (Klenow) fragment of Escherichia coli DNA polymerase I. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[34]  T. Kunkel,et al.  Fidelity of DNA synthesis. , 1982, Annual review of biochemistry.

[35]  M. Gefter,et al.  DNA Replication , 2019, Advances in Experimental Medicine and Biology.