DNA polymerase from mesophilic and thermophilic bacteria. III. Lack of fidelity in the replication of synthetic polydeoxyribonucleotides by DNA polymerase from Bacillus licheniformis and Bacillus stearothermophilus.

Abstract:

1. DNA polymerase from the mesophile Bacillus licheniformis and the thermophile Bacillus stearothermophilus has been used to study the replication of poly(dA-dT)-poly(dA-dT) and poly(dC)-poly(dG) templates at 37, 45, and 55 degrees C. 2. Incorporation of non-complementary deoxyribonucleoside triphosphates (misincorporation) occurred with both enzymes and both templates. Non-specific incorporation (de novo polynucleotide synthesis, random attachment to existing strands, and tritium exchange of nucleotides) accounted for, at most, a small fraction of the total observed misincorporation. The error rates at 37 degrees C for the complete system were as follows:: B. licheniformis: dATP, 1/61; dCTP, 1/830; dGTP, 1/360; dTTP, 1/65; B. stearothermophilus: dATP, 1/68; dCTP, 1/1430; dGTP, 1/440; dTTP, 1/67. For both organisms, the error rate for dCTP and dGTP was independent of incubation temperature; the error rate for dATP and dTTP was 5-50-fold greater than that for dCTP or dGTP and increased significantly from 37 to 55 degrees C. 3. The ratio of dATP to dTTP incorporation with the poly(dA-dT)-poly-(dA-dT) template was independent of temperature and close to unity. The ratio of dCTP to dGTP incorporation with the poly(dC)-poly(dG) template decreased from approx. 0.2 to 0.05 for the mesophile and from approx. 0.06 to 0.03 for the thermophile as the temperature increased from 37 to 55 degrees C.

Polymerases:

Topics:

Source / Purification, Exonuclease Activity, Kinetic Parameters

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
Bli DNA polymerase Stenesh J1977 3-5' Exonuclease (proofreading) No
Bli DNA polymerase Stenesh J1977 Cloned or native Native organism
Bli DNA polymerase Stenesh J1977 Tagged No
Bli DNA polymerase Stenesh J1977 Full length or truncated Full length
Bli DNA polymerase Stenesh J1977 KM 70uM Reaction: Nucleotide incorporation; Substrate: dNTPs
Bst Stenesh J1977 Cloned or native Native organism
Bst Stenesh J1977 Tagged No
Bst Stenesh J1977 Full length or truncated Full length
Bst Stenesh J1977 KM 90uM Reaction: Nucleotide incorporation; Substrate: dNTPs

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