Inactivation of the 5'-3' exonuclease of Thermus aquaticus DNA polymerase.
The gene for Thermus aquaticus (Taq) DNA polymerase enzyme (Taq Pol I) was mutagenized and sixty-two candidate clones were screened for enzyme activity. Two of the clones expressed enzymes (*Taq-3 and *Taq-5) that showed very reduced 5'-3' exonuclease activity and normal DNA polymerase activity. These two enzymes showed heat resitance and storage stability similar to Taq Pol I and had similar effectiveness in PCR. Processivity of the polymerases was compared by measuring the extension of an end-labeled primer annealed to a single stranded DNA, as well as by a PCR method. The processivity of *Taq-3 and *Taq-5 was similar to Taq Pol I (50-80 nucleotides) and more processive than a Taq Pol I deficient in the 5'-3' exonuclease due to absence of the first 290 amino acids (Stoffel fragment). The results indicate two amino acids which are required for normal 5'-3' exonuclease activity in Taq Pol I (Arg-25 and Arg-74).
D003001:Cloning, Molecular; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase / Q000096:biosynthesis / Q000378:metabolism*; D004795:Enzyme Stability; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism*; D005798:Genes, Bacterial; D016296:Mutagenesis; D019384:Nucleic Acid Synthesis Inhibitors; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D019914:Taq Polymerase; D013824:Thermus / Q000201:enzymology* / Q000235:genetics
D017931:DNA Primers; D019384:Nucleic Acid Synthesis Inhibitors; D011994:Recombinant Proteins; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V