Inactivation of the 5′-3′ exonuclease of Thermus aquaticus DNA polymerase

Louise S. Merkens, Sharon K. Bryan, Robb E. Moses

Research output: Contribution to journalArticle

13 Scopus citations

Abstract

The gene for Thermus aquaticus (Taq) DNA polymerase enzyme (Taq Pol I) was mutgenized 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 resistance 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 acid which are required for normal 5′-3′ exonuclease activity in Taq Pol I (Arg-25 and Arg-74).

Original languageEnglish (US)
Pages (from-to)243-248
Number of pages6
JournalBBA - Gene Structure and Expression
Volume1264
Issue number2
DOIs
StatePublished - Nov 7 1995

Keywords

  • (Thermus aquaticus)
  • DNA polymerase
  • Exonuclease
  • Processivity

ASJC Scopus subject areas

  • Structural Biology
  • Biophysics
  • Biochemistry
  • Genetics

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