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 language | English (US) |
---|---|
Pages (from-to) | 243-248 |
Number of pages | 6 |
Journal | BBA - Gene Structure and Expression |
Volume | 1264 |
Issue number | 2 |
DOIs | |
State | Published - Nov 7 1995 |
Keywords
- (Thermus aquaticus)
- DNA polymerase
- Exonuclease
- Processivity
ASJC Scopus subject areas
- Structural Biology
- Biophysics
- Biochemistry
- Genetics