additional information |
the intervening domain of the thermostable Thermus aquaticus DNA polymerase (TAQ: polymerase), which has no catalytic activity, is exchanged for the 3'-5' exonuclease domain of the homologous mesophile Escherichia coli DNA polymerase I (Escherichia coli pol I) and the homologous thermostable Thermotoga neapolitana DNA polymerase (TNE: polymerase). Three chimeric DNA polymerases are constructed using the three-dimensional (3D) structure of the Klenow fragment of the Escherichia coli pol I and 3D models of the intervening and polymerase domains of the TAQ: polymerase and the TNE: polymerase: chimera TaqEc1 (exchange of residues 292-423 from TAQ: polymerase for residues 327-519 of Escherichia coli pol I), chimera TaqTne1 (exchange of residues 292-423 of TAQ: polymerase for residues 295-485 of TNE: polymerase) and chimera TaqTne2 (exchange of residues 292-448 of TAQ: polymerase for residues 295-510 of TNE: polymerase). The chimera TaqEc1 shows characteristics from both parental polymerases at an intermediate temperature of 50°C: high polymerase activity, processivity, 3'-5' exonuclease activity and proof-reading function The chimeras TaqTne1 and TaqTne2 show no significant 3'-5' exonuclease activity and no proof-reading function. The chimera TaqTne1 shows an optimum temperature at 60°C, decreased polymerase activity compared with the TAQ: polymerase and reduced processivity. The chimera TaqTne2 shows high polymerase activity at 72°C, processivity and less reduced thermostability compared with the chimera TaqTne1 |
Thermus aquaticus |