Domain exchange: chimeras of Thermus aquaticus DNA polymerase, Escherichiacoli DNA polymerase I and Thermotoga neapolitana DNA polymerase

The intervening domain of the thermostable Thermus aquaticus DNA polymerase (Taq polymerase), which has no catalytic activity, has been exchanged for the 3'-5' exonuclease domain of the homologous mesophile Escherichia call D NA polymerase I (E.coli pol I) and the homologous thermostable Thermotoga n eapolitana DNA polymerase (Tne polymerase). Three chimeric DNA polymerases have been constructed using the three-dimensional (3D) structure of the Kle now fragment of the E.coli pol I and 3D models of the intervening and polym erase domains of the Taq polymerase and the Tne polymerase: chimera TaqEc1 (exchange of residues 292-423 from Taq polymerase for residues 327-519 of E .coli pol I), chimera TaqTne1 (exchange of residues 292-423 of Taq polymera se 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 showed characteristics from both parental polymerases at an intermediate temperature of 50 degreesC: high polymerase activity, pr ocessivity, 3'-5' exonuclease activity and proof-eading function. In compar ison, the chimeras TaqTne1 and TaqTne2 showed no significant 3'-5' exonucle ase activity and no proof-reading function. The chimera TaqTne1 showed an o ptimum temperature at 60 degreesC, decreased polymerase activity compared w ith the Taq polymerase and reduced processivity. The chimera TaqTne2. showe d high polymerase activity at 72 degreesC, processivity and less reduced th ermostability compared with the chimera TaqTne1.