Domain exchange: chimeras of DNA polymerase, Escherichia coli DNA polymerase I and Thermotoga neapolitana DNA polymerase

• Published in: Protein engineering, design and selection design and selection, 2000-09, Vol.13 (9), p.645
• Main Authors: Villbrandt, B, Sobek, H, Frey, B, Schomburg, D
• Format: Article
• Language: English
• ISSN: 1741-0126; 1741-0134

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 coli DNA polymerase I (E.coli pol I) and the homologous thermostable Thermotoga neapolitana DNA polymerase (Tne polymerase). Three chimeric DNA polymerases have been constructed using the three-dimensional (3D) structure of the Klenow fragment of the E.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 E.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 showed characteristics from both parental polymerases at an intermediate temperature of 50[degrees]C: high polymerase activity, processivity, 3'-5' exonuclease activity and proof-reading function. In comparison, the chimeras TaqTne1 and TaqTne2 showed no significant 3'-5' exonuclease activity and no proof-reading function. The chimera TaqTne1 showed an optimum temperature at 60[degrees]C, decreased polymerase activity compared with the Taq polymerase and reduced processivity. The chimera TaqTne2 showed high polymerase activity at 72[degrees]C, processivity and less reduced thermostability compared with the chimera TaqTne1.