Inteins of Thermococcus fumicolans DNA Polymerase Are Endonucleases with Distinct Enzymatic Behaviors*
- From the ‡Institut de Pharmacologie et Biologie Structurale, IPBS/CNRS, 205 Route de Narbonne, F-31077 Toulouse Cedex, §Appligène-Oncor, Parc d'innovation, BP 72, F-67402 Illkirch Cedex, ‖Ifremer, Laboratoire de Biotechnologie, Centre de Brest, BP 70, F-29280 Plouzané, and ‡Institut National des Sciences Appliquées, Complexe Scientifique de Rangueil, F-31077 Toulouse Cedex, France
Abstract
The DNA polymerase gene of Thermococcus fumicolans harbors two intein genes. Both inteins have been produced in Escherichia coli and purified either as naturally spliced products from the expression of the complete DNA polymerase gene or directly from the cloned inteins genes. Both recombinant inteins exhibit endonuclease activity, with an optimal temperature of 70 °C. The Tfu pol-1 intein, which belongs to the Psp KOD pol-1 allelic family, recognizes and cleaves a minimal sequence of 16 base pairs (bp) on supercoiled DNA with either Mn2+ or Mg2+ as cofactor. It cleaves linear DNA only with Mn2+ and requires a 19-bp minimal recognition sequence. The Tfu pol-2 intein, which belongs to the Tli pol-2 allelic family, is a highly active homing endonuclease using Mg2+ as cofactor. Its minimal recognition and cleavage site is 21 bp long either on linear or circular DNA substrates. Its endonuclease activity is strongly inhibited by the 3′ digestion product, which remains bound to the enzyme after the cleavage reaction. According to current nomenclature, these endonucleases were named PI-TfuI and PI-TfuII. These two inteins thus exhibit different requirements for metal cofactor and substrate topology as well as different mechanism of action.
- bp
- base pair(s)
- pol
- polymerase
- PCR
- polymerase chain reaction
- Received June 17, 1999.
- Revision received November 3, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
