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Nucleic Acids Research, 2000, Vol. 28, No. 21 4391-4396
© 2000 Oxford University Press

The Thy Pol-2 intein of Thermococcus hydrothermalis is an isoschizomer of PI-TliI and PI-TfuII endonucleases

Isabelle Saves1, Heïdy Eleaume1, Jacques Dietrich2 and Jean-Michel Masson1,3,*

1Institut de Pharmacologie et Biologie Structurale, I.P.B.S./C.N.R.S., 205 Route de Narbonne, F-31077 Toulouse Cedex, France, 2IFREMER, Laboratoire de Biotechnologie, Centre de Brest, BP 70, F-29280 Plouzané, France and 3Institut National des Sciences Appliquées, Complexe Scientifique de Rangueil, F-31077 Toulouse Cedex, France

Thy Pol-2 intein, from Thermococcus hydrothermalis, belongs to the same allelic family as Tli Pol-2 (PI-TliI), Tfu Pol-2 (PI-TfuII) and TspTY Pol-3 mini-intein, all inserted at the pol-c site of archaeal DNA polymerase genes. This new intein was cloned, expressed in Escherichia coli and purified. The intein is a specific endonuclease (PI-ThyI) which cleaves the inteinless sequence of the Thy DNA pol gene. Moreover, PI-TliI, PI-TfuII and PI-ThyI are very similar endonucleases which cleave DNA in the same optimal conditions at 70°C yielding similar 3'-hydroxyl overhangs of 4 bp and the reaction is subject to product inhibition. The three enzymes are able to cleave the three DNA sequences spanning the pol-c site and a 24 bp consensus cleavage site was defined for the three isoschizomers. However, the exact size of the minimal cleavage site depends both on the substrate sequence and the endonuclease. The inability of the isoschizomers to cleave the inteinless DNA polymerase gene from Pyrococcus spp. KOD is due to point substitutions on the 5' side of the pol-c site, suggesting that the absence of inteins of this allelic family in DNA polymerase genes from Pyrococcus spp. can be linked to small differences in the target site sequence.

* To whom correspondence should be addressed at Institut de Pharmacologie et Biologie Structurale, I.P.B.S./C.N.R.S., 205 Route de Narbonne, F-31077 Toulouse Cedex, France. Tel: +33 05 61 17 54 76; Fax: +33 05 61 17 59 94; Email: masson@ipbs.fr +Z69882, Y13030, M74198, AJ245819, D29671, U67532, U00707, AP000007


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