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

Thy Pol-2 intein, from Thermococcus hydrothermalis, belongs to the same all elic family as Tli Pol-2 (PI-TliI), Tfu Pol-2 (PI-TfuII) and TspTY Pol-3 mi ni-intein, all inserted at the pol-c site of archaeal DNA polymerase genes. This new intern was cloned, expressed in Escherichia coli and purified. Th e intein is a specific endonuclease (PI-Thyl) which cleaves the inteinless sequence of the Thy DNA pol gene. Moreover, PI-TliI, PI-TfuII and PI-Thyl a re very similar endonucleases which cleave DNA in the same optimal conditio ns at 70 degreesC yielding similar 3'-hydroxyl overhangs of 4 bp and the re action is subject to product inhibition. The three enzymes are able to clea ve the three DNA sequences spanning the pol-c site and a 24 bp consensus cl eavage site was defined for the three isoschizomers. However, the exact siz e of the minimal cleavage site depends both on the substrate sequence and t he endonuclease. The inability of the isoschizomers to cleave the inteinles s DNA polymerase gene from Pyrococcus spp. KOD is due to point substitution s 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.