Inteins of Thermococcus fumicolans DNA polymerase are endonucleases with distinct enzymatic behaviors

The DNA polymerase gene of Thermococcus fumicolans harbors two intein genes . Both inteins have been produced in Escherichia coli and purified either a s naturally spliced products from the expression of the complete DNA polyme rase gene or directly from the cloned inteins genes. Both recombinant intei ns exhibit endonuclease activity, with an optimal temperature of 70 degrees 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 superc oiled 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 h oming endonuclease using Mg2+ as cofactor. Its minimal recognition and clea vage site is 21 bp long either on linear or circular DNA substrate. Its end onuclease activity is strongly inhibited by the 3' digestion product, which remains bound to the enzyme after the cleavage reaction. According to curr ent nomenclature, these endonucleases were named PI-TfuI and PI-TfuII. Thes e two inteins thus exhibit different requirements for metal cofactor and su bstrate topology as well as different mechanism of action.