Inteins of Thermococcus fumicolans DNA Polymerase Are Endonucleases with Distinct Enzymatic Behaviors

• Published in: The Journal of biological chemistry Vol. 275; no. 4; pp. 2335 - 2341
• Main Authors: Saves, Isabelle, Ozanne, Valérie, Dietrich, Jacques, Masson, Jean-Michel
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 28.01.2000; American Society for Biochemistry and Molecular Biology
• Subjects: Base Sequence; DNA-Directed DNA Polymerase - genetics; DNA-Directed DNA Polymerase - metabolism; Endonucleases - antagonists & inhibitors; Endonucleases - genetics; Endonucleases - metabolism; Escherichia coli - genetics; inteins; Nucleic Acid Synthesis Inhibitors; psp gene; RNA Splicing; Substrate Specificity; Tfu gene; Thermococcus - enzymology; Thermococcus fumicolans; Tli gene
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.275.4.2335

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.