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

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 Mn 2؉ or Mg 2؉ as cofactor. It cleaves linear DNA only with Mn 2؉ 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 Mg 2؉ 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.Since the first report of a protein splicing element as an in-frame insertion in the VMA gene of Saccharomyces cerevisiae (1, 2), over 80 putative inteins have been identified.

The Two-step Cleavage Activity of PI-TfuI Intein Endonuclease Demonstrated by Matrix-assisted Laser Desorption Ionization Time-of-flight Mass Spectrometry

“…The purification procedure was unchanged from that previously described (6) and homogeneous fractions of PI-TfuI were obtained after a single step of anion exchange chromatography. These fractions were dialyzed against 10 mM Tris-HCl, pH 7.5, 50% glycerol, 0.1 mM EDTA, 1 mM dithiothreitol, 200 g/ml bovine serum albumin, and 50 mM NaCl, for storage.…”

“…Among the 15 inteins recessed in archaeal DNA polymerase, 14 are potential endonucleases and only one is a mini-intein lacking the central endonuclease domain (1,2). A specific activity of double-stranded DNA cleavage has been demonstrated for 8 of them (3)(4)(5)(6)(7). Besides, six other inteins are known to possess a specific endonuclease activity.…”

“…Section 1734 solely to indicate this fact. (6). That suggests a two-step cleavage of the DNA, giving support to the existence of two distinct active sites in the endonuclease domain of the archaeal intein.…”

“…The endonuclease activity of these 14 inteins was individually studied (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13) and common features are observed. They recognize and cleave long asymmetrical sequences: a 16 to 31-bp sequence spanning the intein insertion site in the inteinless allele of their host gene is the substrate of the intein, the cleavage leaving a 4-base long 3Ј-hydroxyl overhang.…”

“…A previous study (6) showed that PI-TfuI recognizes and cleaves a minimal se-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C.…”