Abstract
PI-TfuI, an intein spliced from the DNA polymerase of Thermococcus fumicolans, is a highly specific endonuclease, whose cleavage efficiency and specificity depend on both the substrate topology and the divalent cation used as cofactor. An open circular intermediate was observed during the cleavage of supercoiled DNA by PI-TfuI, suggesting a two-step cleavage of the DNA. We characterized this nicked intermediate and, through the development of a method of analysis of the cleavage reaction based on matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we demonstrated that the cleavage of DNA by PI-TfuI indeed results from two cleavage events. One step results in the cleavage of the bottom strand, which is independent of the DNA conformation or choice of the metal ion cofactor. A second step, which is slower, leads to the cleavage of the top strand and governs the specific requirements of PI-TfuI concerning the essential cofactor and the DNA topology. These two steps were shown to be independent in optimal conditions of cleavage. These data give support to the existence of two distinct and independent active sites in the endonuclease domain of the archaeal intein.
Highlights
Among the 34 proteins known to harbor inteins, archaeal DNA polymerases are the preferred hosts (Inbase, the New England Biolabs intein data base at www.neb.com/inteins/ intein_intro.html; Inteins-Protein introns web site at bioinfo. weizmann.ac.il/ϳpietro/inteins/)
To address this critical issue, we further studied the mechanism of DNA cleavage by PI-TfuI, an intein endonuclease from the DNA polymerase of Thermococcus fumicolans (GenBankTM accession number Z69882)
As described for other dodecapeptide endonucleases such as PI-SceI and PI-PfuI inteins [8, 14], its specificity of cleavage depends on both the DNA substrate topology and the cation used as a cofactor
Summary
Production and Purification of the PI-TfuI Intein—The recombinant intein was expressed in Escherichia coli, as previously described, after transformation of BL21(De3)(pLys-S) bacteria with the inducible expression vector pET26-Tfu. The supercoiled form of these plasmids or the open circular intermediate of the cleavage were purified from a 1% agarose gel in TBE buffer (90 mM Tris borate, 2 mM EDTA) using the Qiaquick purification kit (Qiagen) and were diluted in water to a concentration of 100 ng/l (56 fmol/l) Endonuclease assays using these substrates were performed in a final volume of 10 l, in 50 mM Tris acetate, pH 8, buffer containing 100 mM NH4OAc and 25 mM MnSO4 or different concentrations of Mg(OAc), at 70 °C. 50 pmol of duplex were incubated with PI-TfuI in 10 l of 50 mM Tris acetate, pH 8, containing 100 mM NH4OAc and 25 mM MnSO4 or 50 mM Mg(OAc) at 70 °C for different incubation times These reaction mixtures were analyzed by MALDI-TOF mass spectrometry as indicated below. Calibration of the spectra was performed using an external calibration with synthetic oligonucleotides F3 and F4
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