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

PI- Tfu I, 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- Tfu I, 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- Tfu I 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- Tfu I 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.