Investigating the endonuclease activity of four Pyrococcus abyssi inteins.
Among the 14 inteins of the Pyrococcus abyssi genome, 10 harbour the LAGLIDADG motifs of dodecapeptide endonucleases. Four of these were cloned, expressed in Escherichia coli and purified to assay their potential endonuclease activity. PabRIR1-2 and PabRIR1-3 are specific endonucleases, named PI-PabI and PI-PabII, respectively, cleaving the sequence spanning their homing site. This is consistent with their size and with the relative positions and sequences of their endonuclease motifs. However, PI-PabI is 10-fold more active than PI-PabII and a discrepancy of the DNA recognition and cleavage mechanisms was observed between the two inteins. In particular, analysis of the DNA cleavage reactions by MALDI-TOF highlighted that while the cleavage of DNA by PI-PabI consists of two steps corresponding to the cleavage of each DNA strand, PI-PabII processes the two DNA strands simultaneously. Furthermore, the two inteins interact differently with DNA. In addition, we did not detect any endonuclease activity for PabLon and PabRIR1-1. Deletions in the intein sequences and mutations in the putative endonuclease motifs probably abolish this activity. Hence, inteins from the same archaebacteria, even if contained in the same host protein, did not evolve uniformly and are presumably at different stages of the invasion cycle.
D020816:Amino Acid Motifs / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D019154:Protein Splicing; D019711:Pyrococcus / Q000201:enzymology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
D004792:Enzyme Precursors; D004720:Endonucleases