Linearization and transposition of circular molecules of insertion sequence IS3

IS3 transposase has been shown to promote production of characteristic circ ular and linear IS3 molecules from the IS3-carrying plasmid; IS3 circles ha ve the entire IS3 sequence with terminal inverted repeats, IRL and IRR, whi ch are separated by a three base-pair sequence originally flanking either e nd in the parental plasmid, whereas linear IS3 molecules have three nucleot ide overhangs at their 5 ' ends. Here, we showed that a plasmid carrying an IS3 derivative, which is flanked by different sequences at both ends, gene rated IS3 circles and linear IS3 molecules owing to the action of transposa se. Cloning and sequencing analyses of the linear molecules showed that eac h had the same 5 '-protruding three nucleotide overhanging sequences at bot h lends, suggesting that the linear molecules were not generated from the p arental plasmid by the two double-strand breaks at both end regions of IS3. The plasmid carrying IS3 with a two base-pair mutation in the terminal din ucleotide, which would be required for transposase to cleave the 3 ' end of IS3, could still generate linear molecules as well as circles. Plasmids be aring an IS3 circle were cleaved by transposase and gave linear molecules w ith the same 5 '-protruding three nucleotide overhanging sequences. These s how that the linear molecules are generated from IS3 circles via a double-s trand break at the three basepair intervening sequence. Plasmids carrying a n IS3 circle with the two base-pair end mutation still were cleaved by tran sposase, though with reduced efficiencies, suggesting that IS3 transposase has the ability to cleave not only the 3 ' end of IS3, but a site three nuc leotides from the 5 ' end of IS3. IS3 circles also were shown to transpose to the target plasmids. The end mutation almost completely inhibited this t ransposition, showing that the terminal dinucleotides are important for the transfer of the 3 ' end of IS3 to the target as well as for the end cleava ge. (C) 1999 Academic Press.