In vitro transposition of ISY100, a bacterial insertion sequence belonging to the Tc1/mariner family

Abstract

The Synechocystis sp. PCC6803 insertion sequence ISY100 (ISTcSa) belongs to the Tc1/mariner/IS630 family of transposable elements. ISY100 transposase was purified and shown to promote transposition in vitro. Transposase binds specifically to ISY100 terminal inverted repeat sequences via an N-terminal DNA-binding domain containing two helix–turn–helix motifs. Transposase is the only protein required for excision and integration of ISY100. Transposase made double-strand breaks on a supercoiled DNA molecule containing a mini-ISY100 transposon, cleaving exactly at the transposon 3′ ends and two nucleotides inside the 5′ ends. Cleavage of short linear substrates containing a single transposon end was less precise. Transposase also catalysed strand transfer, covalently joining the transposon 3′ end to the target DNA. When a donor plasmid carrying a mini-ISY100 was incubated with a target plasmid and transposase, the most common products were insertions of one transposon end into the target DNA, but insertions of both ends at a single target site could be recovered after transformation into Escherichia coli. Insertions were almost exclusively into TA dinucleotides, and the target TA was duplicated on insertion. Our results demonstrate that there are no fundamental differences between the transposition mechanisms of IS630 family elements in bacteria and Tc1/mariner elements in higher eukaryotes