Formation and transposition of the covalently closed IS30 circle: The relation between tandem dimers and monomeric circles

Abstract

In the present study, we demonstrate that a circular IS30 element acts as an intermediate for simple insertion. Covalently closed IS and Tn circles constructed in vitro are suitable for integration into the host genome. Minicircle integration displays all the characteristics of transpositional fusion mediated by the (IS30)2 dimer regarding target selection and target duplication. Evidence is provided for in vivo circularization of the element located either on plasmids or on the genome. It is shown that circle formation can occur through alternative pathways. One of them is excision of IS30 from a hot spot via joining the IRs. This reaction resembles the site-specific dimerization that leads to (IS30)2 establishment. The other process is the dissolution of (IS30)2 dimer, when the element is excised from an IR-IR joint. These pathways differ basically in the fate of the donor replicon: only dimer dissolution gives rise to resealed donor backbone. Analysis of minicircles and the rearranged donor replicons led us to propose a molecular model that can account for differences between the circle-generating processes. Our focus was to the dissolution of IR-IR joints located on the host genome, because these events promoted extensive genomic rearrangements and accompanied mini-circle formation. The results present the possibility of host genome reorganization by IS30-like transposition.