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Transposable elements belonging to the recently identified IS 200/ IS 605 family radically differ from classical insertion sequences in their transposition mechanism by strictly requiring single-stranded DNA substrates. This IS family includes elements encoding only the transposase ( TnpA), and others, like IS Dra 2 from D einococcus radiodurans, which contain a second gene, tnpB, dispensable for transposition and of unknown function to date. Here, we show that TnpB has an inhibitory effect on the excision and insertion steps of IS Dra 2 transposition. This inhibitory action of TnpB was maintained when IS Dra 2 transposition was induced by γ-irradiation of the host cells and required the integrity of its putative zinc finger motif. We also demonstrate the negative role of TnpB when IS Dra 2 transposition was monitored in a heterologous E scherichia coli host, indicating that TnpB-mediated inhibition does not involve D einococcus-specific factors. TnpB therefore appears to play a regulatory role in IS Dra 2 transposition. [ABSTRACT FROM AUTHOR]

We have analysed the transposition and target selection strategy of IS 1655, a typical IS 30 family member resident in Neisseria meningitidis. We have redefined IS 1655 as a 1080 bp long element with 25 bp imperfect inverted repeats (IRs), which generates a 3 bp target duplication and have shown that it transposes using an intermediate with abutted IRs separated by 2 bp. IS 1655 exhibits bipartite target specificity inserting preferentially either next to sequences similar to its IRs or into an unrelated but well defined sequence. IR-targeting leads to the formation of a new junction in which the targeted IR and one of the donor IRs are separated by 2 bp. The non-IR targets were characterized as an imperfect 19 bp palindrome in which the central five positions show slight GC excess and the distal region is AT-rich. Artificial targets designed according to the consensus were recognized by the element as hot spots for insertion. The organization of IS 1655 is similar to that of other IS 30 family members. Moreover, it shows striking similarity to IS 30 in transposition strategy even though their transposases differ in their N-terminal regions, which, for IS 30, appears to determine target specificity. Comparative analysis of the transposases and the evolutionary aspects of sequence variants are also briefly discussed. [ABSTRACT FROM AUTHOR]

The IS1 bacterial insertion sequence family, considered to be restricted to Enterobacteria, has now been extended to other Eubacteria and to Archaebacteria, reviving interest in its study. To analyse the functional domains of the InsAB′ transposase of IS1A, a representative of this family, we used an in vivo system which measures IS1-promoted rescue of a temperature-sensitive pSC101 plasmid by fusion with a pBR322::IS1 derivative. We also describe the partial purification of the IS1 transposase and the development of several in vitro assays for transposase activity. These included a DNA band shift assay, a transposase-mediated cleavage assay and an integration assay. Alignments of IS family members (http://www-is.biotoul.fr) not only confirmed the presence of an N-terminal helix-turn-helix and a C-terminal DDE motif in InsAB′, but also revealed a putative N-terminal zinc finger. We have combined the in vitro and in vivo tests to carry out a functional analysis of InsAB′ using a series of site-directed InsAB′ mutants based on these alignments. The results demonstrate that appropriate mutations in the zinc finger and helix-turn-helix motifs result in loss of binding activity to the ends of IS1 whereas mutations in the DDE domain are affected in subsequent transposition steps but not in end binding. [ABSTRACT FROM AUTHOR]

Insertion of bacterial insertion sequence IS 911 can often be directed to sequences resembling its ends. We have investigated this type of transposition and shown that it can occur via cleavage of a single end and its targeted transfer next to another end. The single end transfer (SET) events generate branched DNA molecules that contain a nicked Holliday junction and can be considered as partial transposition products. Our results indicate that these can be processed by the Escherichia coli host independently of IS 911-encoded proteins. Such resolution depends on the presence of homologous DNA regions neighbouring the cross-over point in the SET molecule. Processing is often accompanied by sequence conversion between donor and target sequences, suggesting that branch migration is involved. We show that resolution is greatly reduced in a recG host. Thus, the branched DNA-specific helicase, RecG, involved in processing of potentially lethal DNA structures such as stalled replication forks, also intervenes in the resolution of partial IS 911 transposition products. [ABSTRACT FROM AUTHOR]

Presents a correction to the article "Host Processing of Branched DNA Intermediates Is Involved in Targeted Transposition of IS911" that was previously published in 2003 issue of the journal "Molecular Microbiology."