Functional domains of the IS1 transposase: analysis in vivo and in vitro

Molecular Microbiology
Bao Ton-HoangMichael Chandler

Abstract

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 ...Continue Reading

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Citations

Dec 12, 2012·Quarterly Reviews of Biophysics·Fred DydaAlison Burgess Hickman
Apr 10, 2007·Journal of Bacteriology·Adeline Achard, Roland Leclercq
Apr 27, 2010·Journal of Bacteriology·Mónika SzabóFerenc Olasz
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Mar 17, 2009·Research in Microbiology·Patricia SiguierMichael Chandler
Feb 7, 2014·FEMS Microbiology Reviews·Patricia SiguierMick Chandler
Oct 8, 2004·Molecular Microbiology·Zita NagyFerenc Olasz
Dec 16, 2020·Journal of Global Antimicrobial Resistance·Yao ZhaiZhancheng Gao
Jun 25, 2015·Microbiology Spectrum·Patricia SiguierMick Chandler

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