Sub-terminal Sequences Modulating IS30 Transposition in Vivo and in Vitro

Mónika Szabó, János Kiss, Zita Nagy, Michael Chandler, F. Olasz

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

Inverted repeats of insertion sequences (ISs) are indispensable for transposition. We demonstrate that sub-terminal sequences adjacent to the inverted repeats of IS30 are also required for optimal transposition activity. We have developed a cell-free recombination system and showed that the transposase catalyses formation of a figure-of-eight transposition intermediate, where a 2 bp long single strand bridge holds the inverted repeat sequences (IRs) together. This is the first demonstration of the figure-of-eight structure in a non-IS3 family element, suggesting that this mechanism is likely more widely adopted among IS families. We show that the absence of sub-terminal IS30 sequences negatively influences figure-of-eight production both in vivo and in vitro. These regions enhance IR-IR junction formation and IR-targeting events in vivo. Enhancer elements have been identified within 51 bp internal to IRL and 17 bp internal to IRR. In the right end, a decanucleotide, 5′-GAGATAATTG-3′, is responsible for wild-type activity, while in the left end, a complex assembly of repetitive elements is required. Functioning of the 10 bp element in the right end is position-dependent and the repetitive elements in the left end act cooperatively and may influence bendability of the end. In vitro kinetic experiments suggest that the sub-terminal enhancers may, at least partly, be transposase-dependent. Such enhancers may reflect a subtle regulatory mechanism for IS30 transposition.

Original languageEnglish
Pages (from-to)337-352
Number of pages16
JournalJournal of Molecular Biology
Volume375
Issue number2
DOIs
Publication statusPublished - Jan 11 2008

Fingerprint

Inverted Repeat Sequences
Transposases
Cell-Free System
Insertional Mutagenesis
Genetic Recombination
In Vitro Techniques
insulin receptor-related receptor

Keywords

  • abutted IRs
  • bent DNA
  • figure-of-eight
  • insertion sequence
  • IR-targeting

ASJC Scopus subject areas

  • Virology

Cite this

Sub-terminal Sequences Modulating IS30 Transposition in Vivo and in Vitro. / Szabó, Mónika; Kiss, János; Nagy, Zita; Chandler, Michael; Olasz, F.

In: Journal of Molecular Biology, Vol. 375, No. 2, 11.01.2008, p. 337-352.

Research output: Contribution to journalArticle

Szabó, Mónika ; Kiss, János ; Nagy, Zita ; Chandler, Michael ; Olasz, F. / Sub-terminal Sequences Modulating IS30 Transposition in Vivo and in Vitro. In: Journal of Molecular Biology. 2008 ; Vol. 375, No. 2. pp. 337-352.
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