Mutants of Escherichia coli integration host factor: DNA-binding and recombination properties

L. M. Hales, R. I. Gumport, J. F. Gardner

Research output: Contribution to journalArticlepeer-review

Abstract

Integration host factor (IHF) is a protein encoded by Escherichia coli which was first discovered as a requirement for bacteriophage λ site-specific recombination. In this study, we characterized mutants of IHF for their ability to bind to varoous IHF binding sites in vivo and to promote recombination of λ in vitro. DNA-binding in vivo was monitored using the challange-phage system. If IHF binds to its DNA-binding site that has been placed into the Pant region of bacteriophage P22, it acts as a repressor of the ant (antirepressor) gene, leading to the formation of lysogens of Salmonella typhimurium. If IHF cannot bind to its site, antirepressor is made leading to cell lysis. Challenge phages containing chimeras of different λ IHF binding sites were constructed to test the contribution to the binding of a dA+dT-rich region, found in the sequence of the H′ site but not in the Hl site. In one case, the binding of mutant IHF proteins was enhanced by the presence of the dA+dT-rich region, indicating that IHF may be affected by neighboring bases and local DNA structure when it binds to its site. A subset of the mutant proteins retained the ability to form a looped attL complex in vivo, representing part of a higher-order protein-DNA complex (the 'intasome'). Additionally, this same subset of proteins also promoted the integration and excision of bacteriophage λin vitro. Thus, these mutant proteins not only retain their DNA-bending ability but make any protein-protein contacts necessary to form a recombination-proficient intasome.

Original languageEnglish (US)
Pages (from-to)1030-1040
Number of pages11
JournalBiochimie
Volume76
Issue number10-11
DOIs
StatePublished - 1994

Keywords

  • DNA-binding
  • challenge phage
  • integration host factor
  • site-specific recombination

ASJC Scopus subject areas

  • Biochemistry

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