Synapsis and DNA cleavage in ¿C31 integrase-mediated site-specific recombination

M. C. A. Smith, R. Till, K. Brady, P. Soultanas, H. Thorpe, Margaret Caroline MacHin Smith

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Abstract

The Streptomyces phage phiC31 encodes an integrase belonging to the serine recombinase family of site-specific recombinases. The well studied serine recombinases, the resolvase/invertases, bring two recombination sites together in a synapse, and then catalyse a concerted four-strand staggered break in the DNA substrates whilst forming transient covalent attachments with the recessed 5' ends. Rotation of one pair of half sites by 180degrees relative to the other pair occurs, to form the recombinant configuration followed by ligation of the DNA backbone. Here we address the nature of the recombination intermediates formed by phiC31 integrase when acting on its substrates attP and attB. We have identified intermediates containing integrase covalently attached to cleaved DNA substrates, attB or attP, by analysis of complexes in gels and after treatment of these complexes with proteinases. Using a catalytically inactive integrase mutant, S12A, the synaptic complexes containing integrase, attP and attB were identified. Furthermore, we have shown that attB mutants containing insertions or deletions are blocked in recombination at the stage of strand cleavage. Thus, there is a strict spacing requirement within attB, possibly for correct positioning of the catalytic serine relative to the scissile phosphate in the active site. Finally, using integrase S12A we confirmed the inability of attL and attR or other combinations of sites to form a stable synapse, indicating that the directionality of integrative recombination is determined at synapsis.

Original languageEnglish
Pages (from-to)2607-2617
Number of pages10
JournalNucleic Acids Research
Volume32
Issue number8
DOIs
Publication statusPublished - May 2004

Keywords

  • LACTOCOCCAL BACTERIOPHAGE TP901-1
  • HUMAN-CELLS
  • EXCISION
  • GENE
  • ELEMENT
  • SYSTEM
  • ATTB

Cite this

Smith, M. C. A., Till, R., Brady, K., Soultanas, P., Thorpe, H., & Smith, M. C. M. (2004). Synapsis and DNA cleavage in ¿C31 integrase-mediated site-specific recombination. Nucleic Acids Research, 32(8), 2607-2617. https://doi.org/10.1093/nar/gkh538