DNA topology and geometry in Flp and Cre recombination

Alexandre A. Vetcher, Alexander Y. Lushnikov, Junalyn Navarra-Madsen, Robert G. Scharein, Yuri L. Lyubchenko, Isabel K. Darcy, Stephen D. Levene

Research output: Contribution to journalArticle

35 Scopus citations

Abstract

The Flp recombinase of yeast and the Cre recombinase of bacteriophage P1 both belong to the λ-integrase (Int) family of site-specific recombinases. These recombination systems recognize recombination-target sequences that consist of two 13 bp inverted repeats flanking a 6 or 8 bp spacer sequence. Recombination reactions involve particular geometric and topological relationships between DNA target sites at synapsis, which we investigate using nicked-circular DNA molecules. Examination of the tertiary structure of synaptic complexes formed on nicked plasmid DNAs by atomic-force microscopy, in conjunction with detailed topological analysis using the mathematics of tangles, shows that only a limited number of recombination-site topologies are consistent with the global structures of plasmids bearing directly and inversely repeated sites. The tangle solutions imply that there is significant distortion of the Holliday-junction intermediate relative to the planar structure of the four-way DNA junction present in the Flp and Cre co-crystal structures. Based on simulations of nucleoprotein structures that connect the two-dimensional tangle solutions with three-dimensional models of the complexes, we propose a recombination mechanism in which the synaptic intermediate is characterized by a non-planar, possibly near-tetrahedral, Holliday-junction intermediate. Only modest conformational changes within this structure are needed to form the symmetric, planar DNA junction, which may be characteristic of shorter-lived intermediates along the recombination pathway.

Original languageEnglish (US)
Pages (from-to)1089-1104
Number of pages16
JournalJournal of Molecular Biology
Volume357
Issue number4
DOIs
StatePublished - Apr 7 2006

Keywords

  • DNA looping
  • DNA topology
  • Protein-DNA interactions
  • Site-specific recombination
  • Tangle analysis

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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    Vetcher, A. A., Lushnikov, A. Y., Navarra-Madsen, J., Scharein, R. G., Lyubchenko, Y. L., Darcy, I. K., & Levene, S. D. (2006). DNA topology and geometry in Flp and Cre recombination. Journal of Molecular Biology, 357(4), 1089-1104. https://doi.org/10.1016/j.jmb.2006.01.037