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J Mol Biol. 2014 Feb 20;426(4):793-815. doi: 10.1016/j.jmb.2013.11.017. Epub 2013 Nov 25.

Organization of DNA partners and strand exchange mechanisms during Flp site-specific recombination analyzed by difference topology, single molecule FRET and single molecule TPM.

Author information

1
Section of Molecular Genetics and Microbiology, Institute of Cell and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA.
2
Microscopy and Imaging Center, Department of Biology and Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843-2257, USA.
3
Department of Chemistry and Biochemistry, Institute of Cell and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA.
4
Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei 112, Taiwan.
5
Section of Molecular Genetics and Microbiology, Institute of Cell and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA. Electronic address: jayaram@austin.utexas.edu.

Abstract

Flp site-specific recombination between two target sites (FRTs) harboring non-homology within the strand exchange region does not yield stable recombinant products. In negatively supercoiled plasmids containing head-to-tail sites, the reaction produces a series of knots with odd-numbered crossings. When the sites are in head-to-head orientation, the knot products contain even-numbered crossings. Both types of knots retain parental DNA configuration. By carrying out Flp recombination after first assembling the topologically well defined Tn3 resolvase synapse, it is possible to determine whether these knots arise by a processive or a dissociative mechanism. The nearly exclusive products from head-to-head and head-to-tail oriented "non-homologous" FRT partners are a 4-noded knot and a 5-noded knot, respectively. The corresponding products from a pair of native (homologous) FRT sites are a 3-noded knot and a 4-noded catenane, respectively. These results are consistent with non-homology-induced two rounds of dissociative recombination by Flp, the first to generate reciprocal recombinants containing non-complementary base pairs and the second to produce parental molecules with restored base pairing. Single molecule fluorescence resonance energy transfer (smFRET) analysis of geometrically restricted FRTs, together with single molecule tethered particle motion (smTPM) assays of unconstrained FRTs, suggests that the sites are preferentially synapsed in an anti-parallel fashion. This selectivity in synapse geometry occurs prior to the chemical steps of recombination, signifying early commitment to a productive reaction path. The cumulative topological, smFRET and smTPM results have implications for the relative orientation of DNA partners and the directionality of strand exchange during recombination mediated by tyrosine site-specific recombinases.

KEYWORDS:

BM; Brownian motion; DNA catenanes; DNA knots; EDTA; EM; electron microscopy; ethylenediaminetetraacetic acid; recombination topology; single molecule fluorescence resonance energy transfer; single molecule tethered particle motion; smFRET; smTPM; tethered DNA substrates; tyrosine recombinases

PMID:
24286749
DOI:
10.1016/j.jmb.2013.11.017
[Indexed for MEDLINE]
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