DNA looping by FokI: the impact of synapse geometry on loop topology at varied site orientations

David Rusling, Niels Laurens, Christian Pernstich, Gijs J. L. Wuite, Stephen E. Halford

    Research output: Contribution to journalArticlepeer-review

    Abstract

    Most restriction endonucleases, including FokI, interact with two copies of their recognition sequence before cutting DNA. On DNA with two sites they act in cis looping out the intervening DNA. While many restriction enzymes operate symmetrically at palindromic sites, FokI acts asymmetrically at a non-palindromic site. The directionality of its sequence means that two FokI sites can be bridged in either parallel or anti-parallel alignments. Here we show by biochemical and single-molecule biophysical methods that FokI aligns two recognition sites on separate DNA molecules in parallel and that the parallel arrangement holds for sites in the same DNA regardless of whether they are in inverted or repeated orientations. The parallel arrangement dictates the topology of the loop trapped between sites in cis: the loop from inverted sites has a simple 180° bend, while that with repeated sites has a convoluted 360° turn. The ability of FokI to act at asymmetric sites thus enabled us to identify the synapse geometry for sites in trans and in cis, which in turn revealed the relationship between synapse geometry and loop topology.

    Original languageEnglish
    Pages (from-to)4977–4987
    Number of pages11
    JournalNucleic Acids Research
    Volume40
    Issue number11
    Early online date23 Feb 2012
    DOIs
    Publication statusPublished - 1 Jun 2012

    Keywords

    • DNA/chemistry
    • DNA Cleavage
    • Deoxyribonucleases, Type II Site-Specific/chemistry
    • Fluorescence Resonance Energy Transfer
    • Models, Molecular
    • Nucleic Acid Conformation
    • Protein Conformation

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