Assembly, translocation, and activation of XerCD-dif recombination by FtsK translocase analyzed in real-time by FRET and two-color tethered fluorophore motion.
The FtsK dsDNA translocase functions in bacterial chromosome unlinking by activating XerCD-dif recombination in the replication terminus region. To analyze FtsK assembly and translocation, and the subsequent activation of XerCD-dif recombination, we extended the tethered fluorophore motion technique, using two spectrally distinct fluorophores to monitor two effective lengths along the same tethered DNA molecule. We observed that FtsK assembled stepwise on DNA into a single hexamer, and began translocation rapidly (∼ 0.25 s). Without extruding DNA loops, single FtsK hexamers approached XerCD-dif and resided there for ∼ 0.5 s irrespective of whether XerCD-dif was synapsed or unsynapsed. FtsK then dissociated, rather than reversing. Infrequently, FtsK activated XerCD-dif recombination when it encountered a preformed synaptic complex, and dissociated before the completion of recombination, consistent with each FtsK-XerCD-dif encounter activating only one round of recombination.
Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford OX1 3PU, United Kingdom;
Peter F J
PF
May
Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom.
Pawel
P
Zawadzki
Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom.
David J
DJ
Sherratt
Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford OX1 3PU, United Kingdom; a.kapanidis1@physics.ox.ac.uk lidia.arciszewska@bioch.ox.ac.uk.
Achillefs N
AN
Kapanidis
Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom a.kapanidis1@physics.ox.ac.uk lidia.arciszewska@bioch.ox.ac.uk.
Lidia K
LK
Arciszewska
D000255:Adenosine Triphosphate / Q000737:chemistry; D001483:Base Sequence; D002876:Chromosomes, Bacterial / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D019426:Integrases / Q000378:metabolism*; D016013:Likelihood Functions; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016011:Normal Distribution; D011485:Protein Binding; D021381:Protein Transport; D011995:Recombination, Genetic
D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; C096452:FtsK protein, E coli; D008565:Membrane Proteins; C473360:XerC protein, E coli; D000255:Adenosine Triphosphate; D004247:DNA; D019426:Integrases; C473361:XerD protein, E coli
DNA translocation; chromosome segregation; single-molecule FRET; site-specific DNA recombination; tethered fluorophore motion