Chi-sequence recognition and DNA translocation by single RecBCD helicase/nuclease molecules.
Major pathways of recombinational DNA repair in Escherichia coli require the RecBCD protein--a heterotrimeric, ATP-driven, DNA translocating motor enzyme. RecBCD combines a highly processive and exceptionally fast helicase (DNA-unwinding) activity with a strand-specific nuclease (DNA-cleaving) activity (refs 1, 2 and references therein). Recognition of the DNA sequence 'chi' (5'-GCTGGTGG-3') switches the polarity of DNA cleavage and stimulates recombination at nearby sequences in vivo. Here we attach microscopic polystyrene beads to biotin-tagged RecD protein subunits and use tethered-particle light microscopy to observe translocation of single RecBCD molecules (with a precision of up to approximately 30 nm at 2 Hz) and to examine the mechanism by which chi modifies enzyme activity. Observed translocation is unidirectional, with each molecule moving at a constant velocity corresponding to the population-average DNA unwinding rate. These observations place strong constraints on possible movement mechanisms. Bead release at chi is negligible, showing that the activity modification at chi does not require ejection of the RecD subunit from the enzyme as previously proposed; modification may occur through an unusual, pure conformational switch mechanism.
Dohoney
K M
KM
Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02454-9110, USA.
Gelles
J
J
D000251:Adenosine Triphosphatases / Q000378:metabolism; D001692:Biological Transport; D004265:DNA Helicases / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D008853:Microscopy; D008863:Microspheres; D011137:Polystyrenes
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D011137:Polystyrenes; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; C468322:exodeoxyribonuclease V, E coli; D000251:Adenosine Triphosphatases; D004265:DNA Helicases