Abstract
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 'χ' (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 ∼30 nm at 2 Hz) and to examine the mechanism by which χ
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 χ is negligible, showing that the
activity modification at χ does not require ejection of the RecD subunit
from the enzyme as previously proposed; modification may occur through an
unusual, pure conformational switch mechanism.