Impediment of E. coli UvrD by DNA-destabilizing force reveals a strained-inchworm mechanism of DNA unwinding.
Escherichia coli UvrD is a non-ring-shaped model helicase, displaying a 3'-5' polarity in DNA unwinding. Using a transverse magnetic tweezer and DNA hairpins, we measured the unwinding kinetics of UvrD at various DNA-destabilizing forces. The multiform patterns of unwinding bursts and the distributions of the off-times favour the mechanism that UvrD unwinds DNA as a dimer. The two subunits of the dimer coordinate to unwind DNA processively. They can jointly switch strands and translocate backwards on the other strand to allow slow (approximately 40 bp/s) rewinding, or unbind simultaneously to allow quick rehybridization. Partial dissociation of the dimer results in pauses in the middle of the unwinding or increases the translocation rate from approximately 40 to approximately 150 nt/s in the middle of the rewinding. Moreover, the unwinding rate was surprisingly found to decrease from approximately 45 to approximately 10 bp/s when the force is increased from 2 to 12 pN. The results lead to a strained-inchworm mechanism in which a conformational change that bends and tenses the ssDNA is required to activate the dimer.
Sun
Bo
B
Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing, China.
Wei
Kong-Ji
KJ
Zhang
Bo
B
Zhang
Xing-Hua
XH
Dou
Shuo-Xing
SX
Li
Ming
M
Xi
Xu Guang
XG
D004265:DNA Helicases / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism*; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D007700:Kinetics; D008954:Models, Biological
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C506746:UvrD protein, E coli; D004265:DNA Helicases