Single-molecule studies reveal dynamics of DNA unwinding by the ring-shaped T7 helicase.
Helicases are molecular motors that separate DNA strands for efficient replication of genomes. We probed the kinetics of individual ring-shaped T7 helicase molecules as they unwound double-stranded DNA (dsDNA) or translocated on single-stranded DNA (ssDNA). A distinctive DNA sequence dependence was observed in the unwinding rate that correlated with the local DNA unzipping energy landscape. The unwinding rate increased approximately 10-fold (approaching the ssDNA translocation rate) when a destabilizing force on the DNA fork junction was increased from 5 to 11 pN. These observations reveal a fundamental difference between the mechanisms of ring-shaped and nonring-shaped helicases. The observed force-velocity and sequence dependence are not consistent with a simple passive unwinding model. However, an active unwinding model fully supports the data even though the helicase on its own does not unwind at its optimal rate. This work offers insights into possible ways helicase activity is enhanced by associated proteins.
Johnson
Daniel S
DS
Department of Physics, Laboratory of Atomic and Solid State Physics, Cornell University, Ithaca, NY 14853, USA.
Bai
Lu
L
Smith
Benjamin Y
BY
Patel
Smita S
SS
Wang
Michelle D
MD
D017123:Bacteriophage T7 / Q000235:genetics*; D001696:Biomechanical Phenomena; D004247:DNA / Q000096:biosynthesis* / Q000235:genetics*; D004265:DNA Helicases / Q000235:genetics*; D004261:DNA Replication / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D040542:Mechanotransduction, Cellular / Q000235:genetics; D008958:Models, Molecular; D020409:Molecular Motor Proteins / Q000235:genetics*; D015394:Molecular Structure; D013314:Stress, Mechanical; D013997:Time Factors
D004277:DNA, Single-Stranded; D020409:Molecular Motor Proteins; D004247:DNA; D004265:DNA Helicases