Single-Molecule Studies Reveal Dynamics of DNA Unwinding by the Ring-Shaped T7 Helicase

Abstract

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.

DNA Looping Mediates Nucleosome Transfer

“…As shown in Fig. 3b, before encountering the nucleosome, the helicase unwound the dsDNA at the expected rate, as reported previously [28][29][30][31][32] . On encountering the nucleosome, the helicase showed a discrete pause, consistent with previous studies that characterized nucleosomes as major barriers for helicase unwinding 33 .…”

“…DNA tethers were formed in flow chambers and were unzipped using an optical trap with a loading rate clamp of 10 pN s À 1 ( Fig. 1 and Supplementary Figs 3 and 5) through the bound protein [10][11][12]15,16,[28][29][30][31]55,[57][58][59][60] . Briefly, chambers were first incubated with antidigoxygenin at 0.2 mg ml À 1 for 5 min, and then the surface was blocked by incubation with casein at 5 mg ml À 1 for 5 min.…”

“…DNA tethers were prepared as described above, and helicase preparation was as described below 28,30,31 . Briefly, 1.5 nM of the helicase monomer was incubated for up to 20 min in the modified nucleosome-unzipping buffer (10 mM Tris (pH 8), 1 mM EDTA, 100 mM NaCl, 3% (v/v) glycerol,1.5 mM MgCl 2 , 1 mM DTT, 0.02% Tween 20, 2 mg ml À 1 AcBSA (Ambion), 1 mg ml À 1 casein and 2 mM dTTP).…”

The importance of surfaces in single-molecule bioscience

“…65 One recent study used this DNA unzipping configuration to study the DNA unwinding activity of the ring-shaped T7 helicase, and revealed that the helicase functioned through a mechanism involving protein induced separation of the DNA strands. 39 Optical tweezers can also measure the forces exerted by molecules. For example, the first studies that measured the forces produced by a transcribing molecule of RNA polymerase relied upon optical tweezers.…”

“…[23][24][25][26] Single-molecule methods have also been used to examine other nucleic acid motors like bacteriophage lambda exonuclease, 27,28 DNA polymerases, [29][30][31][32] Rad54 and Rdh54 DNA translocases, [33][34][35] the chromatin remodeling complexes RSC and Swi/Snf, 36,37 and several different helicases. [38][39][40] A large volume of work has also focused on the conformational dynamics and physical properties of single molecules including the folding, unfolding and strain response of proteins, DNA, and RNA. [41][42][43][44][45][46][47][48] This review is written for the nonspecialist who may be interested in understanding or pursuing single-molecule experiments.…”