Figure 1: Some examples of biological forces and torques. A: During transcription, RNA polymerase must simultaneously translocate along, and rotate around, its helical DNA track. Because of constraints on the transcription machinery, the DNA is overtwisted (positive supercoiling) in front and undertwisted (negative supercoiling) behind, according to the "twin-supercoiled-domain model" proposed by James Wang (Harvard University) .
B: Similar to transcription, DNA replication machinery progresses along the DNA, creating positive supercoiling in front, while the daughter DNA strands behind can be twisted and intertwined, forming positive precatenanes.
Topoisomerases (Types I and II) act during transcription and replication to relieve the torsional stress generated along the DNA.
The Wang Lab
Figure 2: A high-resolution map of histone-DNA interactions within a nucleosome. A: An optical trap was used to unzip through DNA containing a single nucleosome held under constant force. (Artwork by Michael Hall.)
B: A map of histone-DNA interactions within a nucleosome. DNA templates, each containing a single nucleosome, were unzipped from both directions. The resulting dwell-time histogram of the DNA fork along the DNA clearly identified the locations and strengths of histone-DNA interactions. These interactions are highly nonuniform within a nucleosome, with three regions of strong interactions and a finer 5-bp periodicity.
Adapted from Hall, M.A., Shundrovsky, A., Bai, L., Fulbright, R.M., Lis, J.T., and Wang, M.D. 2009. Nature Structural and Molecular Biology 16:124-129.
Figure 3: DNA supercoiling, using a nanofabricated quartz cylinder in an angular optical trap (AOT). A: DNA is supercoiled by the rotation of a trapped quartz cylinder held in an AOT. During an experiment, force, displacement, rotation, and torque are simultaneously measured. (Artwork by Michael Hall.)
B: Quartz cylinders are nanofabricated. The cylinder axis is perpendicular to the extraordinary optical axis of the quartz crystal, and only one end of each cylinder is chemically functionalized for attachment to a DNA molecule.
C: Buckling transition during DNA supercoiling. Under a constant force, the torque and extension of each DNA molecule were directly measured as the DNA underwent a buckling transition (dashed lines) during supercoiling. DNA buckled abruptly, as revealed by a sharp extension drop followed by a torque plateau. This provided a direct measurement of the buckling torque and evidence that the buckling transition is abrupt under thermal agitation.
Adapted from Deufel, C., Forth, S., Simmons, C.R., Dejgosha, S., and Wang, M.D. 2007. Nature Methods 4:223-225; and Forth, S., Deufel, C., Sheinin, M.Y., Daniels, B., Sethna, J.P., and Wang, M.D. 2008. Physical Review Letters 100:148301.
Figure 4: Mechanical disruption of nucleosomes by stretching DNA. A: A single DNA molecule containing 17 positioned nucleosomes was suspended between a microscope coverslip and a trapped micron-sized microsphere. Under feedback control, the coverslip was moved away from the microsphere at a constant speed to disrupt nucleosomes.
B: The resulting force-extension curve displayed a distinct sawtooth pattern containing 17 disruption peaks, which corresponded to the 17 positioned nucleosomes.
Adapted from Brower-Toland, B.D., Smith, C.L., Yeh, R.C., Lis, J.T., Peterson, C.L., and Wang, M.D. 2002. Proceedings of the National Academy of Sciences USA 99:1960-1965.
Figure 5: Two RNA polymerases (RNAPs) work synergistically to overcome a nucleosomal barrier. A: Using Escherichia coli RNAP as a model system, the Wang lab investigated how multiple RNAPs transcribe through a nucleosome. Unzipping through a DNA template accurately located the two RNAPs and the nucleosome, contained therein. This method allowed the lab to determine how the trailing RNAP facilitates the leading RNAP to transcribe through a nucleosome.
B: A cartoon illustrating the mechanism of transcription through a nucleosome. As an RNAP approaches a nucleosome, it encounters obstacles that cause it to pause and, likely, backtrack. The arrival of a trailing RNAP exerts an assisting force on the leading RNAP, rescuing the leader from its backtracked state. The two RNAPs, working together, eventually resume efficient transcription. (Artwork by Michael Hall.)
Adapted from Jin, J., Bai, L., Johnson, D.S., Fulbright, R.M., Kireeva, M.L., Kashlev, M., and Wang, M.D. 2010. Nature Structural and Molecular Biology 17:745-752.
Figure 6: Single-molecule studies of T7 helicase. A: Cartoon illustrating a bacteriophage T7 helicase unwinding a double-stranded DNA (dsDNA) as it translocates along a single-stranded DNA (ssDNA) toward the ssDNA/dsDNA fork junction. An optical trap monitors the position of the helicase on the DNA template. (Artwork by Chris Pelkie and Daniel Ripoll.)
B: The unwinding rate of helicase, along a dsDNA template, is highly sequence-dependent and anticorrelated with the mechanical force to unzip the corresponding dsDNA sequence.
Adapted from Johnson, D.S., Bai, L., Smith, B.Y., Patel, S.S., and Wang, M.D. 2007. Cell 129:1299-1309.
Figure 7: ATP-induced helicase slippage reveals highly coordinated subunits.
A: A single-molecule assay measured the position of the T7 helicase as it unwound the double-stranded DNA (dsDNA) downstream. In the presence of dTTP, helicase did not slip. However, in the presence of ATP, unwinding was interrupted by slippage events, resulting in a remarkable sawtooth pattern in the unwinding trace. This prevented helicase from moving over a substantial distance.
B: A cartoon illustrating how nucleotide binding to T7 helicase subunits regulates processivity, i.e. , the distance that the helicase unwinds the dsDNA before slipping back along single-stranded DNA (ssDNA). The helicase encircles ssDNA as it unwinds the dsDNA. All or nearly all subunits coordinate their nucleotide binding/hydrolysis and their affinity to DNA. When both ATP and dTTP are present in solution, a subunit may be ligated to either an ATP (lower affinity to DNA, open hook) or a dTTP (higher affinity to DNA, closed hook). Helicase will not slip as long as at least one subunit does not release the DNA.
Adapted from Sun, B., Johnson, D.S., Patel., G., Smith, B.Y., Pandey, M., Patel., S.S., and Wang, M.D. 2011. Nature 478:132-135.
