DNA helicases are molecular motor proteins that separate (or unzip) two strands of DNA and run along the DNA by converting chemical energy to mechanical work. Some human helicases, if defective, cause serious diseases whose symptoms include premature aging and higher risk of cancer. Despite extensive study, there are still many open questions about the helicase mechanisms—partly because of the lack of good experimental techniques. Crystal structures provide only the static view of the molecule, and conventional ensemble tools provide the mean values averaged over millions or billions of molecules, missing rare and transient events. Professor Taekjip Ha and postdoctoral research associate Ivan Rasnik (Department of Physics, University of Illinois at Urbana-Champaign) developed and used single molecule fluorescence resonance energy transfer (FRET) to study the unzipping mechanisms of helicases (T. Ha, I. Rasnik, W. Cheng, H. Babcock, G.H. Gauss, T.M. Lohman and S. Chu, "Initiation and re-initiation of DNA unwinding by the Escherichia coli Rep helicase," Nature 419, 638–641 [2002]).

In single molecule FRET, two fluorescent dye molecules are attached to a biological molecule. The interaction between the two dyes can tell us about how far apart they are. This distance information can be used to measure the shape changes of a single molecule during its function. In the DNA unzipping experiment, two dyes are attached to the part of the DNA where the unzipping begins. Then, as the unzipping is initiated by the helicase, the distance between the two dyes increases and the FRET decreases. Using this technique, pauses and re-initiation of unzipping were detected, the first time for any helicase, and their mechanisms were deduced. Such information can be obtained only by using single-molecule measurements, and this technique is generally applicable to many other DNA-protein interactions.

Perhaps one of the most controversial and important questions in the field is how many molecules of the enzyme are required to unzip the DNA. Some argue that the enzyme works solo, and others believe that they function in duo. This single-molecule study shows that this helicase does not work solo. A monomer can move along single-stranded DNA (akin to movement along a one-way street), but once it encounters a junction with double-stranded DNA (a two-way street), it cannot go any farther and displays futile fluctuations and falls off the DNA. Only if another protein binds to the monomer at the junction, forming an active helicase complex, can the complex continue to run along the double-stranded DNA and unzip it. If the complex partially breaks off, the remaining monomer cannot move farther along and gets stalled (pausing; the figure shown above is an artistic rendering of this phenomenon). The binding by another monomer can then restart the unzipping. This study suggests that the low processivity (the ability to go for long distances on the DNA before falling off) observed for this and many other helicases is due to the limited stability of the active helicase complex.

Collaborators on the project were Wei Cheng, George Gauss and Timothy Lohman at Washington University School of Medicine and Hazen Babcock and Steven Chu at Stanford University. The work was funded by the National Institutes of Health, the National Science Foundation, Searle Scholars program, the Research Corporation, and the University of Illinois Research Board. A related news story is available from the UI News Bureau.