Comic book superheroes often combine their supernatural abilities in a coordinated effort to outwit and defeat the villain. Scientists with expertise in different techniques will often follow suit, albeit minus the supernatural abilities. This is exactly what a team of researchers from two different labs at the University of Illinois at Urbana-Champaign (UIUC) have done in order to simultaneously observe the structure and function of proteins crucial for the DNA repair process.
The collaborative labs combined the two cutting-edge techniques of optical tweezers and single-molecule fluorescence energy transfer (smFRET) to directly analyze the structure-function relationship of the UvrD helicase as it acts upon a DNA strand.
Using the UvrD protein from E. coli, the investigators wanted to determine how many UvrD proteins were required to unwind and unzip a DNA strand—a source of contention among scientists for many years.
“The way we answered this was we put a dye molecule on each protein with fluorescence–so we could count them,” explained Yann Chemla, Ph.D., associate professor of physics at UIUC and co-senior author on the studies. “Then we watched the unwinding with an optical trap. We found that a single UvrD helicase can do something—it unwinds the DNA, but not very far. It just goes back and forth a small distance, so we call it 'frustrated'. When we have two UvrD molecules, it seems to unwind much further and doesn't go back and forth as much.”
The findings from these studies were published recently in Science through two articles, the first entitled “Engineering of a superhelicase through conformational control” and the second “Direct observation of structure-function relationship in a nucleic acid–processing enzyme.”
Dr. Chemla’s team was also able to resolve another long standing question concerning the structure-function relationship for UvrD, whether the molecule is organized in either and open or closed positional state.
“This time, we used smFRET,” said Dr. Chemla. “We put two dyes on the molecule, and based on the distance between them, we could see one or another color of light, indicating whether the molecule was in the open or closed position. Then we used an optical trap to observe whether the molecule was unwinding the double-stranded DNA.”
This was an exciting observation for UCIC scientists and Dr. Chemla goes on to explain that “we found that the molecules actually swiveled from open to closed and back again. As it turns out, the closed state unwinds the strands, using a torque wrench action. The open state allows the strands to zip together.”
Working concomitantly with Dr. Chemla’s group, Taekjip Ha, Ph.D., professor of physics at UCIC and co-senior author on the current studies, along with his team engineered a structurally homologous helicase protein, which they dubbed Rep. The researchers were able to lock Rep in either the open or closed position using an additional cross-linking molecule.
Interestingly, the Dr. Ha’s team found that when Rep was locked into the closed positon its activity became exponentially greater, acting as a superhelicase capable of unwinding extremely long stretches of DNA. Conversely, when the molecule was locked in the open position it was devoid of activity.
“The superhelicase we engineered based on our basic, fundamental understanding of helicase function can be used as a powerful biotechnological tool for sensitive detection of pathogenic DNA in remote areas, for example,” concluded Dr. Ha.