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"Laser tweezers" assist UB molecular biochemist in studies of DNA
By LOIS BAKER
Contributing Editor
Piero Bianco produces movies. Not films that chronicle the human condition, à la Hollywood. His subject is human biology at its most basic—the translocation and unwinding of DNA.
Bianco, a single-molecule biochemist, is the first to record on videotape in real time a molecule of a particular DNA motor protein, or nanomachine, in the process of "unzipping" a double strand of bacterial DNA.
If there was an Academy Award for "most important basic science film," Bianco's work surely would be in the running. Knowing how DNA unwinds, copies and repairs itself—what starts it, stops it and why—will make possible major advancements in cancer treatment and is vitally important to the progress of gene therapy and recombinant DNA research.
At UB, where Bianco is an assistant professor in the Center for Single Molecule Biophysics and the Department of Microbiology, he has focused his investigation and his camera on a motor protein more complex than the one captured on his first film and which has a different mode of action.
His goal is to videotape in real time the actions of several motor proteins that accomplish DNA unwinding and DNA repair, and to use this knowledge to answer pressing questions about how cancer drugs work.
Since cancers are caused by uncontrolled cell growth, and DNA motor proteins make this possible by allowing DNA to copy itself, motor proteins are natural drug targets. Researchers know that many cancer drugs stop cell growth, but they don't know precisely how. Bianco is hoping to provide some details.
"We want to find out what happens when you put an anti-tumor drug in the way of the motor protein," Bianco said. "If the drug stops cell growth, we want to find out exactly how it does it. Will the protein start the unwinding with the drug present? If it starts, will it continue? Where will it stop, if at all?"
The resulting movies will show how existing drugs work, and will allow researchers to test the efficacy of new drugs designed to inhibit DNA replication and repair.
Bianco's novel system, developed while a postdoctoral fellow at the University of California at Davis in collaboration with colleagues from Lawrence Livermore National Laboratory, enables him to observe and film in real time the action of a single helicase on a single molecule of DNA. Creating it took several years, culminating in publication of the breakthrough in the journal Nature in January 2001.
Learning how a DNA helicase functions and capturing it on film posed several vexing technical problems at the time: how to snatch a single DNA molecule, stretch it out and hold it stable long enough to watch the helicase in action. Then there was the problem of size: DNA helicases are too small to be observed under a microscope.
Bianco solved these problems in a variety of ways. The crux of the system is an "optical trap," formed by focusing an infrared laser beam through a microscope objective. Under the right conditions, aided by the laws of physics, the laser grabs and holds the DNA molecule long enough for researchers to film the motor protein in action as it unwinds the double helix.
"These 'laser tweezers' allow us to look at one molecule at a time and understand how a protein really works," Bianco said. "When you look at groups of proteins, all of the nuances of individual proteins are lost. With this system, we can pull a DNA molecule out of a solution and actually watch a single DNA helicase molecule take an individual DNA molecule and pull it apart"
To give the tweezers something to grasp, Bianco tethers the DNA molecule to a microscopic polystyrene bead. Next, he attaches a motor protein molecule to the opposite end of the DNA molecule, and tags both bead and DNA with a fluorescent dye. The dye creates an image sufficiently bright to be recorded by a micro-camera designed to perform under very-low-light conditions. Techniques weren't available at the time of his first film to bind florescent dye to the DNA helicase, but Bianco now has that capacity at UB.
Stretching out the molecule and initiating the action occurs in a flow cell—a tiny, custom-made, Y-shaped apparatus the size of a microscope slide. Bianco introduces the bead and its cargo of DNA and motor protein into one channel of the flow cell (one of the arms of the "Y"), ATP—the molecular energy source—into the other channel, and focuses an optical microscope and laser beam on the juncture of the channels.
The stretched-out molecule and its energy source flow in their separate channels into the juncture, where the action begins. The laser beam captures the polystyrene bead in its tweezers-like grip. Manipulating the laser tweezers, Bianco maneuvers the DNA into the path of the ATP, which jump-starts the helicase.
The breakthrough film, described in Nature, features a molecule of Escherichia coli helicase called RecBCD, which acts by unzipping the DNA molecule from one end to the other. That movie shows the stretched-out strands of glowing DNA becoming progressively shorter as the invisible motor protein unzips them, displacing the dye as it goes.
This action is called processive translocation: The effect is that of a string of lights being switched off, one-by-one.
Once DNA strands are separated, the elemental processes of replication or repair can begin.
At UB, Bianco is adapting his novel system to investigate other, more complex bacterial helicases. The present target is called RuvB, a circular nanomachine that drives a critical late step in genetic recombination called branch migration. Unlike RecBCD, this motor protein wraps itself around DNA like a doughnut on a string, carrying out translocation in a different manner. The UB set-up is equipped with two optical traps and is significantly more advanced than his former system, allowing him to work with complicated molecules such as RuvB.
The overarching goal of Bianco's research is to learn how cancer drugs interfere with translocation, which, in turn, will allow drug developers to target chemotherapy drugs to the most effective point in the process. He intends to define and film the action of several motor proteins, then begin working with four specific cancer drugs provided by collaborators at Roswell Park Cancer Institute.
"There is potential here," Bianco said, "to answer questions you could never answer any other way."