BUFFALO, N.Y. -- Piero Bianco produces movies. Not films that
chronicle the human condition, a la Hollywood. His subject is human
biology at its most basic -- the translocation and unwinding of DNA
by a DNA motor protein.
Bianco, an assistant professor in the Center for Single Molecule
Biophysics and the Department of Microbiology, was the first to
record on videotape in real time a molecule of a particular DNA
motor protein in the process of "unzipping" a double strand of
To accomplish this feat, Bianco uses a novel technique he
developed called "laser tweezers." Using this tool, he can grasp
and hold a DNA molecule long enough to capture the action as the
double helix unwinds.
"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 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 (the
motor protein) take an individual DNA molecule and pull it
If there were 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.
Bianco currently is focusing 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 at UB 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 developed his novel system while a postdoctoral fellow at
the University of California at Davis, in collaboration with
colleagues from Lawrence Livermore National Laboratory in
Livermore, Calif. Creating it consumed several years, culminating
in publication of the breakthrough in the journal Nature in January
Learning how a DNA motor protein 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 motor proteins, called helicases, are too
small to be observed under a microscope.
Bianco solved these problems in a variety of ways. The laser
tweezers form the crux of the system. By focusing an infrared laser
beam through a microscope objective and aided by the laws of
physics, he can create an optical trap that stops a DNA molecule in
The molecule itself is too elusive to be caught, however, so
Bianco tethers the DNA molecule to a microscopic polystyrene bead
to give the tweezers something to grasp. 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 earlier to bind fluorescent 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"), inserts 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 beam, 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
This action is called processive translocation. On film, it
looks like 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 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."