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Recombination dynamics observed with single monomer resolution
James E.
Kloeppel, Physical Sciences Editor
217-244-1073; kloeppel@uiuc.edu
8/10/06
|
CHAMPAIGN, Ill. —
Using a sensitive, single-molecule measurement technique, researchers
at the University of Illinois at Urbana-Champaign have observed
the life cycle of RecA, a protein that plays a major role in repairing
damaged DNA.
The protein forms a filament, which grows and shrinks primarily by one
monomer at a time, the researchers report in the August issue of the
journal Cell.
RecA is a DNA recombination protein found in the gut bacterium E. coli.
A human homolog, called Rad51, interacts with many proteins, including
BRCA2, whose mutation increases susceptibility to breast and ovarian
cancers. A better understanding of how these proteins function could
help our understanding of cancer.
“Our measurement technique provides a way of counting the number
of individual monomers bound to DNA in real time,” said Taekjip
Ha, a professor of physics at Illinois and a Howard Hughes Medical Institute investigator. “With
that, we can determine the kinetic rates for reactions occurring at
either end of the protein filament.”
During the recombination process, RecA binds with DNA to form a filament
that spirals around the DNA. The filament can grow in either direction,
and can advance on the DNA by growing at the leading end and dissociating
at the trailing end.
To study the dynamics of RecA, the researchers used a highly sensitive
single-molecule fluorescence resonance energy transfer (FRET) technique
that Ha and colleagues developed.
To use FRET, researchers first attach two dye molecules – one
green and one red – to the molecule they want to study. Next,
they excite the green dye with a laser. Some of the energy moves from
the green dye to the red dye, depending upon the distance between them.
The researchers then measure the brightness of the two dyes simultaneously.
The changing ratio of the two intensities indicates the relative movement
of the two dyes, and therefore the motion of the molecule or its change
in size.
The technique revealed intricate details of how RecA nucleates to form
a filament, how the filament changes shape, and how the filament removes
proteins from DNA.
“Contrary to our initial expectations, both ends of the RecA filament
continually grow and shrink, but a higher binding rate at one end causes
the filament to grow primarily in one direction,” Ha said. “We
also learned that as the filament grows and shrinks, it does so by one
protein unit at a time.”
Following recombination proteins step by step could further help researchers
determine in what ways cancer-causing proteins are defective, and perhaps
find ways to correct them.
With Ha, co-authors are graduate students Chirlmin Joo and Sean A. McKinney,
undergraduate student Muneaki Nakamura, and postdoctoral researchers
Ivan Rasnik and Sua Myong. The work was funded in part by the National
Science Foundation and the National Institutes of Health.
Editor’s note: To reach Taekjip Ha, call 217-265-0717; e-mail: tjha@uiuc.edu.
