Trade.Information

Topic Name: Double helix, : a key role in gene copying

Category: Genetic Engineering

Research persons: Michelle Wang,,Smita Patel,Daniel Johnson, Lu Bai and Benjamin Smith.

Location: Cornell University, Department of Physics,
LASSP, Clark Hall, Ithaca,
NY 14853, United States

Details

Cornell researchers have answered a fundamental question about how two strands of DNA, known as a double helix, separate to start a process called replication, in which genes copy themselves. The research, published in the current issue of the journal Cell, examined the role of an enzyme called a helicase, which plays a major role in separating DNA strands so that replication of a single strand can occur.
Scientists have known that helicases bind to the area of a double helix where the two strands fork away from each other, like the free ends of two pieces of thread wound around each other. The forked area opens and closes very rapidly. But scientists have debated whether helicases actively separate the two strands at the fork or if they passively wait for the fork to widen on its own.
The research found that the helicase appears to actively exert a force onto the fork and separate the two strands.
"A simple passive unwinding mechanism does not explain our data," said Michelle Wang, associate professor of physics and the paper's senior author.
"Defects in helicases are associated with many human diseases, ranging from predisposition to cancer to premature aging," said co-author Smita Patel, a biochemistry professor at the Robert Wood Johnson Medical School in Piscataway, N.J. "Helicases are involved in practically all DNA and RNA metabolic processes."
The researchers made their discovery by anchoring one end of one of the strands in a double helix to the surface of a microscope cover slip. The end of the other strand was attached to a micron-sized plastic bead. They then focused a laser beam on the tiny bead and trapped the bead in place within the beam of light. This setup allowed the researchers to measure the position and force on the bead, creating a very precise sensor of the helicase motion. As the helicase moved toward the fork and the double helix unwound, the tension on the two strands lessened. Using statistical mechanics models, the researchers could then compare actual measurements of movement with predictions based on both active and passive scenarios.
"The unwinding has to have some active component to it, and based on our data, we can tell you exactly how active it is," said Wang. "Basically, it is an active unwinding motor."
While helicases unwind very rapidly in cells, in test tube experiments the unwinding is much slower. The researchers believe that helicases work with other enzymes, where "accessory proteins are helping the helicase out by destabilizing the fork junction," said Wang.
A major part of this work was done by Cornell physics graduate students Daniel Johnson, the lead author, Lu Bai and Benjamin Smith.
 

About Researchers-
Dr. Bai Lu
Laboratory of Cellular and Synaptic Neurophysiology, NICHD
Porter Neuroscience Research Center
Building 35, Room 1C-1004
35 Convent Drive, MSC 3714
Bethesda, MD 20892-3714
Telephone: (301) 435-2970 (office), (301) 496-1777 (fax)
Email: bailu@mail.nih.gov
Michelle D. Wang
Associate Professor of Physics
518 Clark Hall
Cornell University
Ithaca, New York 14853
(607) 255-6414
Email: mdw17@cornell.edu
Smita Patel
Indian Institute of Technology,
Bombay, India
Professor
Research Tower
Room 836
675 Hoes Lane
Piscataway, NJ 08854-5635
Telephone: 732-235-3372
Facsimile: 732-235-4783
E-mail: patelss@umdnj.edu
 

More Research -
Mechanism of Transcription
Transcription is an important process in gene expression. During transcription, RNA polymerase translocates along a DNA template while copying genetic information from DNA to RNA.We study the mechanism by which RNA polymerase moves by tracking the motions of individual RNA polymerase molecules and by theoretically modeling the polymerase kinetics.
Unpacking DNA
Nucleosomes are the fundamental packing units of the DNA in chromosomes.The stability of nucleosomes regulates the accessibility of DNA to many DNA-binding proteins that carry out a variety of cellular functions. We study nucleosomal stability by mechanically unpacking the nucleosomes.
Development of Novel Techniques
Protein-DNA interactions underlie many cellular activities.Our lab develops new physical/biophysical techniques to directly probe these interactions.? Our Unzipping Force Analysis of Protein Association (UFAPA) is a novel and versatile method for detection of the position and dynamic nature of protein-DNA interactions.For some interesting applications of this technique, please see our work on restriction enzymes and DNA repair enzymes.
Our lab also pioneered an angular trapping technique for simultaneous torque generation and detection.? When a birefringent particle is trapped in a polarized laser beam, rotation of the laser polarization allows rotation of the particle while torque exerted on the particle is detected as a change in the polarization.? This technique allows the control and detection of the torque of a biological molecule attached to the particle and has opened new dimensions for applications of optical trapping techniques.
 

In The Images-
1.Michelle D. Wang with UFAPA apparatus in Cornell's Clark
2.Authors of the PNAS article propose this three-stage model for the uncoiling of DNA from nucleosome core particles. At top, a DNA fragment 156 base pairs in length is in its most compact form, coiled 1.6 times around an eight-unit histone protein core. Next, a moderate stretching force releases 76 base pairs of DNA. Additional force releases the other 80 base pairs, and DNA can still reassemble around the histone core. But further force breaks loose the histone core.
3.Diagram explains Wang's strategy for measureing the poweer of a molecular motor, the bead of RNA polymerase that catalyzes the transcription of RNA from the DNA template.
4.DNA_unzip3.72.jpg: Double-strand (ds) DNA is "unzipped" and forced apart as one end of single-strand (ss) DNA is held in place , tethered to a microsphere in a laser's optical trap, while the end of the other single strand is attached to a microscope's moving coverslip. Force analysis lets Cornell biophysicists know which protein (the green oval) is encountered as the DNA unzips. Wang laboratory/Cornell
5.This image shows a DNA double helix (green and purple strands) being separated by a helicase enzyme (yellow globule) at the junction where the two strands fork. To show that helicases actively separate the two strands of DNA, the researchers attached one end of a DNA strand to a microscope cover slip and attached the end of the other DNA strand to a micron-sized plastic bead. The bead was then trapped in a tightly focused laser beam (red), which allowed the researchers to measure the motion of the helicase as it unwound the DNA.