ITHACA, N.Y. -- By using optical tweezers to pull individual strands of chromatin -- the DNA-protein complex that chromosomes are made of -- researchers have seen for the first time how information in fundamental genetic packaging units, called nucleosomes, might become accessible to molecules that "read" it.
The report by physicists and biologists at Cornell University and the University of Massachusetts appears in the current Proceedings of the National Academy of Sciences (Vol. 99, Issue 4), "Mechanical Disruption of Individual Nucleosomes Reveals a Reversible Multistage Release of DNA." It marks the first direct observation of the dynamic structure of individual nucleosomes. Chromosomal DNA is packaged into the compact structure of the nuclesome with the help of specialized proteins called histones. The complex of DNA plus histones in cells of higher organisms is called chromatin.
Michelle D. Wang, assistant professor of physics at Cornell, who led the scientific team, said the researchers are proposing a three-stage model for the way in which nucleosomal units in chromatin open to reveal their DNA to enzymes like RNA polymerase.
The three stages became apparent when a nucleosome was uncoiled as the DNA was stretched with increasing force, says Brent Brower-Toland, lead author of the article and a research associate in Cornell's Laboratory of Atomic and Solid State Physics. Describing the release of DNA from a single nucleosome, he says: "When we pulled on an individual chromatin fiber with increasing force, low force initially released 76 base pairs of DNA per nucleosome, then higher forces yielded 80 more base pairs with the histones still bound to the DNA, followed by histone detachment at still higher forces. But, if we released the fiber before the histones were detached, the nucleosomes were able to reassemble themselves and the whole process could be repeated." "Of course the basic plan -- nucleosomes and higher-order structures condensing DNA into a manageable space -- has been known for some time," Wang says. "But structural and biophysical details of the system have not been clear. Nucleosomes seem to stand in the way of a genetic information-transfer process that happens millions of times a day in the cells of our bodies. We're trying to understand the mechanical barrier presented by the nucleosome and, further, how the nucleosome structure is modified to clear the way for information transfer."
The researchers used optical tweezers to probe the barrier encountered by enzymes like RNA polymerase that prompt DNA in nucleosomes to uncoil from histones. With one end of a nucleosomal DNA strand fastened to a microscope slide and the other end attached to a synthetic microsphere and optically trapped in a laser beam, the researchers could accurately gauge dynamic changes in bond strength and organization as DNA was forced to uncoil from histones. Such precise measurements and observations of individual molecules are not available by classical biochemical techniques, which are more indirect and rely on averaged results from huge populations of molecules.
Under the magnification of an electron microscope, nucleosomes appear to be beads on strings of chromatin. Closer scrutiny shows the beads to be the fundamental organizational units of the genome, occurring, on average, every 200 base pairs along the DNA strands, with 147 base pairs of DNA wrapped 1.65 times around the eight-member clusters of histone proteins. One chromosome's double-stranded fiber of DNA, if stretched into a straight line, would be about two inches long. Were it not for compact storage -- in part due to the coiling of DNA in nucleosomes, which are, in turn, condensed into higher-order structures -- two inches of DNA would be too long to fit inside cells. However, DNA must be decondensed for its genes to be read or copied.
The Cornell and UMass biophysicists conducted their "disruption" experiments with fragments of DNA that were 3,684 base pairs long and accommodated 17 nucleosomes. Their experimental apparatus previously had been designed and used by the Wang laboratory to study the enzymatic action of RNA polymerase as it reads DNA. In the natural process of gene expression, nucleosomes present an obstacle to RNA polymerase as it moves along the DNA molecule transcribing genetic information for use by the cell. So-called chromatin remodeling machines, an entourage of proteins accompanying RNA polymerase, are believed to aid in overcoming these obstacles in various ways. The Cornell-UMass experiments showed more about the nature and magnitude of obstacles faced by RNA polymerase and its entourage. Other authors of the paper are Corey L. Smith and Craig L. Peterson of the University of Massachusetts Medical School's Program in Molecular Medicine; John T. Lis, professor of molecular biology and genetics at Cornell; and Richard C. Yeh, a Cornell graduate student of physics. The study was supported by grants from the National Institutes of Health (NIH) and by the following awards: NIH, Damon Runyon Scholar Award, Beckman Young Investigator Award, Alfred P. Sloan Research Fellow Award and the Keck Foundation's Distinguished Young Scholar Award.
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o PNAS article: http://www.pnas.org/cgi/reprint/99/4/1960.pdf
o PNAS commentary:
o More on optical tweezers:
o Wang lab at Cornell: http://www.physics.cornell.edu/profpages/Wang.html