New gene-editing system enables large-scale studies of gene function.
CAMBRIDGE, Mass.--Twenty years after visualizing a surprising left-handed form of the DNA double helix, a Massachusetts Institute of Technology researcher has found that this altered form of genetic material is involved in some important biological activities, including creating proteins essential for normal brain function.
In the 1970s, when Alexander Rich, William Thompson Sedgwick Professor of Biophysics at MIT, and his colleagues solved for the first time the three-dimensional structure of a fragment of a DNA crystal, they were puzzled to find that instead of looking like the right-handed double helix Watson and Crick had described in 1953, it was a left-handed double helix with an irregular zig-zag backbone.
Is this unusual form of DNA, dubbed Z-DNA by the researchers, an oddity or is it biologically significant? In tomorrow's (June 11) issue of the journal Science, Rich and MIT visiting scientist Thomas Schwartz; Mark Rould, now at the University of Vermont; and MIT research scientists Ky Lowenhaupt and Alan Herbert partly resolve the issue.
They describe how the three-dimensional structure of Z-DNA binds to a portion of an enzyme. The enzyme binds to Z-DNA with great specificity, leading scientists to conclude that the two serve a biological function. The enzyme creates a modified protein that is used by the brain as a receptor for serotonin, among other things.
Furthermore, as yet another striking example of nature's ability to perform many functions with the same materials, the protein bound to Z-DNA is closely related in three-dimensional structure to a family of proteins known to bind to right-handed DNA.
"Twenty years after first visualizing a left-handed form of the DNA double helix, it may now be possible to see ways in which nature uses this altered form of the molecule to carry out some important biological activities," Rich said.
REVERSING THE COIL
Much has been learned about Z-DNA since it was first discovered. It turns out that Z-DNA is found only transiently when genes are actively being transcribed. It occurs mainly in specialized sequences of nucleotides, the building blocks of genetic material, and is stabilized by processes that partially unwind the normal right-handed DNA double helix.
The main process that produces such an unwinding is transcription (the synthesis of messenger RNA), which is used as a template for assembling proteins in biological systems.
The system works this way: When the enzyme making RNA, called RNA polymerase, moves along the DNA double helix, it leaves behind underwound DNA. Selected sequences in this DNA temporarily become left-handed Z-DNA, like a stretched phone cord coiling backwards on itself.
When the RNA polymerase stops moving, other enzymes relax the DNA and it reverts to its normal right-handed form. Like a stretched phone cord that is released, it snaps back into its usual shape.
The researchers discovered that the protein that binds to Z-DNA is an enzyme that changes the genetic message contained in the messenger RNA molecule by changing one of the four nucleotides -- the building blocks of DNA and RNA -- into another. The protein is an editing enzyme that chemically transforms adenine into a molecule that acts like guanine.
The DNA molecule is composed of two complementary strands that are wound around each other and connected by base pairs that look like rungs in a ladder. Each base will pair with only one other: adenine with thymine and guanine with cytosine. The sequence of bases determines the structure -- and therefore the function -- of a protein.
Some of the modified proteins created by the editing enzyme are very important, and several have been found in the brain. Glutamic acid is one of the major excitatory molecules in the brain. The editing enzyme changes the glutamate receptor in a way that has been shown to be important for normal brain function. Without it, animals develop epilepsy and die.
Another change is in the receptor for serotonin. Prozac and similar drugs affect the amount of serotonin, which is involved in transmitting nerve impulses in the brain. Editing the receptor tones down the brain's response to serotonin.
A MOVING TARGET
A few years ago, Alan Herbert developed a system for isolating proteins that bind tightly to Z-DNA. The protein turned out to be the editing enzyme that changes adenine into a guanine-like molecule. The researchers believe that the editing enzyme is acting as a targeting device, picking out an active gene from an inactive one.
Because Z-DNA is only found behind a moving RNA polymerase in the act of transcribing RNA, the presence of Z-DNA differentiates an active gene from an inactive one.
To do its work, the editing enzyme must catch the RNA polymerase in the act of unwinding the DNA double helix and leaving Z-DNA in its wake.
By latching on to transient Z-DNA, the enzyme picks a sure winner. It can then bind to the newly synthesized RNA and edit the message before the RNA is processed any further. This is necessary because the enzyme acts only on double-stranded RNA formed by folding the messenger RNA back on itself.
However, one of the two strands codes for protein while the other may not. The non-coding sequences are removed or spliced out soon after the RNA molecule is transcribed. This means that the editing enzyme has to act before the newly produced message is cleared of the non-coding sequences.
Z-DNA is believed to provide a way of getting the enzyme to the RNA transcript within the small window of opportunity in which the enzyme is able to act.
This work was funded by the National Institutes of Health and the National Science Foundation.