Thousands of times each second along the seven feet of DNA in every cell in our bodies, enzymes are busy moving along the molecule, ferrying the proper chemical bases into the appropriate positions to make copies of the genetic blueprint. This high- speed copying makes all life possible. The enzymes in charge do an incredible job, getting the sequence in humans correct more than 99.999 percent of the time.
How so? Scientists have long assumed that the demand for this high fidelity comes from the chemical bonds --called hydrogen bonds -- between the two rows of bases of DNA. One nucleotide extends a chemical "handshake," and only its appropriate partner can match up with it, forming a pair of bases that take their place as one rung in the twisting ladder of the double helix.
But new evidence from the laboratory of Eric Kool at the University of Rochester shows that the formation of hydrogen bonds is not as important as scientists expected. Instead, shape is paramount; together a pair of bases must fit into its assigned space in the larger DNA molecule so that it can serve as a template for identical molecules. Their latest evidence appears in the June 17 issue of Nature.
"This is the jigsaw-puzzle model of DNA," says Kool, professor of chemistry. "The bases must fit together for a polymerase enzyme to copy them. Shape brings fidelity to the process." The finding is a surprise to many biochemists who have long focused on hydrogen bonds when trying to unravel the operation of polymerase enzymes, which copy DNA.
The Nature paper is the latest in a series of publications and patents in which Kool describes experiments with molecular mimics, synthetic molecules his laboratory creates to substitute for the conventional bases (adenine, cytosine, thymine, guanine) that form the DNA of all known life forms. In the paper, Kool and former postdoctoral associate Tracy J. Matray, now at Geron Corp., designed two radically different shapes for a base pair. With funding from the National Institutes of Health and the U.S. Army, they reduced one base to the smallest chemical entity -- a proton -- and provided it with a partner that was nearly double the size of a normal base. Together the two bases filled about the same space as a conventional pair, fitting within the general structure of DNA, though neither looks anything like a traditional base. It's a little bit like an odd couple that blends into a conventional neighborhood by canceling out each other's foibles.
"It's amazing that although these bases look totally foreign, as long as they fit together properly, like two jigsaw puzzle pieces, enzymes perceive them as part of a DNA molecule and copy them accordingly. Not only do you not need hydrogen bonds to copy DNA, you don't even need the traditional shape of the individual bases," says Kool.
One possible application of the current work, Kool says, is a test for cancer-causing agents that cause mutations by knocking out a single base, which is the most common form of mutation in our bodies. The double-size molecule the team developed is such an effective molecular impostor that polymerase enzymes insert it into any "abasic" site, where a single base is missing, like a party guest who is always looking for an empty chair. The molecule, a pyrene nucleoside triphosphate, fluoresces brightly, flagging mutations and sending an easily visible signal whenever a base is missing.
Kool's vein of research on novel types of DNA and RNA has been adopted by several other laboratories and has a variety of other applications. He has developed "rolling circles" of DNA, providing an inexpensive way to produce hundreds of copies of an RNA molecule by spinning them off like a mimeograph machine. The laboratory has developed loops of DNA and RNA that form a triple helix with single strands, enveloping them like a bun around a hot dog and knocking them out. The loops are specially resistant to enzymes that chew up DNA, and they've shown promise as antisense agents to knock out the proteins involved in diseases like leukemia and HIV. The laboratory has also made molecules that can hone in on different targets depending on chemical conditions.
Another far-off application might be a type of artificial DNA, the creation of several entirely new base pairs that would fit together like conventional DNA and code for proteins much like regular DNA has done for millions of years. Experimenting with alternate genetic codes might provide hints about the type of life that might exist elsewhere in the universe. While such a development sounds like the stuff of science fiction, Kool's work provides one step in that direction. He has already developed several synthetic molecules that can squeeze into natural DNA, and he has overturned decades of dogma by showing that hydrogen bonds are not as vital as shape to the existence and reproduction of DNA.
"The unexpected flexibility of the structure of DNA raises interesting questions about the primordial soup," says Kool. "Why does DNA have the structure it has? How different can we make a molecule and still have it behave like DNA, and still have life?"