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School of Medicine
Mistakes happen, and DNA synthesis is no exception to this universal truth. Errors occur in the genetic code. Fortunately, our cells possess a corrective mechanism—special DNA repair enzymes. Molecular biophysicist James Stivers has dissected the detailed movements of one such enzyme.
DNA repair enzymes fix the
break in a chromosome.
Source: Wikimedia Foundation
February 2012-- Imagine that you work in quality control. Your mission: Check the integrity of billions of widgets as they roll down the assembly line every day, many, many times per day.
Such is the task of a DNA repair enzyme. In this case, the “widgets” it inspects are the 3 billion chemical base pairs of the human genome. DNA repair enzymes look for, and correct, flaws in the DNA sequence that can arise as a result of environmental damage, spontaneous mutation or simple goofs in the DNA replication process. Without such fastidious oversight, DNA damage can cause a cell to expire or to transform into cancer.
Because DNA repair is vital to life, scientists want to understand the mechanism better. In addition, a fuller picture of its function could lead to new drugs or therapies that work at the level of DNA repair.
First, one of the most fundamental questions scientists have asked is how a relatively small number of enzymes perform quality control on such a massive amount of DNA. “This has been a compelling and enigmatic problem that people haven’t been able to put their head around,” says James Stivers, professor of pharmacology and molecular sciences.
Now, a recent study by Stivers and graduate student Joseph Schonhoft sheds further light on the issue. The scientists, as they reported online January 8 in Nature Chemical Biology, have developed a sophisticated biophysical method for documenting the nanosecond-by-nanosecond movements of a DNA repair enzyme.
The cell contains many different types of DNA repair enzymes, each with a different job. Stivers and Schonhoft have focused on one, uracil DNA glycosylase. UNG, as the enzyme is called for short, surveys the DNA sequence and looks for one particular error, the appearance of a nucleotide base called uracil.
Normally, DNA is made up of four nucleotide bases—adenine, thymine, cytosine and guanine—linked together in a particular sequence that varies from one individual to the next. Uracil is another type of nucleotide, but it is normally found in RNA and does not “belong” in DNA. However, every so often—about once in every 100 million DNA base pairs—a mutation or chemical glitch includes a uracil in the DNA chain. UNG’s job is to find these rare uracils, excise them and replace them with the correct nucleotide. But how does UNG accomplish such a needle-in-the-haystack chore?
In extensive studies to date, researchers have used powerful single-molecule imaging methods to address this question. Single-molecule spectroscopy, for instance, allowed scientists to label a single UNG molecule and watch its movements along DNA. Such experiments led scientists to hypothesize that proteins like UNG employ two manners of movement: They “slide” along a strand of DNA, and they “hop” from one section of a DNA strand to another.
But the technology had its limitations, namely low resolution. “It was like seeing a car moving on a road from the vantage point of a passenger in an airplane,” says Stivers. The airplane passenger might be able to tell that the car was moving down the road in a certain direction, but would not be able to discern the topography of the terrain or the small excursions taken by the car during its journey. In much the same way, scientists have been left with many questions about the precise nature of the hop and slide. For example, how long did UNG slide along a strand before hopping off?
So Stivers and Schonhoft took a different approach. Instead of directly observing the enzyme through single-molecule labeling, they decided to use biophysical principles to infer the enzyme’s movements. Basically, the team’s approach relied upon a small molecule “trap,” a molecule that binds to, or traps, UNG enzymes that are dissociated from DNA while leaving alone those UNGs that are bound to DNA. In other words, the trap traps UNGs that have hopped off the DNA strand.
Stivers and Schonhoft used the trap, along with other biophysical methods, to time UNG’s movements. Their findings confirm that the enzyme does indeed slide and hop, but very differently than previously proposed.
UNG attaches to one small part of the genome’s jumble of DNA and then slides back and forth repeatedly along a small (5 base pair) segment, says Stivers. It then hops off that segment and within 50 nanoseconds hops onto another portion of DNA and starts to slide again.
The short slide, followed by a hop, is a perfect strategy, says Stivers. Sliding back and forth repeatedly over a small region enables the enzyme to exhaustively inspect that area for flaws. However, sliding is relatively slow. If the enzyme relied only on sliding, it would never complete its task. The hop, however, enables the enzyme to barrel around the genome.
“The word ‘dynamics’ or ‘dance’ is a good way to describe UNG’s movements,” says Stivers. And the new approach reveals the choreography in far more intricate detail than previous methods achieved. “Our approach,” says Stivers, “allows you to look at that car and be able to tell when the car is moving over potholes and climbing up hills.”
Although the recent study focused on UNG, Stivers says preliminary experiments on other DNA repair enzymes strongly suggest they, too, perform the same hop and slide dance.
In addition to studying the biophysics of that dance, Stivers is also directing several projects that might translate such knowledge into new drugs or therapies. “UNG,” Stivers notes, “is turning out to be a potentially interesting pharmacologic target against HIV-1.” A protein expressed by HIV degrades UNG in some immune cells, he explains. Unable to repair damaged DNA, these cells are then more prone to infection. If researchers designed a drug that blocks the interaction between the HIV protein and UNG, they might be able to protect the immune cells from infection.
In another project, Schonhoft is generating engineered forms of UNG that can slide and hop better than the native enzyme. “We’re asking, can we improve repair?” says Stivers. Such engineered enzymes could be used to understand basic principles of site location and the design of novel DNA modifying enzymes for biotechnology.
At the same time, Stiver’s team has developed a high-throughput platform for design of small molecules that bind and inhibit enzyme targets. He is currently applying this approach to human DNA repair enzymes and to other enzymes that play a role in cancer.
While the prospect of such medical applications stimulates his research, Stivers says what pulls him to the lab every day is the compelling nature of basic science—the urge to expand scientific knowledge. “My philosophy is, Go for the questions you’re interested in, and usually all sorts of interesting and sometimes unanticipated findings come out.”