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The newspaper of The Johns Hopkins University August 20, 2007 | Vol. 36 No. 42
Finding That One-in-a-Billion Error

Cell machinery sniffs out gene damage by trying on DNA for size

By Audrey Huang
Johns Hopkins Medicine

Errors in the genetic code can give rise to cancer and a host of other diseases, but finding these errors can be more difficult than looking for the proverbial needle in the haystack. Now, scientists at Johns Hopkins have uncovered how the tiny protein machines in cells tasked to search for potentially life-threatening genetic damage actually recognize DNA errors.

Appearing on-line this week in Nature, the Johns Hopkins team describes how the UDG enzyme (for uracil DNA glycosylase) scrutinizes the shape of DNA building blocks by holding onto them and testing their fit into a specially sized pocket. The UDG pocket holds onto mistakes only; the enzyme loses its grip on the right building blocks, which fall back in line with the rest of the DNA.

"Locating damage in DNA is critical for a cell's survival. So much can go wrong if damage goes unrepaired; cells can't tolerate any of this going on," said study author James Stivers, professor of pharmacology and molecular sciences in the School of Medicine. "But the question is how these enzymes find the few mistakes among the billions of correct building blocks in DNA."

One typical error that occurs is the DNA building block cytosine being chemically converted to uracil, a similar-looking building block not normally found in DNA. "Even water can cause DNA damage," Stivers said. "It's not a fast reaction, but water does convert the occasional cytosine into an unwanted uracil."

To figure out how the enzyme responsible for cutting unwanted uracils out of DNA works, Stivers and colleagues studied a tiny segment of DNA, asking whether the "breathing" properties of DNA played a role in the search process of UDG. "Although the bases in the DNA double helix resemble the rungs of a ladder, the rungs are not that sturdy," Stivers said. "They actually pop in and out of the helix a bit, randomly."

Each time a base pops out of the helix, it exposes itself to water. So, using a special chemical trick, the researchers magnetically labeled water to allow them to follow its interaction with bases that had randomly popped out of the DNA helix. The team could follow which bases popped out, and for how long, using a strong magnet.

After studying DNA breathing by itself, the researchers added UDG into the mix. They then saw that UDG holds onto the normal DNA building block thymine (T) after it pops out of the DNA on its own. However, because T is not identical to U, UDG then lets it fall back into the DNA helix.

When the DNA contains an unwanted U, the UDG enzyme actually grabs on and pulls it all the way out and holds it in the enzyme's pocket. Once sitting in this pocket, the enzyme clips out the U, leaving a gap in the DNA for other repair machinery to fill in with the correct building block.

"This is the first time we've been able to actually see how an enzyme discrimin- ates between right and wrong bases in DNA," Stivers said. "Our discovery helps us appreciate what properties of DNA itself might lead to errors that are not repaired. The finding may help address how and where diseases like cancer arise in the genome."

The research was funded by the National Institutes of Health and the National Science Foundation.

Authors on the paper are Jared Parker, Mario Bianchet, Daniel Krosky, Joshua Friedman, L. Mario Amzel and Stivers, all of Johns Hopkins.


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James Stivers


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