Radical Thinking

Tullius turned that biochemical intuition into information about the structures of key proteins isolated from these unusual systems. He then turned his attention to DNA, devising a method that uses a highly reactive chemical—the hydroxyl radical—to probe the twists and turns of the double helix. The latter technique, now employed in labs around the world, has allowed Tullius to examine protein–DNA interactions, predict the positioning of nucleosomes, and explore the subtle changes in shape that correlate with gene function across the human genome.

“Tom is a chemist’s chemist who works on biological problems,” says Michael Brenowitz of the Albert Einstein College of Medicine. “He brings to the table a real understanding of chemistry, but he can also talk to biologists. That’s why his methods have been so widely adopted.”

“Innovation always occurs at the interface between fields,” notes Charles Cantor of Sequenom in San Diego. “By making clever use of chemistry to learn things about biology, Tom crosses that bridge.”

Of course sometimes he simply dangles off the bridge to collect samples. Growing up in San Clemente, Calif., Tullius helped to get his high-school chemistry lab certified as a water-quality testing station. “I would get up at 5 in the morning and ride my bike to the end of the jetty,” he says. He and his lab mates would then check the water samples for bacteria. “San Clemente was known for its beautiful beaches,” says Tullius. And for being the home of Richard Nixon’s Western White House. “We used to rent our house to the Secret Service when Nixon was in town.”

But Tullius was more interested in the chemistry than politics. “I’m one of those rare people who started in high school and essentially never changed,” he says. “I’ve been a chemist ever since.” After completing his undergraduate studies at the University of California, Los Angeles in 1973, Tullius headed to Stanford, where a synchrotron was being used “for high-energy physics and for generating Nobel Prizes,” he says. It also produced X-rays that could be used to poke into the structure of biologically interesting molecules, such as proteins. X-rays had been used previously to explore the nooks and crannies of crystallized proteins. But Keith Hodgson and his lab—of which Tullius was a member—were using X-rays in a different way, to characterize the atomic configuration of metal-containing proteins floating free in solution. “It was a tool nobody had ever used before,” says Tullius. “So amazing discovery after amazing discovery happened, because you could basically put any protein with a metal in it into the X-ray beam and get out this beautiful data.”

Tullius used the technique to examine a handful of proteins he’d obtained straight from the source, including xanthine oxidase from buttermilk, plastocyanin from bean leaves, and azurin isolated from Pseudomonas. The latter—a beautiful blue electron-transport protein—offered up the shortest bond ever measured between a copper atom and the sulfur that anchors it to the protein. That diminutive bond allows the protein to rapidly shift electrons, a property key to its biological function. “In some ways this was the low-hanging fruit,” says Tullius. “But it was really interesting, beautiful low-hanging fruit.”

As a postdoc at Columbia University, Tullius starting working with DNA. And by the time he joined the faculty at Johns Hopkins in 1982, Tullius says, “people were starting to think that the structure of DNA could be important for protein recognition. So I started to think about what sort of measurements I could make to see if there are places along natural DNA, like a promoter for a gene, where the helical twist would change.” A slight change in twist, he reasoned, could serve as a signal to attract DNA-binding proteins looking for a place to rest.

“I was sitting at the symphony in Baltimore, listening to music and wondering whether there was some chemistry that could [get at the helical twist],” he says. And then he remembered the hydroxyl radical—a highly reactive molecule capable of breaking the DNA backbone. This probe could be used to detect subtle differences in DNA structure, because even a slight change in twist in the double helix alters the accessibility of the atoms in the backbone. “The experiment worked beautifully,” says Tullius. He and his colleagues were able to measure the helical twist of a piece of viral DNA, and map transcription-factor binding sites—findings published in Science in 1985.

“The real beauty of the method is that it’s cheap and easy,” says Brenowitz, who uses hydroxyl radicals to study the dynamics of RNA folding. “It requires reagents that cost a few pennies and can be done on a bench top—yet it gives you a lot of information.” For example, Tullius used the approach to reexamine the interaction of the lambda repressor with its DNA operator site. That work was “fresh and insightful,” says Brenowitz, “because it revealed details about the protein–DNA interaction that other methods had missed.” The resulting paper—published in PNAS in 1986—has been cited more than 450 times.

“It was a little bit like my early days in bioinorganic chemistry,” says Tullius of the variety of problems he then tackled. “These wonderful systems were out there, ripe for studying with a new structural technique. So every experiment gave us an exciting result.” His team watched DNA wrap around nucleosomes, and transcription factors wrap around DNA. And then Tullius really upped the ante. “I thought, wouldn’t this be great to do, not just on an isolated DNA fragment, but in a whole genome.”

In 1997, Tullius brought that radical plan with him to Boston University, where he built a database containing the cleavage profiles of dozens of DNAs he’d bombarded with hydroxyl radicals. With the help of a bioinformatically-inclined student, Tullius and his team then designed an algorithm that allowed them to look at any sequence of DNA and predict how it would stand up to hydroxyl radical attack, which reveals its underlying structure. “That means that now you don’t have to do this experiment for the entire genome,” says former student Steve Parker of the National Human Genome Research Institute. Although having additional data never hurts, the algorithm as it stands does a bang-up job predicting DNA structure based on sequence. Running that analysis on the entire human genome—as part of the ENCODE project—revealed that our DNA “is not just uniform, canonical B-form DNA”—the structure made famous by Watson and Crick. Instead, Parker says, “the shape of the molecule varies quite dramatically throughout the genome.”

And the biggest surprise: DNA’s shape is even more conserved than its sequence. The ENCODE consortium found many instances where the sequence of a known functional element was not conserved from species to species. “We proposed that if the shape of a DNA segment were important for its function—and different sequences could give you the same shape—then a sequence could change over time but the shape might be conserved,” says Tullius. And that’s exactly what they found.

“The fact that they were able to extract this remarkable result about shape being better conserved than sequence, that’s just really an unexpected and exciting development,” says Howard Hughes Medical Institute investigator Barry Honig of Columbia University. “It places emphasis on the three-dimensional structure of DNA.” Honig and others are finding that subtle variations in shape help transcription factors locate their DNA binding sites. “So we need to go back and start studying DNA as a three-dimensional molecule and not just a string of letters,” adds Honig. “Which is what Tom has been doing for years.”

“Most people think Watson and Crick figured this out 50 years ago and that’s the end of the story,” says Parker. “But it’s not true. This is an exciting area to be studying right now: seeing how genome function could be encoded in the structure of DNA, not just in its primary sequence.”

“He’s had this idea for such a long time,” says former student Jeff Hayes of the University of Rochester Medical Center. “It’s nice to see that it’s come full circle and people are now really appreciating the role of DNA shape in biological processes.”

Next up, Tullius does plan to attack an entire genome inside a live cell—first in E. coli, then in yeast—to see where all the transcription factors and other regulatory proteins sit. “We’ve tried to get this funded twice,” says Tullius. So far without success. “But we’re going to do it, anyway. I think there’s a good chance it will work. Which is why it’s worth a try.”

TOM TULLIUS, F1000 FACULTY MEMBER, CHEMICAL BIOLOGY, SINCE 2002.

The papers Tullius gravitates to are those that present “solutions to problems that have been giant mysteries for a really long time.” One of his favorites presented the crystal structure of a junction between canonical, right-handed B-DNA and its left-handed cousin, Z-DNA (Nature, 437:1183–86, 2005). MIT’s Alex Rich first stumbled across Z-DNA in 1979, a finding Tullius says “shocked Francis Crick.” But several questions remained. Do B-DNA and Z-DNA coexist in cells? If so, how does DNA make the transition from right- to left-handed? Rich’s 2005 structure—26 years in the making—answered those questions at a glance. Not only can the two forms lie side by side, but the switch from one to the other is accomplished by simply flipping out two of the bases, which allows the helix to reverse its twist without kinking. “I think that was the quickest F1000 pick I wrote up,” says Tullius, “because I thought it was so cool.”