A chance meeting in a hallway nearly 10 years ago has lead Stephen Kowalczykowski to new insights into the molecular machines that look after DNA. Now Kowalczykowski's colleagues have named him as the 2005 Distinguished Research Lecturer, the highest award presented by the members of the Davis Division of the Academic Senate.

"I am surprised and honored. The highest honors are those that come from your peers," said Kowalczykowski, a professor in the Section of Microbiology and Director of the Center for Genetics and Development in the Division of Biological Sciences.

Phyllis Wise, dean of the division, said she was pleased about the honor. "Steve's scientific contributions are internationally respected. His work has significantly advanced our understanding of DNA replication and repair. Additionally, as the founding director of the Center for Genetics and Development, Steve has led the development of an outstanding program," she said.

Kowalczykowski will give his lecture on May 10.

The announcement marked the second time last month that he was presented a significant honor by his peers. Kowalczykowski was also among the UC Davis faculty members recognized as a new fellow in the American Academy of Arts and Sciences. (See story on page 3.)

John Roth, chair of the Section of Microbiology, and Martin Privalsky, a professor of microbiology, nominated Kowalczykowski for the distinguished lecturer award. "Much of biology is dedicated to figuring out at a molecular level how some aspect of a cell works. The unique aspect of Steve's research is that the biological function can be visualized directly," Roth said.

DNA repair is one of the most fundamental processes in biology. When DNA repair goes awry, the consequences can be deadly — cancers, mutations and birth defects. Kowalczykowski is particularly interested in the motor proteins that drive the machinery of DNA repair. Among them is RecBCD, a complex of three subunits that travels along the DNA helix, unwinding it as it goes.

"I used to take apart watches and radios as a kid, I like mechanical things of all scales from nanometers to meters. I've always been interested in how things work, and this is uncovering how nature works," Kowalczykowski said.

'Visual biochemistry'

About half the effort in Kowalczykowski's lab is devoted to what he calls "visual biochemistry." Working with Ron Baskin, professor of molecular and cell biology, and others they have developed the technology to watch single DNA repair enzymes such as RecBCD in real time. That work, which has yielded a series of papers in top-ranked journals such as Nature and Cell, grew out of a chance meeting in 1996.

Kowalczykowski was walking down the hallway when Baskin invited him to come and see a device called an optical trap that Baskin had built with Yin Yeh, professor of applied science. An optical trap, or "optical tweezers," uses laser beams to trap and move tiny objects or beads. The device was invented by Steven Chu of Stanford University, awarded the Nobel Prize for his work in 1997. Baskin said that he was thinking of using the device to study motor proteins in muscle fibers.

Kowalczykowski had just returned from a visit to a lab in Oregon, where he had seen some work on attaching DNA strands to surfaces. His lab was also using a fluorescent dye to label DNA and measure the activity of a helicase enzyme that unwinds DNA, dislodging the dye and quenching the fluorescence. The three pieces quickly fell together.

"Within 15 minutes, we'd mapped out a series of experiments to see the RecBCD enzyme travel down DNA," Kowalczykowski said.

But achieving that goal took another five years, with the experimental work led by postdoctoral researcher Piero Bianco, now an assistant professor at the University of Buffalo. Bianco collaborated with Baskin, Yeh and researchers at the Lawrence Livermore National Laboratory to build the first instrument of its kind in the world.

The solution they hit on was to attach one end of a piece of DNA to a tiny polystyrene bead. The DNA was labeled with a dye that fluoresced as long as it was attached to the double-stranded DNA helix.

The beads were placed in a chamber with fluid moving through it, manipulated into mid-stream with the lasers, then held in place in the middle of the flow. The DNA streamed out behind, like a flag in the breeze. Bianco then added the RecBCD helicase enzyme to the chamber.

When RecBCD latched onto the free end of the DNA, it got to work unwinding the double helix, separating the strands and digesting one of them. As the enzyme moved up the strand, it dislodged the dye, switching off the fluorescence. Through a microscope, the researchers could see the glowing "tail" attached to the beads get steadily shorter.

Garnering wide attention

The results were published in the journal Nature in January, 2001 with Bianco, Yeh, Kowalczykowski, and Baskin as authors, together with Laurence Brewer, Michele Corzett and Rod Balhorn from the Lawrence Livermore National Laboratory. The story attracted wide attention. When the San Francisco Chronicle put a movie of the experiment on its Web site, it attracted more viewers than a video clip of President Bush's first inauguration.

That breakthrough lead to a series of new discoveries. For the first time, researchers could study a single biochemical machine at work.

For example, in the bacteria E. coli, a DNA sequence called Chi is known to affect the repair of DNA. Chi is associated with "hotspots" in DNA where genes are most readily exchanged.

Postdoctoral researcher Maria Spies made DNA sequences containing Chi and let RecBCD loose on them. The enzyme made its way up the DNA at the usual rate, but when it reached the Chi sequence, it stopped and then re-started but at a slower speed.

"It's a dramatic finding. It's the first example of a DNA sequence actually controlling the movement of an enzyme along it," Kowalczykowski said. The work was published in the journal Cell in 2003, raising new questions: How exactly does Chi cause the RecBCD enzyme to stop and slow down?

Mark Dillingham, a postdoctoral researcher in the lab, found that both the RecB and RecD subunits are motor units that can function independently. One sits on each strand of the double stranded DNA helix, and together they drive the RecBCD complex in the same overall direction. That work resulted in another paper, published in Nature. The Chi sequence appears to turn off one of the motors, slowing movement of the enzyme complex.

Meanwhile, Mark Singleton, a researcher in Dale Wigley's lab in England, was trying to use X-ray imaging to produce a crystal structure for RecBCD. Growing large proteins as crystals is notoriously difficult. While working in Kowalczykowski's lab, Dillingham, who had some limited success with crystallization, had had the idea of stabilizing RecBCD by attaching it to DNA, and after returning to England he put the idea into effect.

The resulting crystal structure, published in Nature in 2004, elegantly shows RecBCD as a machine for processing DNA. The double stranded helix actually splits over a peg in the protein, and the separated strands are fed into the two motor units. The Chi sequence is read in a hole in the C subunit.

"It's very intellectually satisfying. What everyone loves is when something makes sense," Kowalczykowski said.

Ongoing collaboration

Kowalczykowski is still collaborating with Baskin's lab on the equipment for single-molecule imaging, and developing new questions that can be tested with the instrument. RecBCD is just one of about 25 proteins involved in maintaining DNA in E. coli. Humans have about three times as many proteins doing the same kind of thing, Kowalczykowski said.

"Every week we think of new things to do with this technology," he said.

Kowalczykowski earned his bachelor's degree from Rensselaer Polytechnic Institute in New York in 1972 and a doctorate in chemistry and biochemistry from Georgetown University in Washington, D.C., in 1976. He carried out his postdoctoral research with Professor Peter von Hippel at the University of Oregon, where he became interested in DNA-protein interactions. He was a faculty member at Northwestern University from 1981 to 1991, when he joined UC Davis. He is a Fellow of the American Association for the Advancement of Science and of the American Academy of Microbiology.