Harvard Medical School
Dr. Walter is also a professor of biological chemistry and molecular pharmacology at Harvard Medical School.
Mechanisms of DNA Replication and Repair
If a few of his undergraduate courses had turned out differently, today Johannes Walter might be lecturing on Monet's brushwork or curating a Vermeer retrospective. When he started at the University of California, Berkeley, Walter was torn between studying art history and biology. But he struggled in humanities classes, he recalls, while science classes were "a breeze." Walter remembers being impressed by a demonstration in his introductory biology class that showed phospholipids in water spontaneously arranging themselves into a double layer similar to a cell's membrane. "The idea that the laws of chemistry were the driving force of life was so powerful to me that I decided to study biochemistry."
Since the mid-1990s, Walter has been probing the intricate mechanism that copies a cell's DNA in preparation for division. One reason he's made so much headway on this topic is the technique he devised during his postdoctoral studies at the University of California, San Diego. The method involves preparing highly concentrated nuclear extracts from frog eggs. These extracts recapitulate a wide variety of complex cellular processes, including DNA replication and DNA repair, while allowing extensive experimental manipulation. Walter says he started using the technique to study DNA duplication "almost by accident." He initially intended to use the nuclear extracts for a project he quickly realized wouldn't work, so he had to scramble to find another question to tackle. The experiment he chose challenged the dogma of the day that DNA replication requires an intact nucleus. "I took this nuclear juice and added DNA to it, and lo and behold, the DNA was replicated," he says.
Walter's lab has used the frog egg extracts to uncover new details about the structure and function of a key participant in DNA replication, the MCM2-7 complex. He calls it "the first violin in the orchestra of DNA replication" because it unravels the DNA so that other enzymes can copy it. Walter and colleagues found that each MCM2-7 complex works alone, rather than pairing up with another MCM2-7 complex as some researchers had thought. And their work shows that after the complex attaches to the double helix, it has to go through a major reorganization before it can move along and unravel the DNA.
While studying the binding of MCM2-7 to DNA, his team also identified a surprising mechanism that prevents cells from duplicating their DNA more than once. In our cells, DNA replication usually starts at some 100,000 different sites. To begin the process, each site needs "permission" from a protein called Cdt1, which helps position the MCM2-7 complex on the DNA. Once Cdt1 gives the go-ahead, specific enzymes demolish it, thus preventing a second round of replication. Walter's group discovered how the timing of Cdt1's demolition is controlled so that it occurs only after replication has begun. The researchers showed that Cdt1 latches onto a protein called PCNA that travels with the replication apparatus. The binding of Cdt1 to PCNA flips a switch that promotes Cdt1's demolition. This in turn prevents new MCM2-7 molecules from binding DNA, and replication is thus limited to one round.
After his lab studied DNA replication for several years, an unexpected result expanded Walter's interests into DNA repair. One day, his team added a damaged stretch of DNA to egg extracts, expecting that the DNA injury would halt replication. But it didn't, demonstrating that the extracts can also repair DNA during replication, a topic Walter has been studying ever since. His lab has applied the egg extract technique to explore how cells fix a severe type of DNA damage called an interstrand crosslink (ICL), in which the two DNA strands stick together. Patients with the rare genetic disease Fanconi anemia can't mend ICLs, but researchers weren't sure why.
Walter's team found the glitch. "It was the first molecular description of what is presumably going wrong in these patients," Walter says. Walter's lab has recently turned its attention to understanding how other proteins, such as BRCA1 and BRCA2, coordinate DNA replication with DNA repair. BRCA1 and BRCA2 are of particular interest because defects in these proteins dramatically raise the risk of breast and ovarian cancers.
His team has also branched out into filmmaking—on a very small scale. The researchers have developed a technique they call PhADE to observe an individual enzyme in real time and track its interactions with other molecules at a specific replication site. "We can actually make a molecular movie of DNA replication," he says.
Although Walter long ago gave up his ambition to become an art historian, he says he still tries to bring an artistic sensibility to his work. "In the lab I try to do experiments that are interesting, and I get pleasure from results that are aesthetically pleasing," he says.