Proteins' Behavior May Aid in Understanding Disease
The instant when a cell divides is one of the most dramatic in all of biology. Yet the long period before mitosis--in which cells grow, copy their chromosomes, and grow some more--may be fraught with equally tense moments. Chromosomes are extraordinarily vulnerable to damage from ionizing radiation and other mutagens as they stretch out and are copied. In addition, replication is an exquisitely complex and delicate process that may be interrupted or aborted, leaving a single strand of DNA dangling without a partner. If the damage or failure to replicate is not detected and repaired, the drama of mitosis can turn to tragedy: defective DNA passed on to daughter cells can ultimately result in cancer and other diseases.
Fortunately, the cell has placed molecular monitors at various points during the cell cycle, whose job it is to check for damaged or unreplicated DNA. While biologists have brought to light some of the "checkpoint" proteins involved in detecting damaged and unreplicated DNA, there have been gaps in their knowledge of specific pathways.
One gap may be filling. HMS researchers Kristi Forbes and Tamar Enoch, in collaboration with colleagues at Washington University in St. Louis, report in the Oct. 2 Nature that they have identified not one but two checkpoint proteins involved in sensing unreplicated DNA, each with its own signaling pathway. What is surprising is that one of these proteins, Chk1, is also responsible for signaling the presence of damaged DNA.
"We once had this idea that they were each going to be like little separate circuitry mechanisms. And that's just turned out to be wrong," says Enoch, associate professor of genetics. She and Forbes, who is a graduate student in genetics, believe the cell "is probably being more sophisticated than we originally suspected. We originally thought the cell would flash a yellow light when there was DNA damage and a blue light when there was no DNA replication. In fact, maybe it flashes the yellow light five times when there's no replication and twice when there's DNA damage," Enoch says.
The cell's habit of using similar components in different ways could account for another surprising feature of the discovery. The researchers have found that the second replication-sensitive protein, Cds1, differs in many respects from Chk1, yet they both act on the same protein and, in fact, on the same stretch of amino acids.
The Protein Players
Enoch explains that normally mitosis begins when a protein, the equivalent of a mitosis on-button, is activated by a second protein. It turns out that Chk1 and Cds1 prevent this second protein from acting by phosphorylating it--that is, attaching a phosphate tag at a particular site. The phosphorylated protein is then recognized and sequestered by another set of proteins, which essentially tie its hands, preventing it from pushing the mitotic on-button.
Though the discovery is still confined to the realm of basic research, it has ramifications that someday could extend into the clinic. Many diseases--from the rare ataxia-telangiectasia and Bloom's and Werner's syndromes to more common forms of cancer--have been linked to defects in the checkpoint proteins that govern the cell cycle. These proteins could provide targets for more specialized chemotherapies.
"If cancer cells must lose checkpoint controls to become cancerous, this may be a way to get just at cancer cells," Enoch says. "They'll have certain cell cycle defects that we can exploit in devising chemotherapy." In addition, "keeping those checkpoint proteins functioning might well protect against cancer," she says.
Completing the Scene
The discovery fills in a story that until recently had only a beginning and an end. It started several years ago when Enoch and others discovered a set of genes in fission yeast cells that when mutated, made the yeast cells unable to stop dividing in the presence of unreplicated DNA. One, dubbed rad3, was especially interesting since it produced a protein that was similar to proteins known to be activated directly by DNA.
At the end of the putative replication checkpoint pathway was Cdc2, which had been shown to trigger mitosis when activated by another protein, Cdc25. "If you figured that Cdc2 is the downstream basic part of the cell cycle engine machinery, there must be some piece that connects rad3 with Cdc2," Forbes says.
To find the connecting piece, Forbes overexpressed the cdc25 gene in fission yeast cells, and then exposed them to a drug that would block DNA copying. This created a situation in which mitosis would occur even in the face of non-replicating DNA. She then looked for genes that when overexpressed could suppress the effects of the overabundant Cdc25 protein and prevent mitosis from occurring.
She examined 90,000 genes and found that chk1 and two others belonging to the 14-3-3 family could do the trick. "Now, the 14-3-3 proteins were shown to like binding to a particular sequence in phosphorylated proteins," Enoch says. It turns out that this is the same sequence that was revealed when Chk1 phosphorylated the Cdc25 protein. "So the way Chk1 works is to phosphorylate Cdc25, which then creates a recognition site for the 14-3-3 proteins."
Meanwhile, Forbes had tried mutating both the chk1 and cds1 genes in fission yeast. "What we saw was absolutely striking," Forbes says.
"When both were removed it was like removing the whole checkpoint pathway," says Enoch. Their Washington University colleagues purified large amounts of the Cds1 protein and found that it works by adding a phosphate tag to the same sites on Cdc25 as Chk1 does.
The researchers speculate Cds1 may be used to alert the cell to the presence of unreplicated DNA most of the time, and that Chk1 may act as a kind of pinch hitter. "Proteins often pick up each other's job," Enoch says--especially when the job is important. But if Chk1 can alert the cell to both damage and unreplicated DNA, how does the cell know which emergency to respond to? Enoch explains that perhaps it is not the messenger so much as how the message is sent--again, two flashes versus five--that triggers the appropriate response. "After all, you want to call a fireman when you need a fireman and a paramedic when you need a paramedic," she says.
--Misia Landau