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From telomere study, a new
checkpoint gene

Perserverance and serendipity yield a gene that plays a critical role in the repair of damaged DNA
Joshua Silverman knew Edison’s quote about genius, but he was approaching that 99 percent perspiration mark, and the one percent inspiration wasn’t kicking in. His long, labored experiments, the basis of his doctoral thesis, were not paying off, and his goal of earning a Ph.D. from Rockefeller University was beginning to seem out of reach.
For his graduate studies in Titia de Lange’s Laboratory of Cell Biology and Genetics, Silverman was exploring whether a gene that lengthens the ends of chromosomal caps called telomeres in yeast cells might have a counterpart that plays the same role in human cells. If so, it could lead to clues about how an enzyme called telomerase can selectively add DNA back to chromosomes, lengthening the life span of a cell.
Over the years de Lange and the members of her lab have made fast progress in understanding telomeres, the specialized DNA complexes that, like the plastic sleeves on shoelaces, stop chromosomes from unraveling. To understand how chromosomes are controlled and how they are linked to aging and cancer, de Lange has characterized stuttering sequences, duplicitous enzymes and loopy hooks.
When she and Silverman embarked on their project, they expected a fairly uncomplicated process. To find the human counterpart to their yeast gene, Rif1, they would look through genetic databases. “Then Josh would make antibodies to Rif1, show that the protein is present at chromosome ends, study how it regulates telomeres, and he would get his Ph.D.,” recalls de Lange. “We anticipated this would be a straightforward story.”
But the protein wasn’t on telomeres — and the scientists couldn’t find any evidence it controlled telomere length.
After a year of experiments, Silverman and de Lange were prepared to accept that Rif1 simply wasn’t where they thought it ought to be. In a last-ditch effort to salvage some knowledge from their work, de Lange suggested they see what would happen if they exposed human cells to radiation. “In yeast, Rif1 is part of a pathway that is also involved in a cell’s response to radiation, so we thought this might point us toward the function of Rif1 in human cells,” de Lange explains.
The experiment was a success. It turned out that human Rif1 did do something interesting; they’d just been looking in the wrong place. The radiation-damaged cells showed Rif1 on the sites where the DNA had been broken by the radiation. “We thought Rif1 would be sitting on natural chromosome ends, but we instead found it only binds to ends that are made when DNA is damaged,” de Lange says.
Thanks to that early failure, we now know that Rif1 is closely linked to a cell’s ability to repair DNA, and it is part of a pathway that includes several well-studied cancer susceptibility genes including BRCA1, implicated in breast and ovarian cancers.
“This is one of the most intensely studied pathways in the cell because of its importance to cancer biology, and through completely fortuitous ways, we bumped into an important component of that pathway,” says de Lange.
Encouraged, Silverman tested whether Rif1 was controlled by a master regulator of the DNA damage response known as Ataxia Telangiectasia Mutated (ATM) kinase, named after a faulty gene that predisposes people who inherit it to get cancer or other diseases.
Researchers have found that the ATM kinase blocks a cell’s ability to divide when its DNA is damaged, and that mutations of a few key players within the ATM pathway are implicated in several human cancers, de Lange says. “Downstream of ATM kinase are the disease genes BRCA1, BRCA2, Chk2, Mre11 and Nbs1,” she explains. “So if all the players in this little regulatory neighborhood in the cell are all involved in human cancer predisposition, it doesn’t take much to suspect that Rif1 might also be.”
When they examined cells that lacked ATM kinase, the Rockefeller researchers found  that Rif1 had lost the ability to bind to damaged DNA, showing that Rif1 is a slave to ATMs instructions. “There are several master regulators of the DNA damage response, and they all funnel through the same proteins, so we assumed that if we knocked out ATM, Rif1 might still respond,” says de Lange. “But that wasn’t so. Rif1 only listens to ATM, and is not regulated by anyone else. That makes it very unusual.”
The de Lange group also discovered that Rif1’s role was to halt the replication of a damaged cell so that the DNA could be repaired. Postdoctoral researcher Hiroyuki Takai used small strands of RNA to silence the Rif1 gene, and found that when these cells were exposed to radiation, they continued to synthesize their DNA. “And if chromosomes replicate when they are damaged, you get a real mess, a lot of replication mistakes that could potentially create a cancer cell,” de Lange says.
“The story turned out to be much more significant than we expected,” says de Lange. Not only did the scientists uncover a possible new player in the promotion of cancer, but de Lange expanded the focus of her lab to include this aspect of cancer biology and the project is now continued by postdoctoral fellow Sara Buonomo. And Silverman, for his part, got a cover article in the September 1 issue of Genes & Development and, finally, in June of this year, three new letters after his name

October 8, 2004



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