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
