When do cells with broken
DNA decide to repair themselves or die?
Friday, April 10,
2009
When cells undergo potentially catastrophic damage, for example as
a result of exposure to ionizing radiation, they must make a
decision: either to fix the damage or program themselves for death,
a process called apoptosis.
It's a stark decision that is as mysterious as it is remarkable,
involving what might be described metaphorically as a network of
internal alarms that detect damage to DNA packed tightly in the
cell nucleus. In intricate ways, cells orchestrate a response to
signals from such resident sensors when they are triggered by
exposure to radiation or other toxic processes, which if unchecked
can cause genes to mutate and cancerous tumors to begin
forming.
A team of biologists led by Professor Nicholas Tonks, Ph.D.,
F.R.S., of Cold Spring Harbor Laboratory (CSHL), this week revealed
results of experiments suggesting at least one way in which
cascades of intracellular signals are regulated at what they call a
decision point – as cells decide whether to repair broken DNA
strands or commit suicide following DNA damage.
In a report published online ahead of print in the Journal of
Biological Chemistry, Tonks and colleagues identify a protein with
the unlikely name Eyes Absent, or EYA, as performing a critical
role in setting the damage-repair machinery in motion. Engaged
within the larger context of a complex signaling cascade within the
cell, EYA regulates the formation of specialized microenvironments
on DNA, called gamma-H2A.X foci, which allow the cell to summon
repair enzymes to the site of broken DNA strands. The team's
experiments, conducted by Navasona Krishnan, Ph.D., of the Tonks
lab, showed that when Eyes Absent was not present in damaged cells
grown in culture, such foci were not formed and the cells went the
route of apoptosis—they programmed themselves to die.
'A lovely moment' of discovery
Tonks says the finding is "a powerful example of multiple lines of
research and different kinds of expertise coming together" in what
he describes as "a lovely moment" of synthesis and discovery. An
important collaborator in the work, C. David Allis, Ph.D., of the
Rockefeller University, had recently published a paper in Nature
describing a critical component of the DNA damage-repair signaling
cascade. The Tonks-Allis collaboration, along with contributions
from Seung Jun Kim, Ph.D., a protein crystallographer from the
Korea Research Institute of Bioscience and Biotechnology, led to
the assembly of a puzzle from pieces whose precise relation was not
previously understood.
The parts of the puzzle include a protein called H2A.X, one of a
species of proteins called histones that form structures around
which DNA is "spooled" for dense packing in the cell nucleus.
"David Allis showed us that this particular histone, which is found
at those critical decision points called gamma-H2A.X foci, has
phosphate groups added to its structure at particular points at the
end of the protein. David demonstrated that another kind of protein
--a kinase called WSTF -- attaches phosphate to critical site, a
tyrosine residue, at the extreme end of the protein," Tonks
explains..
"I was interested in the fact that when there is damage to
double-stranded DNA – catastrophic damage, such as when the strand
breaks in two – the phosphate molecule placed at the decision point
by WSTF has to be removed in order for the cell to send out signals
for DNA-repair enzymes to come to the scene."
The removal of phosphate groups from proteins is accomplished by a
family of enzymes called phosphatases – the focus of much research
in Tonks' CSHL laboratory. Tonks is well known for having
characterized the first of what has come to be understood as a
large superfamily of protein tyrosine phosphatases, or PTPs –
enzymes that specifically remove phosphate molecules from amino
acid residues called tyrosines. This function is critical in
regulating cellular signaling in normal and disease conditions. In
effect, important kinds of cellular signals can't be sent without
helper enzymes like PTPs that remove phosphate molecules from
specific locations, and kinase enzymes that perform the reverse
role – that of adding phosphates.
Another vital piece of the puzzle in the current work involves Dr.
Kim, who recently spent 15 months in the Tonks lab at CSHL, working
on growing crystals of protein phosphatases to determine their
structure, including that oddly named protein, Eyes Absent. Why
that one? As Tonks explains, "EYA is known from work on the fruit
fly, or Drosophila, to be a very unusual protein: it's the only one
we know of that acts in the cell nucleus to regulate genes – what
we call a transcription factor – while, in other contexts, is also
known to act as a phosphatase – that is, it can remove phosphate
groups from other proteins."
The role of Eyes Absent in DNA damage repair
EYA was shown to be a phosphatase by three separate groups in 2003.
Although it was thought to be a PTP - i.e., it was thought to take
phosphate groups off tyrosine residues in proteins - the identity
of its target proteins in the cell was unknown. Kim's structure was
important because it revealed a particular distribution of charged
residues on the surface of the protein that suggested to Tonks the
possibility that basic proteins, such as histone H2A.X, may be one
such critical substrate.
This led to the experiments in which Tonks' team showed that Eyes
Absent was in fact the protein that removed the critical phosphate
group from the end of histone H2A.X, thereby allowing the formation
of the so-called gamma-H2A.X foci, which set DNA repair in motion
when double strands were broken. When EYA was experimentally
"knocked out" via a technique called RNA interference, or RNAi,
damaged cells with double-stranded DNA breaks did not repair
themselves; instead, they simply died -- underwent apoptosis.
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Cold Spring Harbor Laboratory
Thanks to Cold Spring Harbor
Laboratory for this article.