Overturning 60 years of scientific presumption, new
evidence from Johns Hopkins scientists shows that enzymes
nibble away at chromosomes when the chromosomes' protective
tips, called telomeres, get too short.
Much like the plastic tips on shoelaces, telomeres
protect the ends of chromosomes. When telomeres get too
short, cells usually die. If they don't, the unprotected
ends drag the chromosomes through an ugly assortment of
fusions that lead to rearrangements, deletions and
insertions that scramble the cell's genetic material and
can lead to cancer. Until now, scientists had presumed that
the fusions were the first thing to happen when telomeres
stop protecting the chromosomes.
"We have always thought that if we can understand how
shortened telomeres create genomic instability, we might be
able to find targets in that process to push abnormal cells
toward death and away from trying to repair themselves,"
said Carol Greider, director of
Molecular Biology and
Genetics at the School of Medicine. "Now it turns out
that what we've always thought was the first step in the
process is not the first step at all."
Writing in the December issue of Molecular and
Cellular Biology, Greider and graduate student Jennifer
Hackett described experiments with yeast that revealed that
instead of just sticking, or fusing, end-to-end,
chromosomes whose telomeres are too short are first nibbled
by enzymes that normally clean up broken chromosomes.
"The fusion pathway was our favorite model of what
goes wrong first when telomeres get too short. All the
papers use that model to describe how loss of telomere
function causes genomic instability," Greider said. "But
just because we see a lot of something doesn't mean it's
the first thing that happens. We were quite surprised to
find that fusion isn't the first effect of short
telomeres."
In the traditional fusion scenario, officially called
the "breakage-fusion-bridge" pathway, a cell interprets
chromosomes with short telomeres as being broken and sets
in motion machinery to "fix" the break by fusing it to
another exposed end. The unintended consequence of this fix
is the connection of two chromosomes. If the fused
chromosomes are pulled to opposite sides of a dividing
cell, they form a bridge that breaks randomly as the cell
divides, and the process begins again.
To test whether this was the correct or only scenario,
Hackett inserted genetic markers into a yeast chromosome to
reveal where genetic damage most often occurs when
telomeres got too short. Instead of random damage, she
discovered that the marker at the very end of the
chromosome was most likely to be lost, and the marker
closest to the chromosome's center the least likely.
"If fusion and breakage was the primary mechanism of
gene loss, the pattern of loss would have been random; each
marker would have been just as likely as the others to be
lost," Greider explained. "The marker loss we saw was not
at all random, so we knew some other mechanism was at
work."
Then, Hackett studied the engineered chromosomes in
yeast missing an enzyme called exonuclease that normally
recognizes and chews up broken chromosomes one strand of
DNA at a time. Without the enzyme, there were fewer
chromosome rearrangements, offering strong evidence that
this enzyme is doing the damage.
"Fusion happens, but it's not the primary mechanism
that triggers gene loss after telomeres get too short,"
Greider said. "Instead, exonuclease activity causes the
bulk of immediate gene loss."
To prove that fusion does indeed result in a random
pattern of marker loss, Hackett made an artificial fused,
or dicentric, chromosome, complete with genetic markers to
identify which segments were destroyed. Since Hackett
engineered it, this fused chromosome could not already have
been "nibbled" by an exonuclease.
"We demonstrated that fused chromosomes do break
randomly, at which point exonucleases attack the exposed
ends," Greider said. "Fusion is a big part of what leads to
major genomic instability when telomeres aren't working,
but it's not the initial problem. Our discovery should
spark researchers in the field to think along new
lines."
Greider cautioned that they still need to verify that
the same mechanism is to blame for genomic instability in
mammalian cells as in yeast. If so, identifying other
proteins that work with exonucleases may offer a target to
block the process and push cells in cancer toward death
instead of genomic instability.
Hackett, who is now a postdoctoral fellow at Harvard
Medical School, was funded by the Johns Hopkins Predoctoral
Training Program in Human Genetics and Molecular Biology
and the National Science Foundation. The studies were
funded by the National Institute of General Medical
Sciences, part of the National Institutes of Health.
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