A new technique for assessing the damage radiation causes to
DNA indicates that the spatial arrangement of damaged sites, or
lesions, is more important than the number of lesions in determining
the severity of the damage. The technique, developed by scientists at
Department of Energy (DOE)'s Brookhaven National Laboratory,
helps reveal why high-energy charged particles such as the heavy ions
in outer space are more potentially harmful than lower-energy forms of
radiation such as x-rays and gamma rays. The research could help
clarify the risks faced by future astronauts flying long-term missions
to the moon or Mars. It will be published in the March 19, 2008 issue
of the journal Nucleic Acids Research.
The technique uses different colored fluorescent "tags"
instead of radioactive ones to monitor repair of damage to DNA, life's
genetic instruction molecule. Because these fluorescent tags reduce the
amount of hazardous waste associated with the research (and its cost)
the Brookhaven scientists, Betsy Sutherland and Brigitte Paap, now at
Arizona State University, have been recognized by DOE's Office of
Science for their "Best in Class" pollution prevention innovation.
"Understanding the effects on humans of radiation exposure -
whether in the natural environment, in outer space, in the workplace,
or due to radiation therapy - requires insight into the induction and
repair of damage to DNA," said Sutherland, an expert in the study of
space radiation. "It's very rewarding to come up with a new technique
that helps us understand this process while at the same time reducing
the waste associated with traditional techniques."
Radiation can damage the DNA "double helix" - a two-stranded,
twisting molecule - in a variety of ways: 1) by knocking off one or
more of the DNA "bases" known by the letters A, T, G, and C, which form
the bonds between the two strands of the double helix; 2) by oxidizing
these bases; or 3) by breaking through one or both strands. All can
result in a failure of the molecule to perform its main task - telling
cells which proteins to make. That can lead to out-of-control cell
growth (cancer) or death.
Cells can often repair radiation-damaged DNA, using
specialized enzymes to excise and patch up the damaged segments. But
damage from ionizing particle radiation appears to be harder to repair
than that caused by lower-energy forms of radiation such as x-rays and
Scientists have long hypothesized that the reason for this
difference was that the high-energy ionizing particles caused more
complex damage containing many lesions close together on the DNA,
leading to slower and less-accurate repair. The technique developed by
Sutherland and Paap allowed them to test this hypothesis.
Using standard techniques of molecular biology, the scientists
created synthetic DNA with known lesions in a variety of spatial
arrangements with a red fluorescent tag attached to one end of the
strand and a green fluorescent tag at the other end. They then applied
a DNA repair enzyme, which clips the DNA at damaged sites. The
scientists then used gel electrophoresis to separate the fragments
according to their length. By looking at the red- and green-tagged
bands, and determining their length, the scientists were able to
measure how well the repair enzyme recognized and repaired the DNA
The results were surprising: Instead of being dependent on the
number of lesions, the ability of the repair enzyme to recognize the
damaged sites appeared to be most affected by the spatial arrangement
of lesions on the DNA strands.
The scientists found that the enzyme readily recognized and
repaired lesions on one of the DNA's two strands that occurred all to
one side of a reference lesion on the opposite strand (think of it as
"upstream"). These upstream lesions were successfully repaired
regardless of whether there were only two or many lesions in the damage.
If the lesions occurred "downstream" from the reference
lesion, however, the repair enzyme was unable to work properly, no
matter whether the clustered damage was a simple, two-lesion cluster,
similar to those caused by x-rays, or a complex multi-lesion cluster
like those induced by space radiation. When the lesions occurred in a
two-sided cluster both up and downstream from the reference lesion,
again the repair enzyme worked poorly.
"Since x-rays produce about half upstream, easily repaired
clusters and about half downstream, repair-resistant clusters, about
half of them would be readily repaired," Sutherland said. "The heavy,
charged particles in space radiation, on the other hand, produce much
more complex, two-sided clusters, containing so many lesions that most
of them are repair-resistant. This directional dependence of the
ability to repair lesions explains why damage from charged-particle
radiation, such as that encountered in outer space, is more harmful,"
The technique using fluorescently labeled synthetic DNA
fragments replaces a technique in which radioactive isotopes are used
as tags. While efficient, radioactive isotopes are more expensive than
the fluorescent tags. Also, using radioactive tracers requires frequent
preparation of freshly labeled DNA, and disposal of the experimental
samples as hazardous waste - which further increases the cost of the
Fluorescently labeled molecules can be stored frozen for long
periods. So the new method minimizes waste generation and improves
worker safety by avoiding the handling of radioactive material.
The technique may now be used throughout the DOE labs and in
universities and industry, and may be considered for other awards,
including the White House "Closing the Circle" award competition.
This research was funded by the Office of Biological and
Environmental Research within the U.S. Department of Energy's Office of
Science; the National Aeronautics and Space Administration (NASA); the
National Space Biomedical Institute; the National Institutes of Health;
and the Brookhaven Lab Pollution Prevention Program.