Ionizing radiation, toxic chemicals, and other agents continually damage the body's DNA, threatening life and health: unrepaired DNA can lead to mutations, which in turn can lead to diseases like cancer.
Intricate DNA repair mechanisms in the cells' nuclei are constantly working to fix what's broken, but whether the repair work happens "on the road" - right where the damage occurs - or "in the shop" - at specific regions of the nucleus - is an unanswered question.
That question may now be closer to an answer. By comparing computer models of damaged human DNA with microscopic images of human cells that reveal focal sites of radiation-induced damage, researchers in the Life Sciences Division (LSD) of the Department of Energy's Lawrence Berkeley National Laboratory, with colleagues at NASA and the Universities Space Research Association, have found evidence that indeed there are specific regions where broken DNA is concentrated for repair.
"NASA has long been interested in the radiation hazards in space," says LSD's Sylvain Costes, who led the study. "On a trip to Mars, astronauts will be exposed to cosmic rays for as long as three years, so NASA has been trying to come up with a mechanistic model of DNA repair to estimate the increased risk of cancer. We are helping to develop such a model."
In many NASA studies cells have been exposed to particles like those found in cosmic rays, such as energetic iron nuclei produced in accelerators at Brookhaven National Laboratory. The goal is to determine how many double-strand breaks (DSBs) , in which both strands of the DNA double helix are severed , occur per gray of radiation. (One gray is equivalent to 100 rads, an older unit signifying "radiation absorbed dose"; a gray equals one joule of energy absorbed per kilogram of matter.)
DSB yield can be measured by pulling apart a cell's radiation-severed DNA using gel electrophoresis: the shorter the fragments in the gel bands, the more frequent the breaks. Interpreting the band patterns thus leads to an estimate of the average number of breaks per cell for a given dose and time following exposure. NASA has developed a computer model based on these measured DSB yields to predict DSB formation in hypothetical human cell nuclei.
On the other hand, gel electrophoresis fails to indicate the severity of the damage, or where in the nucleus the double-strand breaks occur. The default assumption has been that DSBs occur randomly in a homogenous distribution of DNA in the nucleus.
Most gel electrophoresis studies indicate about 25 to 30 DSBs per gray of gamma rays. In microscopic images of real cells, however, the visible sites that might be assumed to correspond to double-strand breaks , sites called "radiation induced foci," or RIF , occur at a lower rate, only about 15 per gray, depending on the cell type.
"What we see through the microscope is not the broken DNA itself but a collection of proteins associated with breaks, which we have labeled with fluorescent stains. These include modified histones, which are part of the chromosomal material, and other proteins that seek out DNA breaks and recruit repair machinery to fix them," says Costes. "The first question we had to answer was how closely RIF are associated with DSBs."
Costes recognized that one way to test the association was to calculate how energy was deposited by different kinds of radiation. A high-energy particle, for example, moves through the nucleus in a straight line and may strike DNA at any point along the way; the pattern of hits along the track should be essentially random.
"Physicists understand very well how energy is deposited in the cell's DNA," says Costes, "but the challenge was to make a predictive model that was consistent with what a biologist sees through the microscope. To test what should be seen, given a specific radiation dose, I set out to turn NASA's high-resolution DNA damage simulator" , a model based on data from gel electrophoresis , "into artificial microscope images of a nucleus damaged by radiation."
To do this, says Costes, "I simulated the optical transformation when imaging events at the nanoscale are viewed with a fluorescent microscope." One optical transformation is blurring, with the result that, in a real microscope, two nearby radiation-induced foci may blend together and look like one.
When such factors were taken into account, Costes's initial model, following the NASA model, confirmed random distribution of double-strand breaks at the micron scale. The frequency of DSBs was radiation dependent, however: in the case of cosmic rays (NASA's main concern), which lead to complex double-strand breaks, there was good agreement with the RIF frequencies seen in the microscope. But in the case of gamma rays, which lead primarily to sparse and noncomplex DSBs, measured frequencies in real microscope images were lower.
This suggests that RIF correspond to sites of complex double-strand breaks in DNA. Moreover , focusing on cosmic rays for the NASA project , although the DSB frequency was the same as predicted by the models, the RIF in real microscope images were distributed very differently.
"For one thing, there is a time effect," says Costes. "Just five minutes after cells are exposed to high-energy particles, microscope images already show a nonrandom distribution of RIF." The RIF occur along straight lines, as expected for a particle track, but are not randomly distributed along the lines. "Even though we have the right foci frequency along a track, many foci appear to repulse each other within the first 30 minutes after irradiation" , a suggestion that they might be moving to specific regions of the nucleus.