Electron accelerator sheds light on gene repair in living cells

With the unlikely but invaluable help of physicists, engineers and an electron accelerator, UW Medical School molecular biologists have found a way to examine how damaged genes are repaired in living cells.

The novel approach has enabled the Wisconsin team to definitively show what many scientists have long suspected but never been able to prove: within the seven-some-feet of the folded chromosome in every cell nucleus, special repair proteins do their work by moving immediately from their home bases to remote gene damage sites. The study is reported in the April 24 Science.

“In the past 24 hours, each chromosome in each cell of our bodies has been damaged by cancer-causing chemicals, ultraviolet radiation or free radicals, which are byproducts of several cellular processes,” said UW Medical School assistant professor of medical genetics John Petrini. “But our cells are equipped with a system that constantly monitors and repairs the damaged DNA.”

Petrini and his colleagues have focused their efforts on the system, a four-protein complex called MR95. In undamaged cells, MR95 also regulates DNA recombination, part of the cell-development cycle at the heart of heredity during which a chromosome that has split is rejoined to itself or another chromosome.

The UW researchers have been motivated by an intrinsic interest in this powerful, fundamental complex, which may also be essential to the development of reproductive cells, as well as the immune system. What’s more, they hope their studies will provide a clearer picture of how chromosomes become unstable, a condition frequently associated with malignancy.

Petrini’s team has made significant progress understanding the protein complex in studies of yeast, which offer a remarkably parallel model to the human DNA repair system. They’ve isolated genes associated with three of the four proteins, and produced mutant yeast and mice to study the way an inability to repair genes may lead to cancer.

For the experiment reported in Science, Petrini enlisted the help of UW–Madison physicists and engineers who work at the university’s Synchrotron Radiation Center, where high-speed propulsion of electrons produces X-rays of varying energy levels. Studies conducted there relating to X-ray lithography may someday result in a smaller, more powerful computer chip.

“We wanted to break chromosomes in a tiny area of the nucleus of a living human cell without blasting it away,” said Petrini. “The electron accelerator at the Synchrotron, which generates so-called ‘ultra-soft’ X-rays that are extremely limited in their effect, allowed us to do that.”

To damage only a small area of the nucleus, the researchers irradiated the cells through a miniscule grid made of gold stripes that protected some parts while not others. After tagging the damaged DNA in the unprotected parts of the nucleus with a fluorescent dye, they compared the areas that had been exposed to those that hadn’t. They found that damaged DNA appeared as distinct stripes that exactly matched the striped pattern of the gold grid.

The scientists also found that one of the MR95 proteins, hMre11, collected at DNA damage sites soon after irradiation, appearing as bright spots within stripes that had been irradiated, whereas no hMre11 was present in regions that were protected from irradiation by the grid. The hMre11 protein remained associated with the damaged nucleus regions until the bulk of repair was complete.

“This confirmed what we and others had previously shown indirectly–that repair proteins assemble quickly at breakage sites as they begin their usual response to DNA damage,” said Petrini, noting that the other possibility would have been for DNA breaks to migrate to specialized nucleus compartments, as happens with some genetic functions. “This implies that DNA repair proteins must be summoned to the damage site in order to effect the repair.”

Experiments to test the signalling implication are under way, he added.

In addition to Petrini, the primary authors on the Science paper are Richard Maser, a genetics department graduate student, and Benjamin Nelms, a post-doctoral trainee in Petrini’s laboratory, formerly of the Medical School’s department of medical physics. Co-authors James MacKay and Max Lagally of the department of materials science and engineering developed the synchrotron and cell irradiation technology.