The XL Factor

Discovery of molecule’s role yields insights into DNA repair, immunodeficiency

By STEPHANIE DUTCHEN October 1, 2018 Research

Man looking in a microscope, with lasers visible in foreground — Co-first author Sean Carney studies DNA break repair in the Loparo lab. Image: Rick Groleau

Sometimes our cells employ meticulous and elegant solutions to fix things that break.

Other times, they slap on some duct tape and keep going.

The human body can apply these quick-and-dirty methods to even its most critical components, such as when both strands of a DNA helix suddenly snap. Such DNA breakages happen hundreds of times a day—whether randomly, from sun exposure or as a result of ordinary activities like breathing—as well as during radiation therapy and chemotherapy for cancer.

Various molecules rush in to hold the DNA ends close until they can be chemically "taped" back together, a process called nonhomologous end joining, or NHEJ.

“It isn’t the smartest of pathways,” said Joseph Loparo, associate professor of biological chemistry and molecular pharmacology at Harvard Medical School, “but it’s the primary one our cells use to fix double-strand breaks.”

Loparo’s lab has been painstakingly identifying molecules needed for NHEJ and revealing how each contributes to the overall repair process. The work helps researchers understand how diseases arise when NHEJ goes awry, and could inform attempts to ensure that deliberate DNA breakages are repaired in the right places when treating cancer or editing the genome.

In their latest effort, published online this month in Nature Structural and Molecular Biology, the team determined the role of a crucial but mysterious molecule known as XLF.

About 10 years ago, researchers discovered that mutations in XLF disrupt DNA repair. People with XLF mutations have immune system deficiencies and DNA that is far more susceptible than normal to radiation damage.

It was clear that XLF does something important. But what exactly?

Using a fluorescence imaging technique that indicates when two tagged molecules come close together, Thomas Graham, a PhD student in systems biology at HMS, and Sean Carney, a postdoctoral researcher in the Loparo lab, observed that XLF forms part of a bridge that brings the broken DNA ends into alignment so they can be reattached.

The team uncovered additional details that overturn a popular theory about the length of the bridge.

Researchers had observed in past experiments that pairs of XLF molecules, like cherries with their stems intertwined, can alternate with pairs of a related molecule, XRCC4—from which XLF gets its name, “XRCC4-like factor”—to create long filaments. Scientists had hypothesized that the bridge they form during NHEJ is similarly long.

In the new study, Loparo’s team demonstrated that the bridge requires only one XLF pair and likely just a couple of XRCC4s.

“We think our work will be especially valuable to those studying the structure of NHEJ factors on DNA breaks,” said Carney. “The long XLF-XRCC4 filaments have been the focus of many structural studies up to this point.”

The team members credit their use of a frog egg extract system with their ability to answer questions that have stymied others. The extract contains a soup of molecules that the scientists can study, leaving room to “see new things that can be missing” in test-tube experiments where researchers add only the molecules they believe are important, said Loparo.

The new study “brings together a lot of different techniques and speaks to the power of trying to visualize biochemistry in real time at the single-molecule level,” said Loparo.

Graham and Carney were co-first authors of the paper. Johannes Walter, professor of biological chemistry and molecular pharmacology at HMS, was also a co-author.

This study was funded by the National Institutes of Health (grant R01GM115487) and the Howard Hughes Medical Institute, where Walter is an investigator.