Dozens of times per day in each of the trillions of dividing cells in our bodies, the double strands that form our DNA may break and need to be fixed. Harvard Medical School scientists have now devised a way to watch how these essential repairs get made in real time and at previously unattainable resolution, allowing them to discover individual steps in the repair process and identify which proteins are involved in each.

The findings, published March 17 in Molecular Cell, could help researchers better understand the molecular underpinnings of diseases involving abnormal DNA double-strand break repair, including cancer, immunodeficiency and neurodegeneration. They could also be applied toward improving technologies and treatments that strategically induce double-strand breaks, such as gene editing and chemotherapy.

Cells have two main ways to repair double-strand breaks: homologous recombination, in which the repair machinery rebuilds the broken DNA using a sister chromatid as a template, and non-homologous end joining, in which the broken DNA ends get reconnected with no template and variable accuracy.

The researchers looked at non-homologous end joining because it is responsible for about 80 percent of double-strand break repairs yet scientists don’t understand it well.

“Until now we’ve had a foggy view of how non-homologous end joining repairs DNA double-strand breaks at the molecular level,” said Joseph Loparo, HMS assistant professor of biological chemistry and molecular pharmacology and senior author of the paper.

“By combining a single-molecule visualization approach with biochemical techniques,” he said, “we were able to begin to better understand how the repair complex assembles on the DNA ends and brings them together.”

(No) reason to FRET

The team conducted its experiments on pieces of DNA bathed in a frog egg extract, or “cell juice,” as first author Thomas Graham, an HMS graduate student in systems biology, calls it.

“That allowed us to achieve higher resolution than we could in a cell,” Graham explained. “Because the same molecular factors are required, we think what we see in extract recapitulates what happens in intact cells.”

DNA strands—and the breaks that occur within them—are too small to see, so the researchers used a tool called fluorescence resonance energy transfer (FRET) microscopy.

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