Seeing is believing: New molecular structure sheds light on DNA repair

If you want to know what the Moon really looks like, you should go visit. Yet most of us will only ever know it as a shiny circle in the sky. It’s like that for biologists. When they look at molecules, they see blobs because most microscopes aren’t powerful enough to discern any structural detail.

But now, thanks to a collaboration between Mount Sinai Hospital’s Lunenfeld-Tanenbaum Research Institute (LTRI), part of Sinai Health System,  and The Hospital for Sick Children (SickKids), Toronto scientists have caught a glimpse of the molecules involved in DNA repair — a process that counteracts DNA damage caused by, for example, radiation or chemicals. Published in the latest issue of Nature, the finding deepens our understanding of how cells see and respond to DNA damage and opens new avenues of research into the process that guards us from mutations that could lead to cancer and other diseases.

Teams led by Drs. Daniel Durocher and Frank Sicheri, of the LTRI, and John Rubinstein, of SickKids, used Electron Cryomicroscopy (Cryo-EM) to see how a protein called 53BP1, a key component of DNA repair machinery, recognizes histones, little balls of proteins that organize our DNA. Cryo-EM is gaining popularity as a high-resolution imaging method of choice, allowing scientists to see atoms that build our body’s molecules.

“This work helps us answer how cells recognize and respond to the alarm caused by damage to our DNA. We were able to see, with this powerful microscope, that 53BP1 decodes the chemical signal that is triggered by DNA damage,” says Durocher, who is also a Professor in the University of Toronto’s Department of Molecular Genetics and Canada Research Chair in Molecular Genetics of the DNA Damage Response.

DNA damage is inevitable – whether it’s harmful products of our own metabolism or environmental factors, all these can cause unwanted genetic changes. Luckily, the cells have evolved efficient repair mechanisms. In one of them, 53BP1 quickly binds to the site of damage and recruits other components of the repair machinery.

Inside the cells, the DNA is tightly wound up around the histones. Should a piece of DNA get broken by, say radiation, the nearby histones become swiftly adorned with chemical tags, which signal to 53BP1 that help is needed.  But how 53BP1, or other cellular machineries, read this “histone code” has been a long-standing question in biology.

“We knew that certain modifications happen near the damaged DNA and we knew that 53BP1 was recruited to the sites of damage, but we really had no idea how 53BP1 recognized those modifications,” says Durocher.

To answer this question, Dr. Marcus Wilson, a postdoctoral researcher in Durocher’s team, teamed up with Dr. Samir Benlekbir from Rubinstein’s group that is among the world-leading in Cryo-EM. Thanks to recent technological improvements, Cryo-EM now stands almost neck-to-neck with the gold standard, but technically challenging, method of X-ray crystallography.

Unlike X crystallography, Cryo-EM requires a small amount of material that is relatively easy to prepare. It works by snap-freezing a small volume of liquid containing the sample so that molecular complexes, including the whole lot — the DNA, wound up around the chemically-tagged histones, with 53BP1 bound to them — are randomly arranged in a thin layer of ice. Then, the complexes are irradiated with an electron beam to capture their images from all possible angles. Finally, software sifts through tens of thousands of snapshots to compute a high-resolution 3D model of the entire molecular structure. It worked so well that it caught the researchers by surprise when they realized they were able to see parts of molecules as small as half a nanometer (that’s half a billionth of a metre!) in size.

“Many in the field will be surprised by how high a resolution we got with our particular hardware - near the X crystallography resolution – and at a sufficient resolution to answer many biological questions about how 53BP1 recognizes the damaged site on the DNA,” says Rubinstein, who is also a U of T Professor at the Departments of Biochemistry and Medical Biophysics and Canada Research Chair in Electron Cryomicroscopy.

The long-awaited 3D model not only lets scientists see exactly how 53BP1 is nestled between the DNA and the histones, but it also underscores the importance of the histone code. If even a single tag is missing, or is in a wrong place, 53BP1 will not bind it.

“This is the first time anyone’s seen how a component of the DNA repair machinery reads and interprets the histone code. It’s a really exciting time for EM and it is allowing us to see how the machinery of the cell works with unprecedented clarity,” says Wilson.

This research was primarily supported by operating grants from the Canadian Institutes of Health Research and the Krembil Foundation and SickKids Foundation.

This paper is an example of how SickKids is contributing to making Ontario Healthier, Wealthier and Smarter.