Development of tools to visualize DNA repair like never before

Researchers from CNIO and Massachusetts General Hospital are developing tools to visualize DNA repair like never before

Images of human cells with their DNA damaged by a laser beam in the laboratory. In green, the classic gamma-H2AX repair protein is bound to fracture generated by the laser beam; in red, the new protein discovered in this study (RNF166) bound to the DNA fragments, which coincided with gamma-H2AX binding; in blue, cell DNA. Credit: CNIO, MGH

Every single one of the trillions of cells that make up the human body suffer from more than 10,000 DNA lesions every day. These damages would be catastrophic if cells were unable to repair them, but a very delicate machinery that detects and repairs genetic damage is at work to prevent DNA mutations and diseases such as cancer. Using machine learning used for high-throughput microscopy, among others, researcher Bárbara Martínez, a member of the Metabolism and Cell Signaling Group led by Alejo Efeyan at the National Cancer Research Center (CNIO), with Raul Mostoslavsky and his team from Massachusetts General Hospital (Boston , USA), has managed to visualize this DNA repair machinery in detail and identified new repair proteins. These results, designed in Boston, developed between Boston and Madrid and published this week in Cell reports, could help develop new cancer treatments.

As soon as there is DNA damage, such as a DNA double-strand break, the cell activates a mechanism called DNA damage response, which acts as a “call to the emergency services,” Martinez explains. Proteins quickly bind damaged DNA to send alarm signals that will be recognized by other proteins that specialize in repairing the damage.

The goal of chemotherapy is to kill tumor cells by inducing DNA lesions, which cause cancer cells to collapse and die. “By knowing how DNA lesions occur and how they are repaired, we want to learn more about how cancer develops and how we can fight it. Any new discovery in DNA repair will help develop better cancer treatments while protecting our healthy cells, ”says Martinez.

The researchers have developed a new method that, using a machine learning analysis method designed by the CNIO Confocal Unit, has made it possible to analyze this process with a degree of detail and precision that has never been achieved before. “Until now, a limiting factor in tracking DNA repair kinetics has been the inability to process and analyze the amount of data generated from images taken with the microscope.”

Researchers have used high-throughput microscopy that allows the acquisition of thousands of images of cells after induction of genetic damage. In the first phase, they introduced more than 300 different proteins into the cells and assessed in a single experiment whether they interfered with DNA repair over time. This technique has led to the discovery of nine new proteins involved in DNA repair.

Video showing how one of the proteins discovered in this work, RNF166, targets DNA breakage. Credit: CNIO, MGH.

But the authors decided to go a step further and visually monitored the 300 proteins after generating genetic damage. To do this, they adapted a classical DNA micro-irradiation technique – which damages DNA with a UV laser – to be used on a large scale for the first time and to analyze the behavior of the 300 proteins examined.

“We saw that many proteins adhered to damaged DNA, and others did the opposite: they moved away from DNA lesions. The fact that they either bind to or remove themselves from damaged DNA, to allow the recruitment of repair proteins to “The lesion is a common feature of DNA repair proteins. Both phenomena are relevant”.

One of the proteins detected is PHF20. The authors showed that this protein moves away from lesions within seconds of damage to facilitate the recruitment of 53BP1, a protein essential for DNA repair. Cells without PHF20 cannot repair their DNA properly and are more sensitive to radiation than normal cells, indicating that PHF20 is important for DNA repair.

These technologies provide new opportunities to study DNA repair and manipulate it. “One advantage is that both platforms are very versatile and can be used to detect new genes or chemical compounds that affect DNA repair. We have evaluated hundreds of proteins in minimal time using techniques that allow direct visualization of DNA repair. “

This work has been funded by the Spanish Ministry of Science and Innovation, the Carlos III Institute of Health, the US National Institutes of Health, the Marie Curie COFUND FP7, the Massachusetts General Hospital, the European Research Council and the Council of Natural Sciences and Engineering Research Canada.

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More information:

Raul Mostoslavsky, Assessment of Kinetics and Recruitment of DNA Repair Factors Using High Content Monitors, Cell reports (2021). DOI: 10.1016 / j.celrep.2021.110176.… 2211-1247 (21) 01676-4

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