Scientists at the University of Copenhagen have discovered how some types of proteins stabilize damaged DNA and thereby preserve DNA function and integrity. This new finding also explains why people with inborn or acquired defects in certain proteins cannot keep their DNA stable and develop diseases such as cancer, according to the team.

Specifically, researchers from the Novo Nordisk Foundation Center for Protein Research (NNFCPR) at the University of Copenhagen found out how certain proteins orchestrate repair of damaged DNA to ensure its stability over generations and to prevent collateral damage to the neighboring unharmed DNA. Their study (“Stabilization of chromatin topology safeguards genome integrity”) has been published in the scientific journal Nature.

“To safeguard genome integrity in response to DNA double-strand breaks (DSBs), mammalian cells mobilize the neighboring chromatin to shield DNA ends against excessive resection that could undermine repair fidelity and cause damage to healthy chromosomes. This form of genome surveillance is orchestrated by 53BP1, whose accumulation at DSBs triggers sequential recruitment of RIF1 and the shieldin–CST–POLα complex. How this pathway reflects and influences the three-dimensional nuclear architecture is not known,” the investigators wrote.

“Here we use super-resolution microscopy to show that 53BP1 and RIF1 form an autonomous functional module that stabilizes three-dimensional chromatin topology at sites of DNA breakage. This process is initiated by the accumulation of 53BP1 at regions of compact chromatin that colocalize with topologically associating domain (TAD) sequences, followed by recruitment of RIF1 to the boundaries between such domains. The alternating distribution of 53BP1 and RIF1 stabilizes several neighboring TAD-sized structures at a single DBS site into an ordered, circular arrangement. Depletion of 53BP1 or RIF1 (but not shieldin) disrupts this arrangement and leads to decompaction of DSB-flanking chromatin, reduction in interchromatin space, aberrant spreading of DNA repair proteins, and hyper-resection of DNA ends.

“Similar topological distortions are triggered by depletion of cohesin, which suggests that the maintenance of chromatin structure after DNA breakage involves basic mechanisms that shape three-dimensional nuclear organization. As topological stabilization of DSB-flanking chromatin is independent of DNA repair, we propose that, besides providing a structural scaffold to protect DNA ends against aberrant processing, 53BP1 and RIF1 safeguard epigenetic integrity at loci that are disrupted by DNA breakage.”

In short, two proteins called 53BP1 and RIF1 engage to build a three-dimensional “scaffold” around the broken DNA strands. This scaffold then locally concentrates special repair proteins, that are in short supply, and that are critically needed to repair DNA without mistakes.

two protein illustration
Source: University of Copenhagen

“It’s a unique discovery. Understanding the body’s natural defense mechanisms enables us to better understand how certain proteins communicate and network to repair damaged DNA. This opens up an opportunity to better design how DNA damage causes disease and design drugs that improve treatment of patients with unstable DNA,” said center director and professor Jiri Lukas, PhD, of the NNFCPR.

Highly advanced super-resolution microscopes were used in this study. This technology enables researchers to zoom in on living cells and visualize objects about the size of one-thousandth of the width of a hair and follow how the protective protein scaffold assembles and grows around the DNA fracture.

 Microscopy of a cell (delimited with blue). The red plaques are areas of DNA damage that are stabilized by proteins.

“This could be compared to putting a plaster cast on a broken leg; it stabilizes the fracture and prevents the damage from getting worse and reaching a point where it can no longer heal,” said postdoc Fena Ochs, PhD, from the NNFCPR.

So why is this discovery so novel? The previous assumption was that proteins such as 53BP1 and RIF1 act only in the closest neighborhood of the DNA fracture. However, with the help of the super-resolution microscopes, scientists were able to see that error-free repair of broken DNA requires a much larger construction.

“Roughly speaking, the difference between the proportions of the protein-scaffolding and the DNA fracture corresponds to a basketball and a pinhead,” said Ochs.

According to the researchers, the fact that the supporting protein scaffold is so much bigger than the fracture underlines how important it is for the cell to not only stabilize the DNA wound, but also the surrounding environment. This preserves the integrity of the damaged site and its neighborhood and increases the likelihood of attracting the highly specialized “workmen” in the cell to perform the actual repair.

These proteins from the so-called shieldin network were also recently identified by researchers from the NNFCPR.

One of the most notable benefits of basic research such as the new study is that it provides scientists with molecular tools to simulate, and thus better understand, conditions that happen during development of a real disease. When the scientists prevented cells to build the protein scaffold around fractured DNA, they observed that large parts of the neighboring chromosome rapidly fell apart. This caused DNA-damaged cells to start alternative attempts to repair themselves, but this strategy was often futile and exacerbated the destruction of the genetic material.

According to the researchers, this can explain why people who lack the scaffold proteins are prone to diseases caused by unstable DNA.

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