Knot good: How cells untie DNA to protect the genome

Not all DNA looks like the familiar twisted ladder. Sometimes, parts of our genetic code fold into unusual shapes. One such structure, the G-quadruplex (G4), looks like a knot. These knots can play important roles in turning genes on or off. But if not untangled in time, they can harm our genome. Now, researchers from the Knipscheer Group, in collaboration with the Karolinska Institutet, have uncovered a surprising mechanism that keeps these knots in check. Their work, published in Science on June 12th, could lead to new ways to treat diseases like cancer.

Our DNA is usually shaped like a double helix. However, under certain conditions, a single strand of DNA can fold into a G-quadruplex (G4) structure, which looks like a knot. These knots often form in regions with many guanine (G) bases. They help regulate important processes like transcription, where DNA is copied into RNA.

But G4s are double-edged swords. While they help with geneA small piece of DNA with a specific function. For example, genes determine which color our eyes have and whether we have curly or straight hair. In a human cell, the DNA contains about 30.000 genes, each having a specific function. Genes are hereditary and can therefore be passed on to offspring.regulation, if they are not untangled in time, they may cause mutationsErrors in the DNA. Mutations can, among other things, arise if the DNA is copied incorrectly or through external influences. For example, tumor cells often contain mutations that are beneficial for their growth., disrupt gene expressionThe activity of a gene or genes. The combination of active genes in a cell determines, amongst other things, the function, shape and size of the cell., and even lead to cancer or early aging. Therefore, cells need tools to untie these knots quickly and efficiently.

Cells use a multi-step process to untie knots (G4 structures) in DNA. First, the G4 knot is recognized as a problem by the cell. Special DNA damage response proteins start the repair by recruiting DNA repair proteins, which help form a loop with RNA, called a G-loop. Next, other DNA repair proteins untie the G4 knot (FANCJ).and break open the loop (XPF-ERCC1). Finally, the normal DNA structure is restored to keep cells healthy. This protects the genome and prevents damage that could harm the cell. Credit: Koichi Sato and Puck Knipscheer. Copyright: Hubrecht Institute

Frog egg extracts to study DNA knots

To study exactly how cells untangle G4 structures, the researchers needed a system that reproduces this process outside living cells. They used protein extracts from frog (Xenopus laevis) eggs. These extracts contain almost everything found inside a real cell, especially proteins needed for DNA replication and repair. This setup allowed the team to introduce DNA with G4 structures and observe the stepwise process of untangling. They could also pinpoint the proteins that drive this mechanism.

A new role for RNA

Using this system, the researchers uncovered a surprising new role for RNA molecules. “With the help of proteins known for their role in DNA repair, RNA binds to the DNA strand opposite the G4 structure, forming a structure called a ‘G-loop’. This G-loop structure is an important intermediate in the untangling mechanism and protects the genomeThe complete set of DNA or genetic material in a cell. from breaking down” says first author Koichi Sato. Although RNA is best known for its function in protein production through translation, this mechanism adds a previously unrecognized role for RNA in genome protection.

Keeping cells healthy

The G-loop acts like a landing pad for additional proteins. These proteins untie the G4 knot, break apart the G-loop and convert the DNA to its normal double helix shape. Thanks to a collaboration with Simon Elsässer and Jing Lyu from the Karolinska Institutet, the team discovered that the G-loop helps untie G4 knots across the entire genome.

“We were surprised to find that G4s are recognized as DNA lesions, even without real DNA damage,” explains group leader Puck Knipscheer. The G-loop brings in proteins that usually fix DNA damage. But here, the cell treats the G4 structure as if it were broken DNA, triggering a DNA damage response. This allows the cell to act fast and prevent serious problems later.

Even better, the process renews the surrounding DNA and removes harmful modifications. With help from Jeroen van den Berg from the Oudenaarden Group, the team shows how important this mechanism is for cell health. When it fails, G4s build up and cause serious problems when the DNA needs to be copied before cell division. This results in DNA breaks and blocks cell growth.

Deploying G4 knots against cancer

The discovery of the G-loop mechanism answers key scientific questions on how cells protect their DNA and could also open doors for future therapies. Many cancers are linked to problems in DNA repair. G4 structures are particularly abundant in cancer cells, and if cells cannot untie them, this will induce DNA damage and cell death.

Targeting the G-loop mechanism could be a smart way to hit cancer cells where they’re weak. For example, by increasing the number of G4 knots or blocking their repair, cancer cells could be killed selectively. However, more research is needed to see if this can truly stop cancer cell growth.

Publicatie

RNA transcripts regulate G-quadruplex landscapes through G-loop formation. Koichi Sato, Jing Lyu, Jeroen van den Berg, Diana Braat, Victoria M. Cruz, Carmen Navarro Luzón, Joost Schimmel, Clara Esteban-Jurado, Maëlys Alemany, Jan Dreyer, Aiko Hendrikx, Francesca Mattiroli, Alexander van Oudenaarden, Marcel Tijsterman, Simon J. Elsässer, and Puck Knipscheer. Science, 2025.

About Puck Knipscheer

Puck Knipscheer is group leader at the Hubrecht Institute, professor by special appointment of Biochemistry of Genome maintenance at the Leiden University Medical Center and Investigator at Oncode Institute.