Frog Egg Extracts Illuminate the Untangling Mechanism of DNA Knots

Not all DNA takes the familiar twisted-ladder shape. In some cases, the genetic code folds into unusual structures. One such shape is the G-quadruplex (G4), which resembles a knot. These structures may play a role in turning genes on or off.

However, if not untangled in time, G4s can damage DNA. In collaboration with the Karolinska Institutet, researchers from the Knipscheer Group have identified a new mechanism that helps control these DNA knots. Their findings, published on June 12, 2025, in Science, could inform new strategies for treating diseases such as cancer.

While DNA is typically found in the form of a double helix, under certain conditions, a single strand can fold into a G4 structure. These knots often form in regions rich in guanine (G) bases and are involved in regulating processes like transcription, the step that converts DNA into RNA.

G4s can be helpful in gene regulation, but they also pose risks. If not resolved quickly, they can lead to mutations, disrupt gene expression, and contribute to diseases such as cancer or premature aging. Cells rely on specific mechanisms to untangle these structures efficiently.

Frog Egg Extracts To Study DNA Knots

To study how cells untangle G4 structures, the researchers needed a system that could mimic the process outside of living cells. They used protein extracts from Xenopus laevis eggs, which contain most of the components found in real cells, including proteins involved in DNA replication and repair.

This setup allowed them to insert DNA containing G4 structures and observe the untangling process step by step. It also made it possible to identify the proteins involved in regulating this pathway.

A New Role for RNA

Through this approach, the researchers uncovered a surprising 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 genome from breaking down.

Koichi Sato, Study First Author, Oncode Institute, Hubrecht Institute-KNAW

While RNA is commonly known for its role in protein production through translation, this study revealed a previously unknown function in maintaining genome stability.

Keeping Cells Healthy

The G-loop acts as a landing site for proteins that help untangle G4 structures. These proteins unwind the G4 knot, break down the G-loop, and restore the DNA to its usual double helix shape. With support from Simon Elsässer and Jing Lyu at the Karolinska Institutet, the researchers found that G-loops assist in resolving G4 knots throughout the genome.

We were surprised to find that G4s are recognized as DNA lesions, even without real DNA damage.

Puck Knipscheer, Group Leader, Hubrecht Institute

The G-loop attracts proteins normally involved in DNA repair. The cell treats the G4 structure as if it were damaged DNA, triggering a damage response. This allows the cell to act quickly and prevent further problems.

This process also helps refresh the surrounding DNA and remove harmful changes. With contributions from Jeroen van den Berg of the Oudenaarden Group, the researchers showed that this mechanism is important for maintaining cell health. When it fails, G4 structures accumulate. This creates complications during DNA replication before cell division, leading to DNA breaks and slowing cell growth.

Deploying G4 Knots Against Cancer

The discovery of the G-loop mechanism helps answer key questions about how cells maintain their DNA. It may also inform future research into new treatments. Many cancers are linked to problems with DNA repair, and G4 structures are especially common in cancer cells. If these structures are not properly resolved, they can lead to DNA damage and cell death.

Targeting the G-loop mechanism could offer a way to affect cancer cells at a vulnerable point. One possible strategy might involve increasing the number of G4 structures or blocking their repair. This could make cancer cells more likely to die. However, more research is needed to confirm whether this approach would effectively slow or stop cancer cell growth.