Knot a Problem: Mechanism Found for DNA Loop Control

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Not all DNA looks like the familiar double helix. Sometimes, parts of our genetic code fold into unusual shapes under certain conditions. One such structure known as a G-quadruplex (G4) looks like a knot. These knot-like conformations can play important roles in turning genes on or off. But if not untangled in time, they can harm our genome.

Researchers in the group of Puck Knipscheer, PhD, at the Hubrecht Institute, in collaboration with scientists at the Karolinska Institute, have now uncovered a surprising mechanism, involving RNA, that keeps these knots in check. The team suggests that findings from the laboratory studies could lead to new ways to treat diseases such as cancer.

Knipscheer is senior author of the team’s published paper in Science, titled “RNA transcripts regulate G-quadruplex landscapes through G-loop formation,” in which the researchers say that their results “… establish an intricate G-loop assembly-disassembly mechanism that controls G4 landscapes and is essential for cellular homeostasis and survival.”

While our DNA is commonly structured as a twisted ladder-shaped double helix, these knots often form in regions with many guanine (G) bases, and help regulate important processes like transcription, where DNA is copied into RNA. “G-quadruplexes (G4s), four-stranded alternative DNA structures formed at G-rich genomic sequences, are thought to be transcriptional regulators enriched in active regulatory regions,” the team noted. “Although mammalian genomes contain more than half a million DNA sequences that can adopt a G4 structure (potential G4-forming sequences, PQSs), only a limited number of G4 structures form, and specific G4 subsets have been detected in different cell types.”

G4s are also double-edged swords. While they help with gene regulation, if they are not untangled in time, they may cause mutations, disrupt gene expression, and even lead to cancer or early aging. “Misregulation of G4 positioning disrupts gene expression and embryonic differentiation and has been linked to neurodegenerative disease, cancer, and accelerated aging,” the scientists continued. Therefore, cells need tools to untie these knots quickly and efficiently. However, they further acknowledged, “Currently, we lack knowledge on how G4s are controlled to prevent transcription dysregulation and genome instability.”

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.

Using their 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’,” stated first author Koichi Sato, PhD. “This G-loop structure is an important intermediate in the untangling mechanism and protects the genome from breaking down.” 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.

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. “Our data establish a stepwise process that controls G4 dynamics through the assembly of a G-loop by the invasion of RNA transcripts across from the G4, followed by G4 unwinding coupled with G-loop disassembly,” the scientists wrote. “Interfering with this G-loop cycle results in severe dysregulation of the transcriptome, extensive genome instability, and proliferation defects, highlighting its vital role in cellular homeostasis and survival.”

Thanks to a collaboration with Simon Elsässer and Jing Lyu from the Karolinska Institute, 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 G4 structure is recognized as a “DNA lesion,” thereby enabling activation of the downstream DNA repair proteins in the absence of DNA damage,” the scientists stated in their report.

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.

The process in addition renews the surrounding DNA and removes harmful modifications. “… this mechanism results in renewal of the non-G4 strand, which allows for erasing potentially mutagenic modifications on the displaced strand, thereby safeguarding the integrity of regulatory DNA motifs,” they added. 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.

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. “Many of the proteins involved in this G-loop cycle, including RAD51, BRCA2, FANCJ, and XPF-ERCC1, also promote canonical DNA repair pathways and are directly linked to human diseases characterized by developmental abnormalities and predisposition to cancer,” the scientists noted.

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.