Natural DNA-Replication Mechanism May Help Fix Genetic Defect in FA, Study Suggests

A natural genetic mechanism may help reverse the genetic defect within the FXN gene — excessive repeats of a portion of DNA, called GAA triplets — that underlie Friedreich’s ataxia (FA), a recent study done in yeast shows.

Researchers have found that a process that occurs while cells are preparing to divide may help remove the excessive repeats in the gene, paving the way for a potential new method for treating the disease.

The study, titled “Large-scale contractions of Friedreich’s ataxia GAA repeats in yeast occur during DNA replication due to their triplex-forming ability,” was published in the journal Proceedings of the National Academy of Sciences.

FA is caused by the repetition of three nucleotides — the building blocks of DNA — called GAA, which occur in the FXN gene. The repeats cause an error that lowers production of the frataxin protein, which is essential for the normal functioning of mitochondria — the structures that provide the energy necessary for cells to work.

Healthy individuals can have from eight to 34 GAA repeats, but FA patients have more than 70, frequently reaching hundreds of repeats.

Evidence suggests that longer repeats are associated with early disease onset and more severe symptoms. The longer mutated FXN gene is known to cause instability to the DNA, meaning the gene structure is fragile and prone to alterations, which include more frail chromosomes. This may cause complex rearrangements of the DNA chains, such as contractions (loss of repeats) or expansions (the addition of repeats).

“The DNA repeats literally gum up the works. They can also cause other mutations in the surrounding DNA, or make chromosomes extremely fragile, breaking into pieces, or rearranging themselves, ” Sergei Mirkin, chair of the department of biology at Tufts University and the study’s lead author, said in a press release.

Researchers are trying to understand how these DNA structures are able to contract, becoming more similar to a healthy gene. That could be a useful strategy for reversing FA.

“If we can shrink the DNA repetition in tissues to levels found in healthy people, we might be able to stabilize the DNA and reduce the effects of disease,” Mirkin said.

To study this, the researchers used a yeast model, Saccharomyces cerevisiae, that they manipulated to carry a long GAA repeat sequence, with 124 repeats.

They were able to select yeast colonies that showed signs of large-scale contractions, losing about 60 GAA repeats — “that is, roughly half of the repeat tract was lost,” the researchers said. In these yeast, they observed that contractions were mostly happening during DNA replication, a process in which the two DNA chains are copied before the cells start to divide.

In particular, the team found that a replication process called “lagging strand synthesis” was responsible for such contraction. From the two DNA chains found in each chromosome, one is copied in a continuous manner, but the other is replicated into small pieces. These small pieces, known as Okazaki fragments, are then stitched together. This is slower and takes more time to complete than continuous replication, giving the process the name “lagging.”

The researchers observed that DNA strands that are chemically prone to form a triplex structure (instead of the usual double helix), also called H-DNA, were those with the most contractions. These complex triple structures are not easily copied. Most of the time, the enzymes responsible for DNA replication just bypassed these regions, forming a copy with fewer GAA repeats.

To confirm these findings, the investigators impaired the function of enzymes related to the lagging strand synthesis, including Pol32, DNA polymerase α and rfa1. They observed that without these proteins working properly, the contraction rate increased significantly. In addition, when they mutated an enzyme involved in the formation of Okazaki fragments, called Rad27, the contraction rate also increased.

“We show that GAA repeats contract during DNA replication, which can explain the high level of somatic [present in all body cells except germ cells] instability of this repeat in patient tissues. We also provided evidence that a triple-stranded DNA structure is at the heart of GAA repeat instability,” the researchers said.

“While these results were uncovered in a yeast model, they do provide us with a clue into the mechanism of DNA repeat instability in Friedreich’s ataxia,” said Alexandra Khristich, a graduate student in Mirkin’s lab and the study’s first author.

“I hope that our discovery would become a starting point for the potential development of therapeutic strategies that tip the balance toward DNA repeat contraction in patient tissues,” she added.