Faulty DNA replication linked to neurological diseases

Lengthy sequences of DNA — with their component triplet of nucleotides repeated hundreds, even thousands of times — are known to be abnormal, causing rare but devastating neurological diseases. But how does the DNA get this way? How does it go haywire, multiplying out of control?

In the current issue of Molecular and Cellular Biology, Sergei Mirkin, professor of biochemistry and molecular genetics at the University of Illinois at Chicago College of Medicine, explains the mechanism, providing an important clue to the origin of these diseases.

Mirkin and Maria Krasilnikova, a research assistant professor in his lab, studied the sequence of a simple repeat of three nucleotides responsible for Friedreich’s ataxia, the most commonly inherited form of ataxias, which causes progressive damage to the nervous system, resulting in symptoms ranging from muscle weakness and speech problems to heart disease.

The DNA triplet that repeats in Friedreich’s ataxia is a guanine and two adenines (GAA) on one DNA strand and the complementary two thymines and a cytosine (TTC) on the opposite strand.

Earlier research had shown that up to 40 repeats of this nucleotide triplet do not cause any symptoms. The DNA is inherited as is, an odd but harmless pattern passed down from one generation to the next.

The problems begin when the repeats exceed 40.

“For Friedreich’s ataxia and other neurological diseases, when the number of repeats exceeds 40, the sequence becomes unstable. That means that as the sequence is passed from one generation to the next, it gets longer. The longer it gets, the more likely it will get still longer. And the longer it gets, the worse the disease,” Mirkin said. “Basically, even if you are more or less okay, there is a probability that your kids will be sick and a still higher probability that your grandkids will be even sicker.”

To study how and why the inherited sequence expands so rapidly, the scientists watched the replication of different lengths of the triplet repeat sequence, using a simple unicellular organism, yeast, as a convenient model. While yeast is far more primitive than humans, its mechanism of DNA replication is remarkably similar.

The researchers found that replication of normal-size repeats proceeded without a hitch.

With larger-length sequences, however, the replication machinery got stuck and replication stalled. According to Mirkin, this temporary stoppage is probably caused by the formation of an unusual three-stranded DNA structure. He first discovered such odd DNA structures during his post-doctoral studies back in 1987, though at the time their significance was unclear.

“I was really delighted to finally find that they have a biological role,” Mirkin said.

In their current study, Mirkin and Krasilnikova found that when replication stalled, the triplet repeat multiplied, creating longer and longer threads of DNA.

“It’s like a car getting stuck in a pothole. You keep spinning the wheels to get out of the pothole, but the more the wheels spin, the more mileage you put on the car, the more repeating units you add to your DNA,” Mirkin said.

The researchers also found that the aberrant lengthening of the sequence was more likely if replication began in one direction rather than the other, starting from the TTC strand rather than the GAA strand.

Mirkin and Krasilnikova believe their results apply to many other neurological diseases linked to lengthy repeats, including myotonic dystrophy, fragile X mental retardation and Huntington’s disease.

“Different genes and different parts of those different genes are involved in these diseases. But there is one common feature: when the number of repetitive units is small, under 40, they are harmless. Over that threshold, the repeats multiply, expanding with each replication and causing rare, but very serious neurological disorders that worsen as the length of the repeats grows,” Mirkin said.

“Individuals can be carriers of relatively long stretches of nucleotide repeats, with no apparent clinical consequences. But then some as yet unknown event triggers the addition of an extra triplet or two, and the threshold is crossed,” Mirkin said. “Once replication stalls, there is no way back.”

Mirkin hypothesizes that the triggering event might be a switch in orientation, “but the million dollar question is what causes that switch.”

The study was funded by the National Institute of General Medical Sciences, one of the National Institutes of Health.