Picking MSH3’s Pocket: New Insights into CAG Repeat Expansion

In HD, DNA repair proteins like MSH3 can sometimes accidentally make the CAG repeat grow longer over time. UK scientists showed that targeting a tiny DNA-binding pocket on MSH3 slows this process, opening a new avenue for therapeutic development.

Huntington’s disease (HD) is caused by a stretch of repeating DNA letters, C-A-G, within the huntingtin gene. We’ve known for a long time that these repeats are harmful, but in recent years we have learned that they are also unstable, and can continue to lengthen over time. This process, known as somatic instability, is increasingly recognized as a potential driver of HD. Growing evidence suggests that as CAG repeats expand within some vulnerable neurons, they eventually reach toxic lengths, contributing to brain cell damage and worsening symptoms.

One big breakthrough in understanding somatic instability has been the discovery that proteins responsible for repairing damaged DNA can also be key drivers of this process. Instead of protecting the genome, they can occasionally help CAG repeats to expand. By targeting a DNA-binding “pocket” of one of these proteins, MSH3, a team of UK scientists led by Sarah Tabrizi showed that they could slow the expansion of CAG repeats. Together, these experiments shed light on how MSH3 drives CAG repeat expansion and suggest it may be possible to interfere with this process while leaving more of MSH3’s normal DNA repair functions intact.

Why do CAG repeats keep growing?

As HDBuzz readers may have noticed, somatic instability has become one of the biggest areas of research in HD, but our understanding of the process is still evolving. Large human genetic studies showed that tiny spelling variations in DNA repair genes can have surprisingly big effects on the age at which HD symptoms begin. Other clues have come from molecular and structural biology, animal models, and experiments in cells, where scientists can remove genes or subtly alter proteins and observe what happens to CAG repeat expansion.

Many of these studies point to the same group of proteins: the cell’s DNA repair machinery. Their job is to patrol our DNA, searching for damage and fixing mistakes before they cause problems. Some of these proteins can band together in teams known as protein complexes, creating molecular DNA repair machines. In HD, however, they seem to be stymied by the expanded CAG repeat, which can form an unexpected, protruding loop. Rather than repair the problem, their attempts to fix it actually make the repeat longer.

*The job of the DNA repair machinery is to patrol our DNA, searching for damage and fixing mistakes before they cause problems. Photo credit:* *Markus Spiske*

We’re still not sure exactly why this happens. Some scientists are trying to understand the underlying biology, while others are focused on finding ways to interrupt the process, often by reducing or blocking key members of this faulty repair crew.

Where MSH3 meets DNA

One of the proteins that has emerged as a key player is called MSH3. It’s part of an important DNA repair complex, and it is good at recognizing unusual loops or bulges in DNA. In HD, this job appears to backfire, helping to drive the addition of extra CAGs.

We already know that lowering MSH3 in cells and animal models of HD can slow the expansion of CAG repeats. In fact, several companies are developing therapies targeting MSH3 levels as a way to slow somatic instability while others are going after the “motor” activity of the MSH3 molecular machine. But another approach is to better understand exactly how MSH3 rubs shoulders with DNA, and whether that interaction can be interrupted without eliminating the protein altogether.

Studies have identified a small DNA-binding pocket on MSH3 where a groove on the protein surface makes contact with wonky DNA loops, like those formed in the presence of too many CAG repeats. In this study, researchers made tiny changes to the surface of the pocket to investigate how impacting DNA binding might alter CAG repeat expansion. If expansion is reduced, perhaps drugs could be designed to block this pocket and stop MSH3 from grabbing onto DNA in the wrong place.

Putting the pocket to the test

To test whether this DNA-binding pocket really matters, the scientists engineered a specialized cell model. These cells carried a piece of huntingtin with an extra-long stretch of CAG repeats (118) that lengthened predictably over time. They also allowed the researchers to control MSH3 levels and make tiny changes to the protein itself.

Proteins are built from twenty different chemical building blocks called amino acids. Two of these, tyrosine (Y) and lysine (K), sit side-by-side within MSH3’s DNA-binding pocket and help it recognize irregular loops of DNA. Using genetic engineering, the researchers replaced these amino acids with different ones to ask what happens when MSH can no longer grip DNA in the same way.

The results were striking. Altering just these two amino acids almost completely stopped CAG repeat expansion. The researchers also showed that this was not because MSH3 had stopped working altogether. The protein was still made normally, and it could still partner with other proteins in the DNA repair complex. Instead, the changes prevented MSH3 from binding to the abnormal DNA loops formed by expanded CAG repeats.

Can we drug the pocket?

Showing that the binding pocket matters is the first step. The next question is whether it can be targeted with a drug. The researchers next turned to a small molecule called CP1. This compound has previously been shown to stick close to MSH3’s pocket and disrupt its ability to bind DNA. CP1 is not a drug meant to treat disease; it’s a long road to make, select, and test molecules which meet that criteria! Instead, you can think of it as a tool compound scientists can use to investigate MSH3.

To test CP1, the researchers set an experimental trap. They created a short piece of DNA containing the type of CAG loop that MSH3 normally recognizes, and used it as molecular “bait.” As expected, MSH3 latched onto the bait, but after treatment with CP1, much less of the protein was caught.

*The researchers used molecular “bait” to test the interaction between MSH3 and DNA and understand how a drug could disrupt it. Credit:* *gurphotogallery*

This wasn’t just a biochemical trick. CP1 also reduced the interaction between MSH3 and CAG-containing DNA loops in living cells, including neurons derived from the stem cells of a person with HD. Most importantly, the CP1 compound slowed somatic instability across all of these experimental systems.

More precise targeting of MSH3

We have known for a while that MSH3 is an attractive target to slow somatic instability, and several companies are already developing therapies designed to lower MSH3 levels. However, because MSH3 normally helps repair damaged DNA, there are understandable concerns about completely shutting down a protein that helps protect our genome. This paper explored a different idea: rather than eliminating MSH3, could scientists simply prevent it from interacting with the abnormal DNA structures that drive CAG repeat expansion?

*The MSH3 DNA-binding pocket may be key to its role in somatic instability. But other tools will be needed to determine whether this is true in the brain, and ultimately in people. Photo credit:* *Enes Beydilli*

The answer appears to be yes. By targeting a small DNA binding pocket, the researchers were able to slow somatic instability while leaving MSH3’s normal function intact, and they demonstrated this in multiple systems.

A few things to keep in your pocket

There are some important caveats to consider. This is still very early stage research. The scientists cleverly adapted existing models and tools, including cells with rapidly expanding CAG repeats, adjustable levels of MSH3, and looping DNA “bait,” to ask important questions about somatic instability. It’s a step towards deeper understanding, but more work will be needed to determine whether the conclusions apply in the complex environment of the brain, and ultimately in people with HD.

The small molecule used in the study also comes with limitations. While CP1 conveniently slips into the MSH3 pocket, it is a research tool, not a drug candidate, and using it at higher doses was actually a bit toxic to cells. Although it clearly disrupted the interaction between MSH3 and CAG containing DNA, it also seems to slightly lower levels of MSH3, making it difficult to completely separate these two effects. More selective compounds would likely be needed to move towards the clinic.

Despite these limitations, this study represents an important advance. It provides evidence that disrupting a specific DNA-binding pocket in MSH3 might be enough to slow CAG repeat expansion. This work identifies the MSH3 DNA-binding pocket as an attractive new therapeutic target—and opens the door to a promising new direction for slowing somatic instability in HD.

Summary

MSH3 is a DNA repair protein that can accidentally make CAG repeats grow longer over time, potentially contributing to Huntington’s disease.
Scientists identified a tiny pocket on MSH3 that helps it grab onto unusual DNA structures formed by expanded CAG repeats.
Changing this pocket or blocking it with a research compound slowed CAG repeat expansion in cells.
This could offer a new way to slow CAG repeat expansion without removing MSH3 entirely, although more research is needed before this approach could become a treatment.

Picking MSH3’s Pocket: New Insights into CAG Repeat Expansion

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