Measuring the rate of DNA repair in HD cells

Blog post by Dr. Tamara Maiuri

Last time, I wrote about how the system to pull down huntingtin and its associated DNA repair proteins works in cells from an HD patient. The drawback to using these cells is that they grow very slowly and don’t yield much protein. So, the last few weeks have been spent stock piling cells. My stash is growing, but it will be several more weeks before I have enough material to send for mass spectrometry, which will give us a list of all the proteins that interact with huntingtin under conditions of oxidative DNA damage.

In the meantime, I’ve been thinking about what we’re going to do with the information we get. What do we want to know about the huntingtin interacting proteins we identify?

Well, we know that DNA repair is an important aspect of disease progression. The age at which people get sick, and other signs of progression including brain structure, are affected by small changes in peoples’ DNA repair genes. What’s more, the huntingtin protein acts as a scaffold for DNA repair proteins. Maybe this job is affected by the expansion that causes HD.

Once we have a list of proteins that interact with huntingtin upon DNA damage, we want to know if, and how, they affect the DNA repair process in HD cells. What we need is a way to measure the DNA repair rates in HD patient cells. Then we can ask: if we tweak the proteins that interact with huntingtin upon oxidative DNA damage, what happens to the repair rates? That way, down the road, we could use those proteins as drug targets to improve the DNA repair situation.

But one step at a time. First we need the DNA repair measuring stick. There are a few options for this, but I recently came across a cool one. It works by first damaging DNA in a test tube, then introducing it into cells, then measuring how well the cells repair the damage in order to express a gene on the DNA. The gene encodes green fluorescent protein (GFP), so you can measure expression (as a proxy for DNA repair) by how many cells are glowing green.

Question 1: Does the system even work?

The first thing I did was to try this in the easy-to-use HEK293 cells (HD patient fibroblasts don’t take up DNA very easily, and this will be a challenge to overcome down the road!). The system worked quite nicely: the cells with damaged DNA didn’t express as much GFP as those with undamaged DNA, as expected. Also, repair of the DNA was slowed down by a drug called Veliparib, which inhibits the DNA repair protein PARP. See the results on Zenodo.

Question 2: Is there a difference in repair rates between normal and HD cells?

Once again, before tackling this question in HD patient cells, I opted for the easier-to-work-with mouse cells while I set up the system. In the first attempt (deposited to Zenodo), there were not enough HD cells recovered. From the few cells recovered, it looked like there might be a decreased DNA repair rate in the HD cells compared to the normal cells.

In the second attempt, enough cells were recovered to tell what was going on. The HD cells did in fact have a lower DNA repair rate, but inhibiting the DNA repair protein PARP had no effect (results on Zenodo). This could mean one of two things: either the difference we see between normal and HD cells is not because of DNA repair rates (which would be a bummer), or PARP inhibition is not working under these conditions. I’m hoping for the latter, and will try some different strategies to make sure we’re dealing with true DNA repair rates here. If we are, then we can use this method to further investigate the huntingtin interacting proteins we identify, and how they cooperate with huntingtin in the DNA repair process.

There are some other ways we can look at DNA repair rates in cells, as well as comparing the dynamics of the huntingtin protein (getting to and from damaged DNA) in normal versus HD cells. I will tackle some of those approaches and report them in the coming weeks.

This work is funded by the HDSA Berman/Topper HD Career Development Fellowship.

2 thoughts on “Measuring the rate of DNA repair in HD cells

Leave a Reply