1. Can you give some background on your research with trinucleotide repeats?
Let’s consider, for example, a pedigree for the human genetic disease, called myotonic dystrophy type 1. In three generations: the grandmother is basically not affected, her daughter got mildly affected in her adulthood, while the grandson has a very severe form of the disease from birth. This phenomenon is called genetic anticipation, and even though this disease is caused by an autosomal dominant mutation, its inheritance is non-Mendelian in a sense that the manifestation of the disease gets worse as a mutant allele passes through generations.
The phenomenon of genetic anticipation was described in 1918, but it was substantiated, in a molecular sense, many years thereafter, when in the early nineties scientists cloned the genes responsible for those diseases. The first success was cloning of the fragile X syndrome gene. That was done by Steve Warren and his group at Emory University. The myotonic dystrophy gene was cloned by Bob Korneluk in Canada and by David Housman here in MIT.
What they found was that in all those cases, the disease is caused by the expansion of a very simple repeat. In the case of myotonic dystrophy, this is the CTG repeat located in the 3’ untranslated region of the DMPK gene, while in the fragile X case it’s the CGG repeat in the 5’ UTR of the FMR1 gene. The rule they found out was very clear-cut. When the number of repeats is between 6 and 50, the individual is healthy. When it’s somewhere between 50 and 200, the person is a carrier. In the disease state, it’s over 200 and up to 3,500 repeats. In other words, the molecular mechanism of genetic anticipation is that the longer the repeat, the more likely it is to lengthen further, the more severe the disease manifestation, and the earlier the onset.
In the DM1 pedigree discussed above, the number of repeats increased tenfold in just three generations. This is what my lab studies. How can a repeat expand so profoundly, and what are the mechanisms of these expansions? Specifically, we concentrate on how the carrier size repeat becomes a disease-size.
All these repeats can form unusual DNA structures that are strikingly different from regular B-DNA. Their individual strands can form imperfect hairpins or even four-stranded G-quartets if they are sufficiently G-C rich. They can also form what’s called slipped-strand DNA, when their complementary strands are annealed out of register. Last but not least, they can form a structure called a DNA triplex, or H-DNA, which I co-discovered in course of my postdoctoral studies. Very early on, back in 1995, Bob Wells had proposed that the formation of these structures during DNA synthesis may account for the repeat instability. His reasoning was as such. If there is a hairpin on the nascent strand during DNA synthesis, then after a round of replication this would result in an expansion. If, in contrast, the same hairpin is on the template strand, then it would end-up in contraction.
So that’s essentially when my lab has jumped in. We first wanted to see whether replication through expandable repeats inside the cell proceeds abnormally. In order to do this, we used a technique called 2-dimensional gel electrophoresis of replication intermediates. One can isolate from a cell all of its DNA, including molecules that are in the process of replication. This DNA is then digested with restriction enzymes and run on the gel across two dimensions, so they are separated by size and shape. This separates DNA molecules at various stages of replication from linear DNA that is not being replicated. If the replication fork is stalled at an abnormal DNA structure, one would observe an accumulation of a particular replication intermediate. Using this approach, we were able to demonstrate that expandable DNA repeats do stall replication in every experimental system studied, including bacterial, yeast and mammalian cells!
After that, we developed a new experimental system to study repeat expansions in baker’s yeast, S. cerevisiae. We took a reporter gene, artificially split it and inserted a carrier-size repeat into the intron. Yeast can not splice out very long introns. Thus, when we inserted 100 repeats, the intron was still short enough to be spliced. When repeats expand, however, the intron gets too longs to be spliced, and we can easily detect this event by the reporter’s inactivation.
Using this system, we were able to carry out genome-wide analysis of the genes that affect the rate of repeat expansions in our system. By the end, we identified roughly 40 genes that are important for the expansion process. As we expected, the majority of them encode the components of DNA replication machinery. Thus, repeat expansions occur during DNA replication! Other genes affect transcription and chromosome maintenance, and we are currently investigating their mechanisms of action.
2. Can you tell me a bit about the courses that you teach? Does the material overlap with your research?
Yeah, there is significant overlap with my Bio 190 course. This is an advanced course called DNA Structure to Function, which is 50% lectures, 50% paper presentations by students. There, we talk a lot about unusual DNA structures, including the structures involved in repeat expansion diseases. We also talk about the problems that they cause for DNA replication, transcription, and recombination. That’s actually directly related to my science.
The second course, which I co-teach with Catherine Freudenreich, is Bio 188. There, we talk about papers that are so important that they were chosen to be awarded with the Nobel Prize. These are really the key papers in modern biology. We give the background to the students and then they present original papers. We then discuss what is so special about these papers, and what makes them worth the highest prize in the natural sciences.
3. What first pulled you toward the study of genetics?
So that has to do with an ancient history. I grew up in the Soviet Union, and for the reasons, which would be too long to describe, genetics was outlawed in the Soviet Union from 1948 on. In 1948, the state made the decision that genetics is a bourgeois science, and as such it should not be taught. And then it was not taught for the next 20 years. So, this ban started when Joseph Stalin was in power, continued through the next country leader Nikita Khrushchev, and ended only when Leonid Brezhnev came to power . Now, you know that DNA structure was unraveled in 1953, so all reasonable people realized right away that not only is genetics a great science, but that there is actually a material carrier of genetic information. Yet this notwithstanding, genetics continued to be outlawed in the Soviet Union.
Anyhow, the state finally allowed genetics to be taught and the first textbook was published sometime around 1968. At the time, I was in the middle school. And I was totally fascinated that this textbook was published and I can read about genetics without worrying for the state repercussions. It was sort of like finally trying the forbidden fruit. I bought myself this college textbook in genetics, and I’m like 12 or 13 years old, so it was very hard for me to comprehend it. But I decided that I’ll read it as many times as needed to before I understand everything it says. So this is how it all started. Then I went to a high school which had a specialization in biology, and afterwards to the Biology Department of the Moscow State University. But the decision was made very early on, when I was essentially still in the middle school.
You grew up here in the US, and you grew up in a different era, so it’s probably hard for you to comprehend how it is to live in a system which is repressive by nature. It prevents you from doing certain things, it prevents you from reading certain books, watching certain movies, or studying certain scientific topics. It’s almost impossible for someone who grew up in this country to comprehend it. But such a system has a fundamental flaw, as people always try to do things that are forbidden: to read the forbidden books, watch the forbidden movies, do the forbidden science. And ultimately, when a critical mass of people is doing forbidden stuff, the system collapses. That’s what happened with the Soviet Union. But in my particular case, this repressive Soviet system made me a geneticist, which I’m kind of grateful for. That said, it required a lot of drive and resilience on my part.
When I teach my course, I always tell my students “there is science, and there is life.” Scientists do not live in an ivory tower. Life intervenes, and sometimes in a very brutal ways. Whether it’s like with genetics, which was not allowed to be taught in the Soviet Union, or before that during the Nazi years in Germany, when top German scientists of Jewish descent immigrated to the United States, which ultimately made US science great. The interplay between science and life is certainly worth discussing. In my case, as I said, this interplay made me study genetics. Were it not forbidden, who knows?
You can read more about Dr. Mirkin’s research at http://ase.tufts.edu/biology/labs/mirkin/, or register for one of the courses he teaches, Biology 188: Seminar in Molecular Biology & Genetics (Fall) and Biology 190: DNA: Structure to Function (Spring). If you would like to have your own research featured on the TuftScope blog, you can reach Kurtis at firstname.lastname@example.org