DNA is the genetic material that codes the “Secret of Life” in most living organisms.  The central thesis of molecular biology is that information present on DNA is transcribed into an RNA intermediate and ultimately gets translated into a protein message. Proteins are molecular workhorses that ensure smooth functioning of the cell.  Hence it is crucial that information present on the DNA be stably maintained for proper cellular functions.  

The DNA is wrapped around a proteinaceous material, and this unit is collectively referred to as the chromosome.  The genome, which is all of an organism’s DNA, is composed of non-repetitive and repetitive sequences (the Human Genome Project has estimated that the repetitive DNA occupies ~50% of the human genome).  Repetitive DNA sequences are like a double-edged sword — they are crucial for genome stability but their unchecked spread can interfere with normal cellular functions as is seen in some genetic disorders and cancers.

Despite being a stable structure, DNA sometimes accumulates damage called mutations.  Unless faithfully repaired by the cell’s defense system, the damage could persist and result in serious malfunctioning of the cell.  Our laboratory studies one such DNA damage, mutations called “Trinucleotide Repeat Expansions.”  The research is important because different trinucleotide repeat sequences expand to cause genetic diseases in humans.  Since the discovery of this mutation in 1991, expansion of CAG/CTG trinucleotide repetitive DNA has been shown to be the basis of 13 genetic diseases such as Huntington’s disease (a degenerative neurological disease) and Myotonic dystrophy (a muscle-wastage syndrome).  

DNA is composed of four nucleotide bases labeled A, T, G and C.  CAG trinucleotide DNA is defined as a sequence of DNA in which the three DNA bases C, A, and G are repeated as a unit numerous times (CAGCAGCAGCAG, for instance, would be referred to as four CAG repeats). These repetitive DNA elements are highly unstable and can expand, contract, and break.  They can also form irregular structures on DNA that are capable of jamming the cellular machinery.  The CAG expansions can range anywhere from 6 to 35 repeats in a normal individual or expand to more than 38 repeats to cause the disease.  These mutations are unique because unlike typical DNA mutations where the damage is faithfully transmitted to offspring, these repeats undergo dynamic mutations, where the repeat accumulates further expansions when transmitted to the next generation.  This forms the basis of a phenomenon called “genetic anticipation” where a longer repeat sequence causes an earlier onset of the disease in the offspring, in other words, a father with an intermediate repeat length may have a child with a substantially longer repeat.  Further, expanded repeats correlate with increasing severity of the disease in subsequent generations.   

We know that these repeat tracts accumulate structural damage.  An individual’s DNA repair machinery is vital to fix any DNA damage.  Under extraordinary circumstances where the cell’s defense is unable to efficiently deal with anomalous sequences like these, the damage might persist and result in further complications.  This damage could be mis-repaired and in instances where the damage persists, it can result in breaks on the chromosome, and lead to loss of the entire chromosome.  Chromosomal breaks and loss are frequently observed in cancers.  

Our research is focused on understanding the genetic basis of the CAG/CTG trinucleotide repeat expansion disease.  More specifically, we characterize the role of proteins that repair damaged DNA in ensuring stability of DNA with repeat sequences. Understanding how, when, why the repeats expand and how the cell copes with such a mutation, is crucial to finding a cure for the diseases.

To answer some of these questions, we are experimenting with baker’s yeast (Saccharomyces cerevisiae), where the CAG/CTG repeats are integrated into the yeast chromosome.  Yeast, a simple unicellular organism, is a useful tool to address the most complex of problems in human biology because most of the cellular processes between these two life forms are conserved.  In genetics, one way to determine the role of a gene within a cell is to delete or “knock out” a gene and subsequently analyze the effects of loss of that gene’s function.  Recall that DNA comes in the form of a double helix that comprises of two strands of DNA.  If one strand breaks for some reason, it is a single strand break.  If both the strands break, it is called a double strand break.  Using a simple genetic assay that measures the rate of breakage of the CAG/CTG repeat sequences on the yeast chromosome, we have been able to show that double strand break repair (DSB) proteins are important for preventing DNA breaks at the expanded CAG/CTG trinucleotide repeats.  Polymerase Chain reaction (PCR) is a molecular biology technique where a small segment of DNA can be amplified exponentially in a test tube.  In this example, the PCR technique is used to amplify DNA that contains the repeats to monitor additions and deletions to CAG repeat tract.  With the PCR-based assay that measures the frequency of repeat expansions and contractions in a strain of yeast that lacks certain repair proteins, we show that efficient repair is important for preventing CAG/CTG expansions as well.  We have also shown that reduced efficiency of double stranded break repair of expanded CAG/CTG tracts has dire consequences on cell survival.  

One common example of a trinucleotide repeat expansion disorder is Huntington’s disease.  Brain cells in patients undergo extensive cell death in an aggravated form of the disease. This neurodegeneration translates into involuntary, jerky body movements referred to as chorea as well as a reduction in various cognitive abilities including thinking and speech.  Diseases like Huntington’s chorea are complicated because they could have multiple causes and multiple effects.  The diseases caused by this mutation definitely deserve a holistic approach to finding a cure and DNA repeat expansion is just the beginning.  Efforts are ongoing to understand other aspects of the disease, namely processes acting at the RNA and protein levels that might lead to the disease.  

This challenge, the apparent chaos and the internal mystery of the functioning of living cell, is what makes biological research an exhilarating and satisfying experience and I am excited to be a part of this intellectual revolution.

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