Tara N. Lyons-Darden, Ph.D.

Dissertation research performed under the guidence of Michael D. Topal

ABSTRACT
    Triplet repeats are repetitive sequences of DNA that vary in sequence and length among individuals.  These sequences are normally found throughout mammalian genomes.  In certain families, the number of triplet repeats in particular genes can drastically increase from one generation to the next.  Expansions beyond a threshold repeat length can result in several hereditary neurodegenerative and neuromuscular disorders such as Huntington’s disease, myotonic dystrophy, and Fragile X.  Triplet repeat expansions have been shown to occur during meiosis as well as mitosis, but it is uncertain what initiates the mechanism of triplet repeat expansion.
    The instability of DNA may provide a plausible explanation for triplet repeat expansion.  DNA instability can be represented by alterations in endogenous environmental factors as well as DNA damage.   A minimal in vitro DNA replication assay was utilized in these studies.  The results show that in vitro triplet repeat expansion was induced by higher temperatures, optimal Mg2+ concentrations, increased triplet repeats in the template, a higher AT-rich DNA composition, and the presence of DNA damage.  The expanded products were double-stranded and sequencing revealed the presence of excess triplet repeats.  DNA instability appeared to induce triplet repeat expansion in vitro.  Additionally, these expanded triplet repeat products were stabilized by cross-linkers and visualized by electron microscopy.  The products had a double-stranded appearance and contained both the primer and template.  This indicated that the primer was likely forming a secondary structure with the excess triplet repeats and it appeared that the structures increased over time to produce large expansion products.  The results from this dissertation provided support for a mechanism of triplet repeat expansion that involves DNA slippage in combination with the formation of intermediate secondary structures.  This situation could occur in the nascent strand of lagging strand synthesis or even after a DNA nick during a DNA repair event.  The information gained may provide important insights into the possible mechanisms and causes of triplet repeat expansions in vivo, which result in triplet repeat disorders.