Year of Publication

2011

Degree Name

Doctor of Philosophy (PhD)

Document Type

Dissertation

College

Medicine

Department

Toxicology

First Advisor

Dr. Guo-Min Li

Abstract

CAG/CTG repeat instability is associated with at least 14 neurological disorders, including Huntington’s disease and Myotonic dystrophy type 1. In vitro and in vivo studies have showed that CAG/CTG repeats form a stable hairpin that is believed to be the intermediate for repeat expansion and contraction.

Addition of extra DNA is essential for repeat expansion, so DNA synthesis is one of the keys for repeat expansion. In vivo studies reveal that 3’ CTG slippage with subsequent hairpin formation (henceforth called the 3’ CTG slippage hairpin) occurs during DNA synthesis. It is proposed that hairpin tolerance machinery is activated because prolonged stalling of DNA polymerase triggers severe DNA damage. As a means toward studying the hairpin-mediated expansion, we created a special hairpin substrate, mimicking the 3’ CTG slippage hairpin, to determine which polymerase promotes hairpin bypass. Our studies reveal polymerase β (pol β) is involved in the initial hairpin synthesis while polymerase δ (pol δ) is responsible for the resumption of DNA synthesis beyond the hairpin (extension step). Surprisingly, we also found that the pol δ can remove the short CTG hairpin by excision of the hairpin with its 3’ to 5’ exonuclease activity.

Besides repairing the hairpin directly, resolving the hairpin is an alternative pathway to maintain CAG/CTG repeat stability. With limited understanding of which human helicase is responsible for resolving CAG/CTG hairpins, we conducted a screening approach to identify the human helicase involved. Werner Syndrome Protein (WRN) induces the hairpin repair activity when (CTG)35 hairpin is formed on the template strand. Primer extension assay reveals that WRN stimulates pol δ synthesis on (CAG)35/(CTG)35 template and such induction was still found in the presence of accessory factors. Helicase assay confirms that WRN unwinds CTG hairpin structures.

Our studies provide a better understanding of how polymerases and helicases play a role in CAG/CTG repeat instability. Considering CAG/CTG repeat instability associated disorders are still incurable, our studies can provide several potential therapeutic targets for treating and/or preventing CAG/CTG repeat associated disorders.

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