ELUCIDATE THE MECHANISM OF DYSREGULATED DNA DAMAGE REPAIR AND PARYLATION IN HUNTINGTON DISEASE

Abstract

Aberrant DNA damage repair (DDR) and poly(ADP-ribosyl)ation (PARylation) have been implicated in the pathogenesis of neurodegenerative disorders, including Huntington disease (HD). HD is an autosomal dominant neurodegenerative disorder caused by the expansion of the CAG repeat in the huntingtin gene. Multiple genome-wide association studies have implicated DNA damage repair (DDR) mechanisms as a major modifier of disease. Others have also shown elevated levels of DNA damage and dysregulated PAR signaling in HD, implicating a role of the huntingtin protein in DNA damage repair. In this dissertation, I characterized a potential mechanism of HD pathology through the deficient PAR and XRCC1 signaling. In patient-derived fibroblast cell lines, we investigated the recruitment of PARP and the formation of PAR chains at DNA damage sites. Live cell quantification of PARP and PAR formation showed elevated levels of PARP1 recruitment in homozygous HD cells compared to wild-type cells, while levels of PAR formation were significantly reduced in heterozygous HD cells and control cells with huntingtin knockdown. This implicates a regulatory role of wildtype huntingtin in PARP1 recruitment and PAR formation. Compared to the control, HD cells also had lower basal energy levels and lower energy levels under oxidative stress conditions. Additionally, HD cells exhibited a higher susceptibility to genotoxic stress, indicating impairments in their DNA damage response. XRCC1 is an important scaffolding protein involved in single-stranded break repair and is regulated by PARylation. Under basal conditions and under oxidative stress, XRCC1 and phospho-XRCC1 levels were also reduced in HD cells. The unrepaired single-stranded breaks, coupled with energy deficiency, could result in the higher susceptibility to cell death seen in HD. Collectively, these results characterize the impact of the dysregulated DNA damage repair response in HD and implicate the hypoactivation of XRCC1 in HD pathology. This dissertation also outlines two additional chapters on the work conducted in collaboration with the Chatterjee lab. First, the design and validation of an algorithm named PepMLM, which generates protein binders using amino acid sequences and machine learning language models. The binders generated were attached to the catalytic domain of the E3 ubiquitin ligase to selectively degrade proteins of interest. I validated this system for the degradation of the huntingtin protein and MSH3, a genetic modifier of HD. When expressed in cells, the constructs were able to significantly reduce the levels of huntingtin and MSH3. Second, the design and validation of a flexible gene editing system through CRISPR. The Chatterjee lab designed a Cas9 protein, SpRYc, that has high editing efficiency with a flexible PAM recognition sequence, enabling flexible experimental design. I validated this system through its utilization to introduce a CAA interruption in the CAG track of the huntingtin protein with about 30% editing efficiency.