Trinucleotide repeat (TNR) expansions are the cause of over 40 human neurodegenerative diseases, and are linked to DNA damage and base excision repair (BER). We explored the role of DNA damage and BER in modulating TNR instability through analysis of DNA structures, BER protein activities, and reconstitution of repair using human BER proteins and synthesized DNA containing various types of damage. We show that DNA damage and BER can modulate TNR expansions by promoting removal of a TNR hairpin through coordinated activities of BER proteins and cofactors. We found that during repair in a TNR hairpin, coordination between the 5’-flap endonuclease activity of flap endonuclease 1 (FEN1), 3’-5’ exonuclease activity of AP endonuclease 1 (APE1), and activity of DNA ligase I (LIG I) can resolve the double-flap structure produced during BER in the hairpin loop. The resolution of the double-flap structure resulted in hairpin removal and prevention or attenuation of TNR expansions and provides the first evidence that coordination among BER proteins can remove a TNR hairpin. We further explored the role of BER cofactors in modulating TNR instability and found that the repair cofactor proliferating cell nuclear antigen (PCNA) facilitates genomic stability by promoting removal of a TNR hairpin. Hairpin removal was accomplished by altering dynamic TNR structures to allow more efficient FEN1 cleavage and DNA polymerase β (pol β) synthesis and stimulating the activity of LIG I. This study provides the first evidence that a DNA repair cofactor plays an important role in modulating TNR instability. Finally, we explored the effects of sugar modifications in abasic sites on activities of BER proteins and BER efficiency during repair in a TNR tract. We found that an oxidized sugar inhibits the activities of BER enzymes, interrupting their coordination and preventing efficient repair. Inefficient repair results in accumulation of repair intermediates with DNA breaks, contributing to genomic instability. Our results indicate that DNA base lesions and BER play a crucial role in modulating TNR instability. The research presented herein provides a molecular basis for further developing BER as a target for prevention and treatment of neurodegenerative diseases caused by TNR expansion.

Trinucleotide repeat (TNR) instability including repeat expansions and repeat deletions is the cause of more than 40 inherited incurable neurodegenerative diseases and cancer. TNR instability is associated with DNA damage and base excision repair (BER). In this dissertation research, we explored the mechanisms of BER-mediated TNR instability via biochemical analysis of the BER protein activities, DNA structures, protein-protein interaction, and protein-DNA interaction by reconstructing BER in vitro using synthesized oligonucleotide TNR substrates and purified human proteins. First, we evaluated a germline DNA polymerase β (pol β) polymorphic variant, pol βR137Q, in leading TNR instability-mediated cancers or neurodegenerative diseases. We find that the pol βR137Q has slightly weaker DNA synthesis activity compared to that of wild-type (WT) pol β. Because of the similar abilities between pol βR137Q and WT pol β in bypassing a template loop structure, both pol βR137Q and WT pol β induces similar amount of repeat deletion. We conclude that the slightly weaker DNA synthesis activity of pol βR137Q does not alter the TNR instability compared to that of WT pol β, suggesting that the pol βR137Q carriers do not have an altered risk in developing TNR instability-mediated human diseases. We then investigated the role of DNA synthesis activities of DNA polymerases in modulating TNR instability. We find that pol βY265C and pol ν with very weak DNA synthesis activities predominantly promote TNR deletions. We identify that the sequences of TNRs may also affect DNA synthesis and alter the outcomes of TNR instability. By inhibiting the DNA synthesis activity of pol β using a pol β inhibitor, we find that the outcome of TNR instability is shifted toward repeat deletions. The results provide the direct evidence that DNA synthesis activity of DNA polymerases can be utilized as a potential therapeutic target for treating TNR expansion diseases. Finally, we explored the role of post-translational modification (PTM) of proliferating cell nuclear antigen (PCNA) on TNR instability. We find that ubiquitinated PCNA (ub-PCNA) stimulates Fanconi associated nuclease 1 (FAN1) 5’-3’ exonucleolytic activities directly on hairpin structures, coordinating flap endonuclease 1 (FEN1) in removing difficult secondary structures, thereby suppressing TNR expansions. The results suggest a role of mono-ubiquitination of PCNA in maintaining TNR stability by regulating nucleases switching. Our results suggest enzymatic activities of DNA polymerases and nucleases and the regulation of the activities by PTM play important roles in BER-mediated TNR instability. This research provides the molecular basis for future development of new therapeutic strategies for prevention and treatment of TNR-mediated neurodegenerative diseases.