bi300410d_si_001.pdf (58.47 kB)
Download file

The Nucleotide Sequence, DNA Damage Location, and Protein Stoichiometry Influence the Base Excision Repair Outcome at CAG/CTG Repeats

Download (58.47 kB)
journal contribution
posted on 16.12.2015, 21:21 by Agathi-Vasiliki Goula, Christopher E. Pearson, Julie Della Maria, Yvon Trottier, Alan E. Tomkinson, David M. Wilson, Karine Merienne
Expansion of CAG/CTG repeats is the underlying cause of >14 genetic disorders, including Huntington’s disease (HD) and myotonic dystrophy. The mutational process is ongoing, with increases in repeat size enhancing the toxicity of the expansion in specific tissues. In many repeat diseases, the repeats exhibit high instability in the striatum, whereas instability is minimal in the cerebellum. We provide molecular insights into how base excision repair (BER) protein stoichiometry may contribute to the tissue-selective instability of CAG/CTG repeats by using specific repair assays. Oligonucleotide substrates with an abasic site were mixed with either reconstituted BER protein stoichiometries mimicking the levels present in HD mouse striatum or cerebellum, or with protein extracts prepared from HD mouse striatum or cerebellum. In both cases, the repair efficiency at CAG/CTG repeats and at control DNA sequences was markedly reduced under the striatal conditions, likely because of the lower level of APE1, FEN1, and LIG1. Damage located toward the 5′ end of the repeat tract was poorly repaired, with the accumulation of incompletely processed intermediates as compared to an AP lesion in the center or at the 3′ end of the repeats or within control sequences. Moreover, repair of lesions at the 5′ end of CAG or CTG repeats involved multinucleotide synthesis, particularly at the cerebellar stoichiometry, suggesting that long-patch BER processes lesions at sequences susceptible to hairpin formation. Our results show that the BER stoichiometry, nucleotide sequence, and DNA damage position modulate repair outcome and suggest that a suboptimal long-patch BER activity promotes CAG/CTG repeat instability.