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

Abstract

Expansion of CAG/CTG repeats is the underlying cause of >fourteen 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 as to 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, repair efficiency at CAG/CTG repeats and at control DNA sequences was markedly reduced under the striatal conditions, likely due to the lower level of APE1, FEN1 and LIG1. Damage located towards the 5’ end of the repeat tract was poorly repaired accumulating incompletely processed intermediates as compared to an AP lesion in the centre or at the 3’ end of the repeats or within a control sequences. Moreover, repair of lesions at the 5’ end of CAG or CTG repeats involved multinucleotide synthesis, particularly under the cerebellar stoichiometry, suggesting that long-patch BER processes lesions at sequences susceptible to hairpin formation. Our results show that BER stoichiometry, nucleotide sequence and DNA damage position modulate repair outcome, and suggest that a suboptimal LP-BER activity promotes CAG/CTG repeat instability.

Tissue specificity in DNA repair: lessons from trinucleotide repeat instability

“…This simple inverse correlation was substantiated by in vitro assays in which varying the amount of BER proteins to mimic the striatal or cerebellar situations modulated the processing of a CAG/CTG repeat by LP-BER and thereby the instability [58]. Together these experiments strongly suggest that the amount of BER proteins can account for the tissue-specificity of instability.…”

Repeat instability during DNA repair: Insights from model systems

“…In the HD mouse model, the stoichiometry of proteins involved in BER has been suggested to explain why the striatum is more prone to expansion than the cerebellum. Specifically, a low ratio of proteins like FEN1, APE1, and LIG1 relative to Polβ would favor strand displacement that would lead to expansions (Goula et al ., 2009, 2012a). Whether this correlation holds up when a larger range of tissues are tested remains to be seen.…”

Impact of alternative DNA structures on DNA damage, DNA repair, and genetic instability

“…The binding affinity and repair efficiency of hOGG1 protein on 8-oxoG in a hairpin structure was significantly reduced compared to 8-oxoG in B-DNA [133]. In the striatal tissue of HD mice carrying expanded CAG repeats, abasic sites located at the 5’ ends of the CAG repeats in a hairpin conformation were repaired less efficiently than abasic sites in B-DNA [134]. Purified MSH2-MSH3 complex can bind to CAG repeats in a hairpin structure, but the ATP hydrolysis activity of MSH2-MSH3 was reduced upon binding to a hairpin structure that contained A-A mis-paired bases in the stem.…”