Abstract
Expansion of trinucleotide repeats (TNRs) is associated with progression of several human neurological diseases including Huntington's disease. The proposed mechanism of TNR expansion involves replication‐associated slippage, leading to the formation of TNR hairpins. Thus, factors preventing or removing TNR hairpins are predicted to inhibit disease progression and promote TNR stability. To determine how human cells process TNR hairpins, circular plasmid DNA substrates containing a (CAG)25 or (CTG)25 hairpin were constructed and analyzed for repair by human nuclear extracts. We show that human cells catalyze error‐free repair of (CAG)25 and (CTG)25 hairpins in a nick‐directed PCNA‐dependent manner. The repair is initiated by incisions specifically targeting the repeat sequences in the nicked strand, followed by repair DNA synthesis using the continuous strand as a template, thereby ensuring TNR stability. Depletion of PCNA blocks repair prior to TNR incision. However, distinct incision products were observed in reactions containing TNR hairpins with different strand locations and secondary structures, indicating utilization of distinct endonucleases in different hairpin repair. Our findings on TNR hairpin repair will provide significant insight into the mechanisms of TNR expansion and the etiology of human diseases caused by TNR expansions.
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