Repair of CAG/CTG repeat loop in human cells

Abstract

Trinucleotide repeat (TNR) expansion is the genetic basis for certain neurological and neurodegenerative disorders, including Huntington's disease. Since TNR sequences, e.g., (CAG)n, form a loop or hairpin structure, DNA loop repair is considered a major mechanism for TNR stability. However, the molecular mechanisms of DNA loop repair and protein components involved in this pathway are poorly understood. Using a biochemical approach, where human nuclear extracts were examined for their ability to process a plasmid DNA containing a (CAG)25 or (CTG) 25 loop, we show here that repair of CAG or CTG loop in human cells occurs in a nick‐ or loop‐directed manner. The repair is initiated by an incision at the top of the loop, followed by additional incision and excision, limited repair DNA synthesis, and ligation. Several biochemical mutants derived from fractionations allow us to demonstrate that PCNA, RPA, and FEN1 are essential components involved in TNR loop repair. We also show that MutSβ, a mismatch recognition protein that was previously thought to promote CAG expansion in transgenic mouse model by binding to the CAG loop and inhibiting its repair, has no inhibitory effect on TNR loop repair in human extracts, suggesting that the transgenic animal model may not reflect TNR instability in human cells. Our findings provide molecular insight into the mechanism by which cells maintain TNR stability.