Processing of various sizes of (CAG)n/(CTG)n hairpins in human nuclear extracts

Abstract

Expansion of CAG/CTG trinucleotide repeats (TNRs) in humans is associated with a number of neurological and neurodegenerative disorders, including Huntington's disease. Increasing evidence suggests that formation of a stable DNA hairpin within CAG/CTG repeats during DNA metabolism leads to TNR instability. However, the molecular mechanism by which cells recognize and repair CAG/CTG hairpins is largely unknown. Recent studies have shown that human cells possess a repair system that can remove DNA hairpins containing a sized of 20–25 CAG/CTG repeats. The repair system specifically targets the repeat tracts for incisions in the nicked strand in a nick‐directed and error‐free manner. To determine the substrate spectrum of the hairpin repair system and its ability to process smaller hairpins, which may be the intermediates for CAG/CTG expansions, we constructed a series of CAG/CTG hairpin heteroduplexes containing different numbers of repeats (from 5 to 25) and examined their repair in human nuclear extracts. We show here that although repair efficiencies slightly differ among these substrates, removal of the individual hairpin structures all involve endonucleolytic incisions within the repeat tracts in the nicked DNA strand, a pattern consistent with the repair of 25 repeat‐containing hairpins. Analysis of the repair intermediates allowed us to precisely determine the incision site for each substrate. These results suggest that the hairpin repair system in human cells prevents CAG/CTG stability by processing various sizes of CAG/CTG hairpin structures during DNA metabolism.