Large-scale contractions of Friedreich's ataxia GAA repeats in yeast occur during DNA replication due to their triplex-forming ability.

Abstract

Friedreich's ataxia (FRDA) is a human hereditary disease caused by the presence of expanded (GAA)_n repeats in the first intron of the <i>FXN</i> gene [V. Campuzano <i>et al.</i>, <i>Science</i> 271, 1423-1427 (1996)]. In somatic tissues of FRDA patients, (GAA)_n repeat tracts are highly unstable, with contractions more common than expansions [R. Sharma <i>et al.</i>, <i>Hum. Mol. Genet.</i> 11, 2175-2187 (2002)]. Here we describe an experimental system to characterize GAA repeat contractions in yeast and to conduct a genetic analysis of this process. We found that large-scale contraction is a one-step process, resulting in a median loss of ∼60 triplet repeats. Our genetic analysis revealed that contractions occur during DNA replication, rather than by various DNA repair pathways. Repeats contract in the course of lagging-strand synthesis: The processivity subunit of DNA polymerase δ, Pol32, and the catalytic domain of Rev1, a translesion polymerase, act together in the same pathway to counteract contractions. Accumulation of single-stranded DNA (ssDNA) in the lagging-strand template greatly increases the probability that (GAA)_n repeats contract, which in turn promotes repeat instability in <i>rfa1</i>, <i>rad27</i>, and <i>dna2</i> mutants. Finally, by comparing contraction rates for homopurine-homopyrimidine repeats differing in their mirror symmetry, we found that contractions depend on a repeat's triplex-forming ability. We propose that accumulation of ssDNA in the lagging-strand template fosters the formation of a triplex between the nascent and fold-back template strands of the repeat. Occasional jumps of DNA polymerase through this triplex hurdle, result in repeat contractions in the nascent lagging strand.