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)nrepeats in the first intron of theFXNgene [V. Campuzanoet al.,Science271, 1423–1427 (1996)]. In somatic tissues of FRDA patients, (GAA)nrepeat tracts are highly unstable, with contractions more common than expansions [R. Sharmaet al.,Hum. Mol. Genet.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)nrepeats contract, which in turn promotes repeat instability inrfa1,rad27, anddna2mutants. 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.