Inverted repeat-stimulated sister-chromatid exchange events are RAD1-independent but reduced in a msh2 mutant

Abstract

Inverted repeats (IRs) and trinucleotide repeats (TNRs) that have the potential to form secondary structures in vivo are known to cause genome rearrangements. Expansions of TNRs in humans are associated with several neurological disorders. Both IRs and TNRs stimulate spontaneous unequal sister-chromatid exchange (SCE) in yeast. Secondary structure-associated SCE events occur via double-strand break repair. Here we show that the rate of spontaneous IR-stimulated unequal SCE events in yeast is significantly reduced in strains with mutations in the mismatch repair genes MSH2 or MSH3, but unaffected by a mutation in the nucleotide excision-repair gene RAD1. Non-IR-associated unequal SCE events are increased in both MMR- and rad1-mutant cells; however, SCE events for both IR- and non-IR-containing substrates occur at a higher level in the exo1 background. Our results suggest that spontaneous SCE occurs by a template switching mechanism. Like IRs, TNRs have been shown to generate double-strand breaks (DSBs) in yeast. TNR expansions in mice are MSH2-dependent. Since IR-mediated SCE events are reduced in msh2 cells, we propose that TNR expansion mutations arise when DSBs are repaired using the sister or the homolog as a template.

Figure 1

Unequal SCE assay. (A) The his3 unequal SCE substrate. The his3-Δ3′ construct is marked with a tail, and the his3-Δ5′ construct is marked with an arrowhead. The shaded region indicates the region of homology between the two truncated his3 fragments. A wild-type HIS3 gene can form by unequal exchange (B) or by a non-reciprocal gene conversion event (C).

Figure 2

Consequences of replication fork stalling at secondary structures. (A) Pausing at the replication fork can lead to template switching. Template switching can form a wild-type HIS3 gene. The large loop must be preserved from mismatch repair until the next round of DNA replication, or else be repaired in favor of loop retention, to generate a His⁺ cell. (B). A DSB formed due to nicking of the secondary structure within the triplet repeat sequence may be repaired by an intra-chromosomal single-strand annealing-like event, leading to contraction of the repeat tract. The hatch marks represent the repeated units.