The role of break-induced replication in large-scale expansions of (CAG)n/(CTG)n repeats

Abstract

Expansions of (CAG)n•(CTG)n trinucleotide repeats are responsible for over a dozen neuromuscular and neurodegenerative disorders. Large-scale expansions are typical for human pedigrees and may be explained by iterative small-scale events such as strand slippage during replication or repair DNA synthesis. Alternatively, a distinct mechanism could lead to a large-scale repeat expansion at a step. To distinguish between these possibilities, we developed a novel experimental system specifically tuned to analyze large-scale expansions of (CAG)n•(CTG)n repeats in Saccharomyces cerevisiae. The median size of repeat expansions was ~60 triplets, though additions in excess of 150 triplets were also observed. Genetic analysis revealed that Rad51, Rad52, Mre11, Pol32, Pif1, and Mus81 and/or Yen1 proteins are required for large-scale expansions, whereas proteins previously implicated in small-scale expansions are not involved. Based on these results, we propose a new model for large-scale expansions based on recovery of replication forks broken at (CAG)n•(CTG)n repeats via break-induced replication.

Role of recombination and replication fork restart in repeat instability

“…Replication during BIR is highly mutagenic as it proceeds via a migrating bubble of conservative DNA synthesis (108, 109). Recent work has shown that large-scale CAG/CTG expansions (defined as addition of more than 20 repeats) of a (CAG) 140 repeat tract in a yeast model utilizes traditional HR machinery as well as Pol32 and Pif1, implicating BIR as a mechanism for generation of large-scale repeat expansions (Figure 2F) (110). These large-scale expansions depended on replication, therefore they could also be generated though a mechanism similar to broken fork repair (111) or HR-dependent fork restart that occurs at protein barriers to replication (33) (Figure 1A).…”

RNA biology of disease-associated microsatellite repeat expansions

“…One intriguing mechanism for rapid and large repeat accumulation is break-induced replication (BIR) [148, 176]. BIR is a homologous recombination pathway that can rescue collapsed or broken replication forks [195].…”

Close encounters: Moving along bumps, breaks, and bubbles on expanded trinucleotide tracts

“…In this case, a single end can invade the homologous chromosome and copy to the chromosome end [81,183,185]. In the engineered yeast system harboring large CAG expansions [185], loss of Rad51, Rad52, Mre11, Pif1, and Mus81 and/or Yen1 proteins suppressed TNR expansion, all of which are proteins of BIR and recombination pathways [183,184]. Pif1 prevents replication- fork stalling at G-quadruplexes [186].…”

“…YEN-1 is a Holliday junction resolvase, which promotes template switching during BIR [187]. Notably, defects in the replicative DNA polymerases δ and ε strongly increased rates for both repeat expansions, but mutants that impair translesion DNA polymerases have no effects, indicating the specificity of the reaction for B family polymerases for the expansions [184]. These results are consistent with previous reports that DNA polymerases η and ζ lack significant effects on triplet repeat instability [188], that Mre11-Rad50-Xrs2 complex promotes trinucleotide repeat expansions independently of homologous recombination [189], and the fact that the catalytic subunit of DNA PK has no impact on expansion in mice [108].…”