title: Replication stalling and heteroduplex formation within CAG/CTG trinucleotide repeats by mismatch repair.
abstract: Trinucleotide repeat expansions are responsible for at least two dozen neurological disorders. Mechanisms leading to these large expansions of repeated DNA are still poorly understood. It was proposed that transient stalling of the replication fork by the repeat tract might trigger slippage of the newly-synthesized strand over its template, leading to expansions or contractions of the triplet repeat. However, such mechanism was never formally proven. Here we show that replication fork pausing and CAG/CTG trinucleotide repeat instability are not linked, stable and unstable repeats exhibiting the same propensity to stall replication forks when integrated in a yeast natural chromosome. We found that replication fork stalling was dependent on the integrity of the mismatch-repair system, especially the Msh2p-Msh6p complex, suggesting that direct interaction of MMR proteins with secondary structures formed by trinucleotide repeats in vivo, triggers replication fork pauses. We also show by chromatin immunoprecipitation that Msh2p is enriched at trinucleotide repeat tracts, in both stable and unstable orientations, this enrichment being dependent on MSH3 and MSH6. Finally, we show that overexpressing MSH2 favors the formation of heteroduplex regions, leading to an increase in contractions and expansions of CAG/CTG repeat tracts during replication, these heteroduplexes being dependent on both MSH3 and MSH6. These heteroduplex regions were not detected when a mutant msh2-E768A gene in which the ATPase domain was mutated was overexpressed. Our results unravel two new roles for mismatch-repair proteins: stabilization of heteroduplex regions and transient blocking of replication forks passing through such repeats. Both roles may involve direct interactions between MMR proteins and secondary structures formed by trinucleotide repeat tracts, although indirect interactions may not be formally excluded.
authors.affiliation: Institut Pasteur, Unité de Génétique Moléculaire des Levures, Département Génomes & Génétique, 25 rue du Dr Roux, F-75015 Paris, France; Sorbonne Universités, UPMC Univ Paris 06, IFD, 4 Place Jussieu, 75252 Paris Cedex 05, France; CNRS, UMR3525, F-75015 Paris, France.
authors.forename: David
authors.initials: D
authors.lastname: Viterbo
authors.affiliation: Current address: King Abdullah University of Science and Technology (KAUST), Biological and Environmental Sciences and Engineering Division (BESE), Thuwal 23955-6900, Kingdom of Saudi Arabia.
authors.forename: Grégoire
authors.initials: G
authors.lastname: Michoud
authors.affiliation: Institut Pasteur, Unité de Génétique Moléculaire des Levures, Département Génomes & Génétique, 25 rue du Dr Roux, F-75015 Paris, France; Sorbonne Universités, UPMC Univ Paris 06, IFD, 4 Place Jussieu, 75252 Paris Cedex 05, France; CNRS, UMR3525, F-75015 Paris, France.
authors.forename: Valentine
authors.initials: V
authors.lastname: Mosbach
authors.affiliation: Institut Pasteur, Unité de Génétique Moléculaire des Levures, Département Génomes & Génétique, 25 rue du Dr Roux, F-75015 Paris, France; Sorbonne Universités, UPMC Univ Paris 06, IFD, 4 Place Jussieu, 75252 Paris Cedex 05, France; CNRS, UMR3525, F-75015 Paris, France.
authors.forename: Bernard
authors.initials: B
authors.lastname: Dujon
authors.affiliation: Institut Pasteur, Unité de Génétique Moléculaire des Levures, Département Génomes & Génétique, 25 rue du Dr Roux, F-75015 Paris, France; Sorbonne Universités, UPMC Univ Paris 06, IFD, 4 Place Jussieu, 75252 Paris Cedex 05, France; CNRS, UMR3525, F-75015 Paris, France. Electronic address: gfrichar@pasteur.fr.
authors.forename: Guy-Franck
authors.initials: GF
authors.lastname: Richard
mesh_terms: D053843:DNA Mismatch Repair; D004261:DNA Replication; D004271:DNA, Fungal / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051719:MutS DNA Mismatch-Binding Protein / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D019680:Trinucleotide Repeat Expansion; D018911:Trinucleotide Repeats / Q000235:genetics*
chemical_list: D004271:DNA, Fungal; D051719:MutS DNA Mismatch-Binding Protein
keywords: Heteroduplex; Mismatch repair; Replication pause; Trinucleotide repeats