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Expansion of a CGG-repeat tract in the 5’ untranslated region of the FMR1 gene causes the fragile X-related disorders (FXDs; aka the FMR1 disorders). The expansion mechanism is likely shared by the 35+ other diseases resulting from expansion of a disease-specific microsatellite, but many steps in this process are unknown. We have shown previously that expansion is dependent upon functional mismatch repair proteins, including an absolute requirement for MutLγ, one of the three MutL heterodimeric complexes found in mammalian cells. We demonstrate here that both MutLα and MutLβ, the two other MutL complexes present in mammalian cells, are also required for most, if not all, expansions in a mouse embryonic stem cell model of the FXDs. A role for MutLα and MutLβ is consistent with human GWA studies implicating these complexes as modifiers of expansion risk in other Repeat Expansion Diseases. The requirement for all three complexes suggests a novel model in which these complexes co-operate to generate expansions. It also suggests that the PMS1 subunit of MutLβ may be a reasonable therapeutic target in those diseases in which somatic expansion is an important disease modifier., Author summary Repeat Expansion Diseases, including the fragile X-related disorders, are a large group of human genetic disorders caused by a mutation in a disease-specific tandem repeat or microsatellite. This mutation increases the number of repeats in that microsatellite. Unusual features of this mutation include its high frequency and its absolute requirement for proteins involved in Mismatch Repair, some of the very proteins that normally protect against classical microsatellite instability. Proteins known to be essential for this mutation include MLH3, the MLH1 binding partner in MutLγ, one of the three mammalian MutL complexes. Here we show that PMS2 and PMS1, MLH1-binding partners in the remaining two MutL complexes, MutLα and MutLβ respectively, are also required for expansion in a cell-based model of the fragile X-related disorders. This has interesting implications for the mechanism of repeat expansion. It also identifies another potential therapeutic target for reducing the mutation responsible for these diseases.

The Fragile X-related disorders (FXDs) are Repeat Expansion Diseases resulting from an expansion of a CGG-repeat tract at the 5’ end of the FMR1 gene. The mechanism responsible for this unusual mutation is not fully understood. We have previously shown that mismatch repair (MMR) complexes, MSH2/MSH3 (MutSβ) and MSH2/MSH6 (MutSα), together with Polβ, a DNA polymerase important for base excision repair (BER), are important for expansions in a mouse model of these disorders. Here we show that MLH1/MLH3 (MutLγ), a protein complex that can act downstream of MutSβ in MMR, is also required for all germ line and somatic expansions. However, exonuclease I (EXO1), which acts downstream of MutL proteins in MMR, is not required. In fact, a null mutation in Exo1 results in more extensive germ line and somatic expansions than is seen in Exo1+/+ animals. Furthermore, mice homozygous for a point mutation (D173A) in Exo1 that eliminates its nuclease activity but retains its native conformation, shows a level of expansion that is intermediate between Exo1+/+ and Exo1-/- animals. Thus, our data suggests that expansion of the FX repeat in this mouse model occurs via a MutLγ-dependent, EXO1-independent pathway, with EXO1 protecting against expansion both in a nuclease-dependent and a nuclease-independent manner. Our data thus have implications for the expansion mechanism and add to our understanding of the genetic factors that may be modifiers of expansion risk in humans., Author summary The Fragile X-related disorders arise from expansion of a tandem repeat or microsatellite consisting of CGG-repeat units. The expansion mutation is not well understood, but our previous data suggests that MutSα and MutSβ, mismatch repair (MMR) proteins that normally protect the genome against microsatellite instability, are actually responsible for these mutations in a knockin mouse model of these disorders. In this manuscript we describe the role in expansion of two proteins that act downstream of the MutS proteins in MMR, MutLγ and EXO1. Our data suggests that expansion occurs via a MutLγ-dependent, EXO1-independent pathway, with EXO1 playing both a nuclease-dependent and a nuclease-independent role in preventing expansions.