Background: The Repeat Expansion Diseases are caused by increases in the number of repeat units in a specific tandem repeat in a single gene. The Fragile X-related disorders (FXDs) arise from expansion of a CGG.CCG-repeat in the 5' UTR of the X-linked FMR1 gene. Carriers of alleles with 55-200 repeats, so-called premutation (PM) alleles, are at risk for a neurodegenerative disorder, Fragile X-associated tremor-ataxia syndrome (FXTAS), and a form of ovarian dysfunction known as FX-associated primary ovarian insufficiency (FXPOI). Furthermore, in females, the PM allele can undergo expansion on intergenerational transfer that can result in their children having alleles with >200 repeats. This expanded allele is known as a full mutation (FM) and, with very few exceptions, all individuals who inherit such alleles have Fragile X syndrome (FXS), the leading heritable cause of intellectual disability and autism. FXS symptoms arise because repeat expansion leads to gene silencing and the subsequent absence of FMRP, the FMR1 gene product, a protein important in many pathways including insulin signaling. The mechanism by which is expansion occurs is thought to differ from the generalized microsatellite instability (MSI) seen in many different cancers in that the instability is confined to a single genetic locus, it shows a strong expansion bias and our work has now shown that genes that normally protect against MSI are actually required to generate the FX mutation. Expanded alleles are also associated with a folate-sensitive fragile site that is coincident with the repeat on the X chromosome. There is reason to think that this fragile site is responsible for the high frequency loss of the affected chromosome resulting in Turner syndrome (45, X0) in female carriers of a FM allele. Progress report: Our previous work using a FX mouse model we developed (Entezam et. al., 2007), has shown that Polbeta together with the mismatch repair complexes, MutSbeta, MutSalpha and MutLgamma are actually responsible for the mutation that results in the FXDs (Lokanga et. al., 2012; 2015; Zhao et. al., 2014; 2015; 2016; Zhao and Usdin, 2015; Zhao et. al., 2018).In contrast, we showed that EXO1 and FAN1, 2 5'-3' exonucleases protect against expansion (Zhao et. al., 2018; Zhao and Usdin, 2018a). We have also shown that LIG4, a DNA ligase essential for Non-Homologous End-Joining (NHEJ), a form of double-strand break repair, also protects against expansion (Gazy et. al, 2018). This provided the first evidence for a double-strand break (DSB) intermediate in the expansion process and thus for the role of a form of DSB repair other than NHEJ in generating expansions. Many of these same genes have now been implicated in expansion in other Repeat Expansion Diseases including Huntington Disease and myotonic dystrophy type 1. This lends weight to the idea that our mouse model is relevant for understanding the unusual mutational mechanism responsible for these diseases. In this reporting period, we have extended our studies to examine other proteins in an attempt to better understand how normal DNA repair processes become subverted to produce these mutations. Some of this work was facilitated by the development of the first tissue-culture model suitable for studying the expansion mechanism in vitro (Gazy et. al., 2020). This model uses mouse embryonic stem cells derived from our FXD mouse model. Expansion in these cells shows a dependence on the same genetic factors important for expansion in vivo. These cells also can show 1-2 expansion events/week, a frequency that allows us to test the effect of different genetic factors much more rapidly than was previously possible. We used these cells and a CRISPR-Cas9-mediated gene editing approach to make a point mutation in the nuclease-domain of the MLH3 subunit of MutLgamma. This work demonstrated that the MLH3 nuclease activity is essential for the expansion process (Hayward et. al., 2020). We also used a CRISPR-Cas9 gene disruption approach to show that, in addition to MutLgamma, both of the other mammalian MutL complexes, MutLalpha and MutLbeta, also required for expansion (Miller et. al., 2020). This has interesting implications for our understanding of the expansion mechanism. It may also provide novel insights into normal mechanisms of mismatch repair. Of particular interest is the importance of MutLbeta, a protein that is much more abundant than MutLgamma, but whose normal function is still not well understood at all.