Investigating the Role of Genome Repair Mechanisms in the Instability of Myotonic Dystrophy Type 2-Causing CCTG Repeats

Genome maintenance is essential for the healthy development and survival of all organisms. Various molecular mechanisms are in place to prevent and repair mutations to the genome. However, some of these mechanisms lead to instability of expansion-prone microsatellite DNA repeats, which are the cause of over 40 genetic diseases in humans. While trinucleotide repeats (TNRs) have been well studied, information on tetranucleotide repeats is sparse. CCTG repeat instability in the CNBP gene leads to Myotonic Dystrophy Type II (DM2), a neuromuscular disease. In this study, we utilized Saccharomyces cerevisiae cells containing an engineered URA3 reporter with CCTG repeats to investigate the effect of genetic and environmental factors on large-scale CCTG repeat contractions. First, we identified several candidate genes involved in DNA replication, repair, and recombination events, as well as osmotic and replicative stressors for use in the study. Then we constructed knockout and separation-of-function mutants with the use of CRISPR-Cas9. Finally, we conducted assays to investigate the mutation rate of CCTG repeats in these mutants. Our results showed a significant effect of several proteins involved in DNA repair, such as Sgs1, Msh3, Msh6, Mlh1, Mlh2, Rad51, and Rad52, among other mutants, on CCTG repeat contraction rates. Though the helicase Srs2 did not show a significant effect on CCTG contractions, treatment with replicative stressors revealed a protective effect. Overall, however, the results suggest that homologous recombination during repair of double-strand breaks and mitotic crossover events is one of the major drivers of large-scale CCTG repeat contractions. We propose that the results of our study will aid in the development of therapeutic solutions for DM2 patients, that reduce instability of or induce contraction in CCTG repeats.