Visualizing the Protein-DNA interactions of human MutSbeta and MutLgamma in Trinucleotide Repeat Expansion using Atomic Force Microscopy

DNA mismatch repair (MMR) is a process essential to maintaining genomic stability by correcting errors that occur during DNA replication. Surprisingly, two MMR homologs MutSb and MutLg have been found to promote a mutation known as trinucleotide repeat (TNR) expansion, which causes the onset of various neurodegenerative diseases, most notably Huntington’s disease. In this work, atomic force microscopy (AFM) was used to characterize the protein-DNA interactions between MutSb, MutLg, and DNA substrates with an extrahelical (CAG)₁ loop in different nucleotide conditions. We found that MutSb has a high binding specificity to (CAG)₁loops, but the addition of ATP facilitates the formation of sliding clamps and the association of multiple MutSb complexes to a single DNA strand. In the presence of MutLg, 1:1 MutSb-MutLg complexes form at the loop independently of ATP, which implies that crucial ATP-mediated conformational changes may be getting bypassed. Moreover, ATP appears to promote nonspecific interactions between MutLg and DNA. Additional control experiments without MutSb, without DNA, and at varying timepoints will help us better understand the extent of these unexpected interactions and demystify why these proteins promote instead of prevent TNR expansion.