Investigating the Intersection between DNA Mismatch Repair and Triplet Nucleotide Repeat Expansion
Authors: Kutlu Selin , Case Brandon , Hingorani Manju 
Published: November 2023

Mismatch repair (MMR) is a highly effective system dedicated to correcting errors produced during DNA replication. Errors arise when DNA polymerases incorporate incorrect nucleotides, leading to base pair mismatches, or slip from the primer‐template junction, leading to insertion‐deletion loops (IDLs). The proteins of interest in this study are the eukaryotic MutS homologs Msh2–Msh6 and Msh2–Msh3, which recognize single base pair mismatches or small IDLs, and larger IDLs, respectively1. Polymerases slip more easily at repeat sequences, which can lead to the formation of triplet nucleotide repeat (TNR) hairpins during replication. Msh2–Msh3 is known to recognize and promote the expansion of these TNR hairpin structures, which in turn is associated with neurological disorders such as Huntington's disease2. The goal of my research project is to understand how Msh2–Msh6 and Msh2–Msh3 interact with IDLs compared to TNRs. Gel mobility shift assays (GMSA) are being used to determine if there are differences in Msh2–Msh6 or Msh2–Msh3 complexes bound to these DNA structures. Preliminary results indicate that Msh2–Msh6 binds CAG repeat TNR hairpins with high affinity, similar to Msh2–Msh3. This leads to the question of why Msh2–Msh6 binding does not affect the TNR structure nor lead to its repair, while Msh2–Msh3 binding leads to its expansion. The answer may lie in differences in the affinity and dynamics of interaction between the proteins and DNA, or their ATPase activity, which is essential for the response to an error in the DNA3. In previous pre‐steady state ATPase studies of Msh2–Msh6, a rapid burst in hydrolysis was observed in the presence of duplex DNA3. In the presence of a mismatched DNA, however, this burst phase is absent, allowing Msh2–Msh6 to adopt a dual ATP‐bound form capable of sliding away from the mismatch, which is critical for signaling mismatch repair3. Due to the presence of ‘A‐A’ mismatches in the hairpin stem, it is possible that Msh2–Msh6 is recognizing this structure as error containing DNA. This hypothesis can be tested by measuring the ATPase response and related actions of Msh2–Msh6 on the TNR hairpin. Currently, a malachite green phosphate release assay is being used to measure the steady state ATPase activity of Msh2–Msh6, to determine any changes in the absence or presence of the TNR and to find optimal conditions for pre‐steady state experiments. In addition, a fluorescence‐based assay is being developed to measure the kinetics of Msh2–Msh6 interaction with the TNR and determine if the protein can adopt an ATP‐bound sliding clamp form on this DNA substrate. It is anticipated that the results of this kinetic analysis will help us understand in a quantitative manner how and why Msh2–Msh6 may not promote TNR expansion, while Msh2–Msh3 does. This mechanistic knowledge may help us find ways to avoid and possibly mitigate the devastating effects of TNR‐induced neurological disorders.

Support or Funding Information

NSF grant MCB 1022203; ASBMB Undergraduate Research Award