Specific Measurement of MutSβ and MutSα DNA Mismatch Repair Activity Using a Frameshift Reporter Assay

Abstract

This study introduces a lentiviral frameshift reporter to investigate the distinct roles of MutSα (MSH2/MSH6) and MutSβ (MSH2/MSH3), complexes that recognise base-base mismatches and loopouts to initiate DNA mismatch repair, in microsatellite instability in human cells. We demonstrate that MSH3 deficiency amplifies tetranucleotide repeat instability, with a strong contraction bias. Conversely, MSH6 deficiency increases mutation rates at mononucleotide repeats. These observations are consistent with their known roles in repair of loopout structures of different size and sequence. Deficiency of MLH1, the core component of MutL complexes that act downstream of MutS to coordinate repair, increases instability at both tetra- and mononucleotide repeats. Our reporter shows promise as an efficient tool for assessing the impact of genetic and chemical intervention on distinct MMR pathways. This is particularly important as MutSβ is an emerging therapeutic target in repeat expansion diseases, but therapies must avoid the oncogenic consequences of off target MutSα depletion.

Keywords: DNA mismatch repair (MMR), Microsatellite instability (MSI), Frameshift reporter, MutSα (MSH2/MSH6), MutSβ (MSH2/MSH3), Tetranucleotide repeat instability, Mononucleotide repeats, MLH1 deficiency, Loopout structures, Therapeutic target