Human PMS1-dependent non-canonical mismatch repair engages with MBD4 to repair methylated CpG deamination.

CpG dinucleotides are mutational hotspots due to spontaneous deamination of 5-methylcytosine (5mC), resulting in T:G mismatches that can lead to CpG>TpG transitions. These mutations are a hallmark of aging and cancer and play a central role in the evolution of vertebrate genomes. We have previously uncovered MBD4 as the primary base excision repair (BER) glycosylase responsible for 5mC deamination repair. Here, we employ an APOBEC1 deaminase fused to a catalytically dead Cas9 to induce targeted 5mC deamination independently of DNA replication and track its repair in human cells. This approach reveals that MBD4 elicits a coordinated repair response with a non-canonical branch of mismatch repair (MMR) involving complexes MutLβ (MLH1-PMS1) and MutSα (MSH2-MSH6). We uncover the physical interaction between MBD4 and MutLβ and demonstrate that MBD4-mediated repair requires MLH1. We show that PMS1 deficiency phenocopies the CpG>TpG hypermutation signature characteristic of MBD4 loss, establishing 5mC deamination repair as a key function of human PMS1. In alignment with our experimental data, we show that the CpG>TpG mutational burden in MMR-deficient tumors is partly explained by replication-independent processes. Altogether, we uncover a novel function of non-canonical MMR that underscores its interplay with BER in safeguarding genomic integrity against damage to methylated DNA.