Human PMS1-dependent non-canonical mismatch repair converges with MBD4 to repair 5-methylcytosine deamination

CpG dinucleotides are hotspots for mutagenesis by spontaneous deamination of 5-methylcytosine (5mC) into thymine, resulting in T:G mismatches that can lead to C>T transitions. These mutations are a hallmark of aging and cancer and a major force shaping the evolution of vertebrate genomes. We have previously uncovered MBD4 as the primary base excision repair (BER) glycosylase responsible for 5mC deamination repair. In this study, we employ a cytosine base-editing system, comprising an APOBEC1 deaminase fused to a catalytically dead Cas9, to induce targeted cytosine deamination in native chromatin and track its repair. This approach reveals that MBD4 cooperates with a non-canonical branch of mismatch repair (MMR) to elicit 5mC deamination repair. We demonstrate that MBD4 activity depends on MMR complexes MutS (MSH2-MSH6) and MutL{beta} (MLH1-PMS1), but not on post-replicative MMR elicited by the MutL (MLH1-PMS2) complex. We find that PMS1 loss phenocopies the genome-wide CpG>TpG hypermutated profile associated with MBD4 deficiency, uncovering that 5mC deamination repair may represent one of the primary functions of the MutL{beta} complex. The mutational landscape of MMR-deficient tumors aligns with our experimental results, showing that replication-independent CpG>TpG mutagenesis partly contributes to the mutational burden of tumors inactivated for MLH1, MSH2 and MSH6. Finally, using structural predictions alongside biochemical validation, we show that MBD4 physically interacts with MutL{beta} in an MLH1-dependent manner, illuminating the structural basis for the convergence of the BER and MMR pathways. 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.