Mbd4 and MutSα protect cells from spontaneous deamination of 5-methylcytosine

5-Methylcytosine (5mC) is a common source of somatic mutations. Deamination of 5mC to thymine generates a G/T mismatch, which occurs spontaneously and must be repaired prior to DNA replication to avoid mutation. We generated genetically engineered mice and cell lines to define DNA repair pathways that protect against 5mC deamination. We observed a low background mutation rate in mouse bone marrow or colon, typically 0.2-0.5 CG > TG mutations/genome/day. This increased 3-7 fold in cells lacking the glycosylase methyl-binding domain 4 (Mbd4), one of the few glycosylases capable of excising thymine from G/T mismatches. We found no role for thymine DNA glycosylase (Tdg) in the repair of 5mC deamination in these tissues. Instead, our results flag potential cooperation between Mbd4 and the mismatch repair (MMR) complex MutSα (Msh6:Msh2), evident through elevated rates of CG > TG mutations in Msh6-deficient cells: 2.6-4.8 CG > TG mutations/genome/day in primary cells and 13.9 CG > TG mutations/genome/day in cell lines. Loss of Mbd4 results in a specific increase in DNA damage from 5mC deamination, whereas the broader functionality of MutSα explains why mutational signatures linked to Msh6 deficiency are more variable and reflect the replicative history of the cell. Our findings support the emerging view that MutSα has broader DNA repair activity outside of replication.