Human MutLα activates methylpurine DNA glycosylase to induce alkylation damage cytotoxicity.
Alkylation chemotherapy is commonly used against tumors such as glioblastoma, yet resistance often develops through downregulation of mismatch repair (MMR). Previous work has established that loss of MMR prevents the excision of the thymine-containing strand across O 6meG-T mismatches, thereby avoiding the futile repair cycle that ultimately leads to cell death. Here, we provide an alternative explanation to this prevailing mechanism of chemoresistance by MMR loss. We found that the MMR protein MutLα physically and functionally interacts with the base excision repair (BER) enzyme methylpurine DNA glycosylase (MPG), which processes common alkylation adducts, such as 7meG and 3meA. Biochemical reconstitution demonstrates that MutLα activates MPG glycosylase activity by promoting MPG substrate binding, and enhancing MPG release from the abasic site product, thereby facilitating enzyme turnover. This glycosylase stimulation requires ATP hydrolysis as well as the MLH1-interacting region on MPG. Both MutLα or its ability to interact with MPG promote the generation of alkylation-induced abasic sites in cells, which contribute to the cytotoxicity of methyl methanesulfonate (MMS), an SN2 alkylating agent that does not produce O 6meG. Our results provide new insight into the mechanism of alkylation chemoresistance and uncover an unappreciated cross-talk between MMR and BER.