Termination of exonuclease 1‐catalyzed mismatch excision requires physical interaction between exonuclease 1 and MutLα

The human mismatch repair (MMR) reaction was previously reconstituted in vitro using purified recombinant proteins that include MutSα or MutSβ, MutLα, RPA, EXO1, HMGB1, PCNA, RFC, polymerase δ, and DNA ligase I. Although MutLα is not essential for 5′ nick‐directed excision, it promotes termination of mismatch‐provoked EXO1‐ catalyzed DNA excision upon mismatch removal. However, the mechanism of the excision termination reaction is unknown. This study examines whether the physical interaction between the MLH1 subunit of MutLα and EXOI, which is mediated by a conserved motif in EXO1 known as an MLH1 interacting protein (MIP)‐box, is responsible for the excision termination. For this purpose, alanine substitution mutations were engineered at residues F506 and F507 in the MIP‐box of EXO1, and the resulting FF‐EXO‐AA mutant protein was tested for its role in MMR in vitro . As expected, FF‐EXO1‐AA behaves similarly as wild type EXO1 in catalyzing 5′‐directed excision and interacting with MutSα, but the EXO1 mutant protein fails to interact with MutLα. Our in vitro MMR assay show that mismatch‐provoked excision is efficiently terminated in reactions containing wild type EXO1, but not in reactions containing FF‐EXO1‐ AA. These observations strongly suggest that the termination of EXO1‐catalyzed mismatch excision is executed via physical interaction between MutLα and EXO1.