SN1 methylating agents are therapeutics that methylate the O6 position of guanine (O6meG) forcing mispairing with thymine during DNA replication. The mismatch repair (MMR) system recognizes O6meG:T mispairs and activates DNA damage response (DDR). Exonuclease I (EXO1) functions during MMR by excising the damaged strand; however, how MMR mechanistically contributes to DDR activation and if EXO1 is required to activate MMR-dependent response remains unknown. Here we show that Exo1 knockdown in mouse embryonic fibroblasts (MEFs) results in decreased G2/M arrest and checkpoint response (ie, Cdc2/Chk1 phosphorylation), and increased cell proliferation and viability following exposure to the SN1 methylator MNNG. Moreover, ectopic expression of full-length human hEXO1 restored normal response to MNNG. We next examined gamma-H2AX foci formation as a surrogate marker of MMR-dependent DNA repair. 12h after MNNG treatment, 40% of MEFs scored positive for multiple gamma-H2AX foci and this population further increased with prolonged treatment. In contrast, Exo1 depleted MEFs displayed a significantly diminished number of gamma-H2AX foci-positive cells following MNNG. Increased association of Msh2/Msh6 with chromatin was observed in MNNG treated MEFs; however, Exo1 depletion resulted in no upregulation of MMR proteins within the chromatin fraction. Furthermore, MNNG-induced Msh2/Chk1 association was disrupted upon Exo1 depletion. Taken together, we conclude that Exo1 is required to activate DDR following SN1 methylator exposure. Data gathered suggests that Exo1 functions in activating MMR-dependent DNA repair, promoting assembly of MMR complexes with alkylated chromatin, and enhancing the association of MMR components with critical checkpoint mediators. Experiments designed to determine if Exo1 functions in a structural (ie, scaffolding) or catalytic (ie, nucleolytic) manner during SN1 methylator-induced DDR are currently underway.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2542. doi:1538-7445.AM2012-2542