Exonuclease 1 (Exo1) is required for activating response to SN1 DNA methylating agents

Evgeny Izumchenko, John Saydi, Kevin D. Brown

Research output: Contribution to journalArticlepeer-review

Abstract

SN1 DNA methylating agents are genotoxic agents that methylate numerous nucleophilic centers within DNA including the O6 position of guanine (O6meG). Methylation of this extracyclic oxygen forces mispairing with thymine during DNA replication. The mismatch repair (MMR) system recognizes these O6meG:T mispairs and is required to activate DNA damage response (DDR). Exonuclease I (EXO1) is a key component of MMR by resecting the damaged strand; however, whether EXO1 is required to activate MMR-dependent DDR remains unknown. Here we show that knockdown of the mouse ortholog (mExo1) in mouse embryonic fibroblasts (MEFs) results in decreased G2/M checkpoint response, limited effects on cell proliferation, and increased cell viability following exposure to the SN1 methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), establishing a phenotype paralleling MMR deficiency. MNNG treatment induced formation of γ-H2AX foci with which EXO1 co-localized in MEFs, but mExo1-depleted MEFs displayed a significant diminishment of γ-H2AX foci formation. mExo1 depletion also reduced MSH2 association with DNA duplexes containing G:T mismatches in vitro, decreased MSH2 association with alkylated chromatin in vivo, and abrogated MNNG-induced MSH2/CHK1 interaction. To determine if nuclease activity is required to activate DDR we stably overexpressed a nuclease defective form of human EXO1 (hEXO1) in mExo1-depleted MEFs. These experiments indicated that expression of wildtype and catalytically null hEXO1 was able to restore normal response to MNNG. This study indicates that EXO1 is required to activate MMR-dependent DDR in response to SN1 methylating agents; however, this function of EXO1 is independent of its nucleolytic activity.

Original languageEnglish (US)
Pages (from-to)951-964
Number of pages14
JournalDNA Repair
Volume11
Issue number12
DOIs
StatePublished - Dec 1 2012
Externally publishedYes

Keywords

  • Cell cycle checkpoints
  • DNA damage response
  • Mismatch repair

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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