Exonuclease 1 (Exo1) is required for activating response to S(N)1 DNA methylating agents.
S(N)1 DNA methylating agents are genotoxic agents that methylate numerous nucleophilic centers within DNA including the O(6) position of guanine (O(6)meG). Methylation of this extracyclic oxygen forces mispairing with thymine during DNA replication. The mismatch repair (MMR) system recognizes these O(6)meG: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 S(N)1 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 S(N)1 methylating agents; however, this function of EXO1 is independent of its nucleolytic activity.
Department of Biochemistry and Molecular Biology and the UF-Shands Cancer Center, University of Florida College of Medicine, Gainesville, FL 32610, USA.
Eugene
E
Izumchenko
John
J
Saydi
Kevin D
KD
Brown
D000477:Alkylating Agents; D000818:Animals; D020137:Base Pair Mismatch; D049109:Cell Proliferation; D002470:Cell Survival; D000071877:Checkpoint Kinase 1; D002843:Chromatin / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D019175:DNA Methylation / Q000187:drug effects*; D053843:DNA Mismatch Repair; D045643:DNA Repair Enzymes / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D059565:G2 Phase Cell Cycle Checkpoints; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006657:Histones / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology*; D051379:Mice; D051718:MutS Homolog 2 Protein / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D025941:Protein Interaction Mapping; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D000477:Alkylating Agents; D000071877:Checkpoint Kinase 1; D002843:Chromatin; D045643:DNA Repair Enzymes; D018076:DNA, Complementary; D005090:Exodeoxyribonucleases; D006657:Histones; D008769:Methylnitronitrosoguanidine; D051718:MutS Homolog 2 Protein; D011494:Protein Kinases; D011994:Recombinant Proteins; C000605867:CHEK1 protein, human; C000605869:Chek1 protein, mouse; C114331:EXO1 protein, human; C497173:Msh2 protein, mouse; C475961:gamma-H2AX protein, mouse