title: Exo1 independent DNA mismatch repair involves multiple compensatory nucleases.
abstract: Functional DNA mismatch repair (MMR) is essential for maintaining the fidelity of DNA replication and genetic stability. In hematopoiesis, loss of MMR results in methylating agent resistance and a hematopoietic stem cell (HSC) repopulation defect. Additionally MMR failure is associated with a variety of human malignancies, notably Lynch syndrome. We focus on the 5'→3' exonuclease Exo1, the primary enzyme excising the nicked strand during MMR, preceding polymerase synthesis. We found that nuclease dead Exo1 mutant cells are sensitive to the O6-methylguanine alkylating agent temozolomide when given with the MGMT inactivator, O6benzylguanine (BG). Additionally we used an MMR reporter plasmid to verify that Exo1(mut) MEFs were able to repair G:T base mismatches in vitro. We showed that unlike other MMR deficient mouse models, Exo1(mut) mouse HSC did not gain a competitive survival advantage post temozolomide/BG treatment in vivo. To determine potential nucleases implicated in MMR in the absence of Exo1 nuclease activity, but in the presence of the inactive protein, we performed gene expression analyses of several mammalian nucleases in WT and Exo1(mut) MEFs before and after temozolomide treatment and identified upregulation of Artemis, Fan1, and Mre11. Partial shRNA mediated silencing of each of these in Exo1(mut) cells resulted in decreased MMR capacity and increased resistance to temozolomide/BG. We propose that nuclease function is required for fully functional MMR, but a portfolio of nucleases is able to compensate for loss of Exo1 nuclease activity to maintain proficiency.
authors.affiliation: Department of Pharmacology, University Hospitals Seidman Cancer Center and Case Western Reserve University, United States; Division of Hematology/Oncology, Center of Stem Cell and Regenerative Medicine, University Hospitals Seidman Cancer Center and Case Western Reserve University, United States.
authors.forename: Amar
authors.initials: A
authors.lastname: Desai
authors.affiliation: Department of Pharmacology, University Hospitals Seidman Cancer Center and Case Western Reserve University, United States; Division of Hematology/Oncology, Center of Stem Cell and Regenerative Medicine, University Hospitals Seidman Cancer Center and Case Western Reserve University, United States; Case Comprehensive Cancer Center, University Hospitals Seidman Cancer Center and Case Western Reserve University, United States. Electronic address: slg5@case.edu.
authors.forename: Stanton
authors.initials: S
authors.lastname: Gerson
mesh_terms: D000818:Animals; D002460:Cell Line; D053843:DNA Mismatch Repair; D045643:DNA Repair Enzymes / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D003606:Dacarbazine / Q000031:analogs & derivatives / Q000633:toxicity; D004706:Endodeoxyribonucleases / Q000235:genetics*; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D006147:Guanine / Q000031:analogs & derivatives / Q000633:toxicity; D000076228:MRE11 Homologue Protein; D051379:Mice; D008810:Mice, Inbred C57BL; D064251:Multifunctional Enzymes; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D000077204:Temozolomide; D015854:Up-Regulation
chemical_list: D004268:DNA-Binding Proteins; C512689:Mre11a protein, mouse; D064251:Multifunctional Enzymes; D009687:Nuclear Proteins; C064976:O(6)-benzylguanine; D006147:Guanine; D003606:Dacarbazine; D004706:Endodeoxyribonucleases; D004720:Endonucleases; C583623:Exo1 protein, mouse; D005090:Exodeoxyribonucleases; C000596972:Fan1 protein, mouse; D000076228:MRE11 Homologue Protein; C473337:Dclre1c protein, mouse; D045643:DNA Repair Enzymes; D000077204:Temozolomide
keywords: DNA repair; Exonuclease 1; Mismatch repair; Temozolomide