title: Restoration of G1 chemo/radioresistance and double-strand-break repair proficiency by wild-type but not endonuclease-deficient Artemis.
abstract: Deficiency in Artemis is associated with lack of V(D)J recombination, sensitivity to radiation and radiomimetic drugs, and failure to repair a subset of DNA double-strand breaks (DSBs). Artemis harbors an endonuclease activity that trims both 5'- and 3'-ends of DSBs. To examine whether endonucleolytic trimming of terminally blocked DSBs by Artemis is a biologically relevant function, Artemis-deficient fibroblasts were stably complemented with either wild-type Artemis or an endonuclease-deficient D165N mutant. Wild-type Artemis completely restored resistance to γ-rays, bleomycin and neocarzinostatin, and also restored DSB-repair proficiency in G0/G1 phase as measured by pulsed-field gel electrophoresis and repair focus resolution. In contrast, cells expressing the D165N mutant, even at very high levels, remained as chemo/radiosensitive and repair deficient as the parental cells, as evidenced by persistent γ-H2AX, 53BP1 and Mre11 foci that slowly increased in size and ultimately became juxtaposed with promyelocytic leukemia protein nuclear bodies. In normal fibroblasts, overexpression of wild-type Artemis increased radioresistance, while D165N overexpression conferred partial repair deficiency following high-dose radiation. Restoration of chemo/radioresistance by wild-type, but not D165N Artemis suggests that the lack of endonucleolytic trimming of DNA ends is the principal cause of sensitivity to double-strand cleaving agents in Artemis-deficient cells.
authors.affiliation: Department of Pharmacology and Toxicology, Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, USA.
authors.forename: Susovan
authors.initials: S
authors.lastname: Mohapatra
authors.forename: Misako
authors.initials: M
authors.lastname: Kawahara
authors.forename: Imran S
authors.initials: IS
authors.lastname: Khan
authors.forename: Steven M
authors.initials: SM
authors.lastname: Yannone
authors.forename: Lawrence F
authors.initials: LF
authors.lastname: Povirk
mesh_terms: D001761:Bleomycin / Q000633:toxicity; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D004268:DNA-Binding Proteins; D004706:Endodeoxyribonucleases / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D004720:Endonucleases; D016193:G1 Phase; D006801:Humans; D009154:Mutation; D009687:Nuclear Proteins / Q000032:analysis / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D000071156:Promyelocytic Leukemia Protein; D011836:Radiation Tolerance; D014157:Transcription Factors / Q000032:analysis; D025521:Tumor Suppressor Proteins / Q000032:analysis; D009353:Zinostatin / Q000633:toxicity
chemical_list: D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D000071156:Promyelocytic Leukemia Protein; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D001761:Bleomycin; C070576:PML protein, human; D009353:Zinostatin; C426344:DCLRE1C protein, human; D004706:Endodeoxyribonucleases; D004720:Endonucleases