title: Synergistic decrease of DNA single-strand break repair rates in mouse neural cells lacking both Tdp1 and aprataxin.
abstract: Ataxia oculomotor apraxia-1 (AOA1) is an autosomal recessive neurodegenerative disease that results from mutations of aprataxin (APTX). APTX associates with the DNA single- and double-strand break repair machinery and is able to remove AMP from 5'-termini at DNA strand breaks in vitro. However, attempts to establish a DNA strand break repair defect in APTX-defective cells have proved conflicting and unclear. We reasoned that this may reflect that DNA strand breaks with 5'-AMP represent only a minor subset of breaks induced in cells, and/or the availability of alternative mechanisms for removing AMP from 5'-termini. Here, we have attempted to increase the dependency of chromosomal single- and double-strand break repair on aprataxin activity by slowing the rate of repair of 3'-termini in aprataxin-defective neural cells, thereby increasing the likelihood that the 5'-termini at such breaks become adenylated and/or block alternative repair mechanisms. To do this, we generated a mouse model in which APTX is deleted together with tyrosyl DNA phosphodiesterase (TDP1), an enzyme that repairs 3'-termini at a subset of single-strand breaks (SSBs), including those with 3'-topoisomerase-1 (Top1) peptide. Notably, the global rate of repair of oxidative and alkylation-induced SSBs was significantly slower in Tdp1(-/-)/Aptx(-/-) double knockout quiescent mouse astrocytes compared with Tdp1(-/-) or Aptx(-/-) single knockouts. In contrast, camptothecin-induced Top1-SSBs accumulated to similar levels in Tdp1(-/-) and Tdp1(-/-)/Aptx(-/-) double knockout astrocytes. Finally, we failed to identify a measurable defect in double-strand break repair in Tdp1(-/-), Aptx(-/-) or Tdp1(-/-)/Aptx(-/-) astrocytes. These data provide direct evidence for a requirement for aprataxin during chromosomal single-strand break repair in primary neural cells lacking Tdp1.
authors.affiliation: Genome Damage and Stability Centre, University of Sussex, Brighton, BN1 9RQ, UK. smfame20@sussex.ac.uk
authors.forename: Sherif F
authors.initials: SF
authors.lastname: El-Khamisy
authors.forename: Sachin
authors.initials: S
authors.lastname: Katyal
authors.forename: Poorvi
authors.initials: P
authors.lastname: Patel
authors.forename: Limei
authors.initials: L
authors.lastname: Ju
authors.forename: Peter J
authors.initials: PJ
authors.lastname: McKinnon
authors.forename: Keith W
authors.initials: KW
authors.lastname: Caldecott
mesh_terms: D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism*; D002166:Camptothecin / Q000494:pharmacology; D002478:Cells, Cultured; D053904:DNA Breaks, Single-Stranded / Q000187:drug effects*; D004260:DNA Repair / Q000235:genetics*; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000502:physiology*; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D009687:Nuclear Proteins / Q000502:physiology*; D010727:Phosphoric Diester Hydrolases / Q000502:physiology*
chemical_list: D000972:Antineoplastic Agents, Phytogenic; C514665:Aptx protein, mouse; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D010727:Phosphoric Diester Hydrolases; C524959:Tdp1 protein, mouse; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin