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.

Fig. 1

Deletion of Tdp1 in Aptx^−/− neural cells uncovers a requirement for aprataxin during repair of “oxidative” DNA strand breaks. (A) Primary quiescent cortical astrocytes from wild-type, Aptx^−/−, Tdp1^−/−, and Tdp1^−/−/Aptx^−/− littermates were mock treated (−H₂O₂) or treated (+H₂O₂) with 75 μM H₂O₂ and levels of DNA strand breakage were quantified by alkaline comet assays. Inset: Cartoon depicting the approach employed here to increase the sub-fraction of 5′-AMP breaks, by slowing the rate of repair of 3′-termini at SSBs via co-deletion of Tdp1 in aprataxin-defective neural cells. (B) Following treatment with H₂O₂ as indicated above, cells were incubated for the indicated repair periods in drug-free medium and mean fraction of DNA strand breaks remaining at the indicated time points were quantified by alkaline comet assays. Mean tail moments were quantified for 100 cells/sample/experiment and data are the average for three independent experiments (±S.E.M.). Asterisks denote statistically significant (P < 0.05; t-test) differences between Tdp1^−/− and Tdp1^−/−/Aptx^−/− histograms at the indicated time points (C) Representative scatter plots from one of the experiments included in (B), showing comet tail moments of 100 individual cells per sample at the time points indicated.

Fig. 2

Defective repair of alkylation-induced DNA single-strand breaks in Tdp1^−/−/Aptx^−/− double knockout neural cells. (A) Primary quiescent cortical astrocytes of the indicated genotypes were mock treated or treated with the indicated concentrations of MMS for 10 min at 37 °C. DNA strand breakage was then quantified by alkaline comet assays and data for 100 cells/sample/experiment were averaged from three independent experiments (±S.E.M.). Asterisks denote statistically significant (P < 0.05; t-test) differences between wild-type and Tdp1^−/−/Aptx^−/− histograms. (B) Representative scatter plots from one of the experiments included in (A).

Fig. 3

Accumulation of similar levels of camptothecin-induced DNA single-strand breaks in Tdp1^−/− and Tdp1^−/−/Aptx^−/− double knockout neural cells. Primary quiescent cortical astrocytes were mock treated or treated with 14 μM camptothecin (CPT) for 1 h at 37 °C and DNA strand breakage was then quantified by alkaline comet assays. Mean tail moments were plotted ±S.E.M.

Fig. 4

Normal rates of DNA double-strand break repair in Tdp1^−/−/Aptx^−/− neural cells. Primary quiescent cortical astrocytes from wild-type and Tdp1^−/−/Aptx^−/− littermates (A) or quiescent wild-type (1BR), h-Tert immortalized AOA1 (FD104 and FD105), and AOA1 fibroblasts stably expressing recombinant human aprataxin (FD104 m-21 and FD105 m-21) (D) were mock (0) or γ-irradiated (3Gy) on ice and then incubated for the indicated repair periods at 37 °C (A, B, and D) or incubated with the indicated concentrations of MMS for 10 min at 37 °C (C). The mean number of γH2AX foci was quantified from 50 cells/sample/experiment and data represent the average of three independent experiments (±S.E.M.). (B) The mean fraction of γ-ray-induced γH2AX foci remaining at the indicated repair periods were quantified ±S.E.M. Lig4^−/− mouse embryonic fibroblasts were included in parallel for comparison.