ATM deficiency does not impact on the accumulation of Top1-single-strand breaks.

<p>(<b>a</b>) Human lymphoblastoid cells (LCLs) derived from a normal individual ‘WT’, spinocerebellar ataxia with axonal neuropathy ‘SCAN1’, or ataxia telangiectasia ‘A–T’ patients, and mouse embryonic fibroblasts (MEFs) or quiescent cortical astrocytes from control ‘WT’ or <i>Tdp1-/-</i> mice were incubated with DMSO (Mock) or 30 µM camptothecin (CPT) for 40 min with or without pre-incubation with 10 µM ATM inhibitor KU-55933 (ATMi) for 2 hours at 37°C. Top1-single-strand breaks ‘Top1-SSBs’ were quantified by alkaline comet assays (ACAs). Mean tail moments were calculated for 50 cells/sample/experiment and data are the average of <i>n</i> = 3 biological replicates ± s.e.m. (<b>b</b>) Top1-SSBs were analysed in quiescent cortical astrocytes derived from wild-type ‘WT’, <i>Atm-/-</i> or <i>Tdp1-/-</i> mice following incubation with DMSO (Mock) or 30 µM camptothecin (CPT) and quantified as described above.</p

ATM deficiency results in accumulation of Top1-CCs in human cells.

<p>(<b>a</b>) Top1-SSBs and Top1-CCs were quantified in wild-type 1BR3 ‘WT’ or ATM deficient AT-1BR ‘A–T’ human primary fibroblasts by ACAs and MACAs, respectively. Cells were mock incubated with DMSO ‘Mock’ or with the 30 µM CPT for 40 min at 37°C and DNA strand breaks quantified from 50 cells/sample/experiment. Data are the average of <i>n</i> = 3 biological replicates ± s.e.m. (<b>b</b>) Primary human fibroblasts were grown to confluency and serum starved for 3 days, and Top1-SSBs/Top1-CCs were quantified as described in (a).</p

Reactive oxygen species scavengers reduce the accumulation of Top1-CCs in <i>Atm-/-</i> cells.

<p>(<b>a</b>) Top1-CCs were analysed in WT or <i>Atm-/-</i> quiescent astrocytes with and without incubation with 30 µM CPT ± a prior 2-hour incubation with 50 µM of the transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole ‘DRB’. Top1-CCs were quantified by MACA from 50 cells/sample/experiment and data represent the average of <i>n</i> = 3 biological replicates ± s.e.m. (<b>b</b>) Top1-CCs were analysed as in (a) with or without prior incubation with the reactive oxygen species (ROS) scavengers mannitol (50 mM) or N-Acetyl cysteine ‘NAC’ (10 mM) for 17-hours. 50 cells/sample/experiment were analysed and data represent the average of <i>n</i> = 3 biological replicates ± s.e.m. P values indicate the statistical difference between WT and <i>Atm-/-</i> cells (student t-test). <b><i>Note that prior incubation with ROS scavengers reduces the endogenous level of Top1-CCs in Atm-/- cells to that observed in control cells</i></b><b>.</b></p

Loss of <i>Atm</i> results in accumulation of Top1-CCs in cortical neural cells.

<p>(<b>a</b>) Endogenous steady-state level of Top1-SSBs and Top1-CCs were quantified in quiescent wild type ‘WT’ and <i>Atm-/-</i> cortical astrocytes by ACAs and MACAs, respectively. Neural cells were subsequently subjected to 30 µM CPT for 40 min at 37 °C and the level of Top1-SSBs and Top1-CCs were quantified as above from 50 cells/sample/experiment. Data are the average of <i>n</i> = 3 biological replicates ± s.e.m. <b><i>Inset</i></b>: astrocytes were incubated with DMSO ‘Mock’ or CPT ‘CPT’ and the expression of Top1 was measured by anti-Top1 immunoblotting. Anti-actin was employed as a loading control. (<b>b</b>) WT or <i>Atm-/-</i> quiescent astrocytes were mock incubated with DMSO ‘Mock’ or with the 30 µM CPT for 40 min at 37°C with or without a prior 2-hour incubation with the proteasome inhibitor MG132 ‘PI’. Top1-CCs were quantified by MACA from 50 cells/sample/experiment and the average of <i>n</i> = 3 biological replicates ± s.e.m is presented (<b>c</b>) Cortices from WT or Atm-/- mice were harvested at P6 and cells were dissociated and immediately subjected to MACA analyses. Data are the average of n = 3 biological replicates ± s.e.m. (<b>d</b>) <b>Left</b>: scheme depicting the biochemical fractionation of Top1-CCs. Blue circles are Top1 covalently bound to DNA (Top1-CCs) and yellow circles are Top1 non-covalently bound to DNA. <b>Right</b>: Wild-type ‘WT’ or <i>Atm-/-</i> quiescent cortical astrocytes were mock incubated or incubated with 30 µM CPT for 60 min at 37 °C. Neural cells were lysed in denaturing buffer and lysates fractionated on CsCl gradients. Fractions were slot blotted onto nitrocellulose and immunblotted with anti-Top1 monoclonal antibodies. A representative experiment from 3 biological replicates is shown. P values indicate the statistical difference between WT and <i>Atm-/-</i> cells (student t-test).</p

Modification of the alkaline comet assay uncovers un-degraded Top1-DNA cleavage complexes (Top1-CCs).

<p>(<b>a</b>) Scheme depicting the major differences between Top1-CCs and Top1-SSBs: Top1 relaxes DNA supercoiling by introducing a reversible nick to which Top1 becomes covalently attached (Top1-CCs). Stalling of Top1-CCs through collision with the transcription machinery or oxidative DNA damage triggers proteasomal degradation of Top1, resulting in Top1 single-strand breaks (Top1-SSBs). Repair of Top1-SSBs is initiated by removal of Top1 peptide by TDP1 followed by subsequent ligation. <b><i>Note that un-degraded ‘Top1-CCs’ are not detected by the ‘classical’ alkaline comet assays (ACA) due to the reversible nature of these intermediates and the reduced ability of covalently bound Top1 on DNA to produce measurable tail upon electrophoresis.</i></b> (<b>b</b>) Control ‘WT’ or SCAN1 LCLs ‘SCAN1’ harbouring the TDP1 catalytic mutation H493R were incubated with 20 µM camptothecin “CPT” with or without a prior 2-hr incubation with 30 µM proteasome inhibitor MG132 ‘PI’. Cells were divided into two fractions for the comparative detection of Top1-SSBs and Top1-CCs using the ACAs and modified ACAs ‘MACA’, respectively. Mean tail moments were calculated for 50 cells/sample/experiment and data are the average of <i>n</i> = 3 biological replicates ± s.e.m. <b><i>Note that inhibiting the proteasome resulted in a reduction of Top1-SSBs (as measured by ACA) to near background levels with minimal impact on Top1-CCs (as measured by MACA).</i></b></p