ATM deficiency results in accumulation of DNA-Topoisomerase I covalent intermediates in neural cells

Accumulation of peptide-linked DNA breaks contributes to neurodegeration in humans. This is typified by defects in tyrosyl DNA phosphodiesterase 1 (TDP1) and human hereditary ataxia. TDP1 primarily operates at single-strand breaks (SSBs) created by oxidative stress or by collision of transcription machinery with topoisomerase I intermediates (Top1-CCs). Cellular and cell-free studies have shown that Top1 at stalled Top1-CCs is first degraded to a small peptide resulting in Top1-SSBs, which are the primary substrates for TDP1. Here we established an assay to directly compare Top1-SSBs and Top1-CCs. We subsequently employed this assay to reveal an increased steady state level of Top1-CCs in neural cells lacking Atm; the protein mutated in ataxia telangiectasia. Our data suggest that the accumulation of endogenous Top1-CCs in Atm-/- neural cells is primarily due to elevated levels of reactive oxygen species. Biochemical purification of Top1-CCs from neural cell extract and the use of Top1 poisons further confirmed a role for Atm during the formation/resolution of Top1-CCs. Finally, we report that global transcription is reduced in Atm-/- neural cells and fails to recover to normal levels following Top1-mediated DNA damage. Together, these data identify a distinct role for ATM during the formation/resolution of neural Top1-CCs and suggest that their accumulation contributes to the neuropathology of ataxia telangiectasia

SETDB1, HP1 and SUV39 promote repositioning of 53BP1 to extend resection during homologous recombination in G2 cells

Recent studies have shown that homologous recombination (HR) requires chromatin repression as well as relaxation at DNA double strand breaks (DSBs). HP1 and SUV39H1/2 are repressive factors essential for HR. Here, we identify SETDB1 as an additional compacting factor promoting HR. Depletion of HP1, SUV39, SETDB1 or BRCA1 confer identical phenotypes. The repressive factors, like BRCA1, are dispensable for the initiation of resection but promote the extension step causing diminished RPA or RAD51 foci and HR in irradiated G2 cells. Depletion of the compacting factors does not inhibit BRCA1 recruitment but at 8 h post IR, BRCA1 foci are smaller and aberrantly positioned compared to control cells. BRCA1 promotes 53BP1 repositioning to the periphery of enlarged foci and formation of a devoid core with BRCA1 becoming enlarged and localised internally to 53BP1. Depletion of the compacting factors precludes these changes at irradiation-induced foci. Thus, the repressive factors are required for BRCA1 function in promoting the repositioning of 53BP1 during HR. Additionally, depletion of these repressive factors in undamaged cells causes diminished sister chromatid association at centromeric sequences. We propose a model for how these findings may be functionally linked

TDP1 deficiency sensitizes human cells to base damage via distinct topoisomerase I and PARP mechanisms with potential applications for cancer therapy

Base damage and topoisomerase I (Top1)-linked DNA breaks are abundant forms of endogenous DNA breakage, contributing to hereditary ataxia and underlying the cytotoxicity of a wide range of anti-cancer agents. Despite their frequency, the overlapping mechanisms that repair these forms of DNA breakage are largely unknown. Here, we report that depletion of Tyrosyl DNA phosphodiesterase 1 (TDP1) sensitizes human cells to alkylation damage and the additional depletion of apurinic/apyrimidinic endonuclease I (APE1) confers hypersensitivity above that observed for TDP1 or APE1 depletion alone. Quantification of DNA breaks and clonogenic survival assays confirm a role for TDP1 in response to base damage, independently of APE1. The hypersensitivity to alkylation damage is partly restored by depletion of Top1, illustrating that alkylating agents can trigger cytotoxic Top1-breaks. Although inhibition of PARP activity does not sensitize TDP1-deficient cells to Top1 poisons, it confers increased sensitivity to alkylation damage, highlighting partially overlapping roles for PARP and TDP1 in response to genotoxic challenge. Finally, we demonstrate that cancer cells in which TDP1 is inherently deficient are hypersensitive to alkylation damage and that TDP1 depletion sensitizes glioblastoma-resistant cancer cells to the alkylating agent temozolomide

Deficiency in origin licensing proteins impairs cilia formation: implications for the aetiology of meier-gorlin syndrome

Mutations in ORC1, ORC4, ORC6, CDT1, and CDC6, which encode proteins required for DNA replication origin licensing, cause Meier-Gorlin syndrome (MGS), a disorder conferring microcephaly, primordial dwarfism, underdeveloped ears, and skeletal abnormalities. Mutations in ATR, which also functions during replication, can cause Seckel syndrome, a clinically related disorder. These findings suggest that impaired DNA replication could underlie the developmental defects characteristic of these disorders. Here, we show that although origin licensing capacity is impaired in all patient cells with mutations in origin licensing component proteins, this does not correlate with the rate of progression through S phase. Thus, the replicative capacity in MGS patient cells does not correlate with clinical manifestation. However, ORC1-deficient cells from MGS patients and siRNA-mediated depletion of origin licensing proteins also have impaired centrosome and centriole copy number. As a novel and unexpected finding, we show that they also display a striking defect in the rate of formation of primary cilia. We demonstrate that this impacts sonic hedgehog signalling in ORC1-deficient primary fibroblasts. Additionally, reduced growth factor-dependent signaling via primary cilia affects the kinetics of cell cycle progression following cell cycle exit and re-entry, highlighting an unexpected mechanism whereby origin licensing components can influence cell cycle progression. Finally, using a cell-based model, we show that defects in cilia function impair chondroinduction. Our findings raise the possibility that a reduced efficiency in forming cilia could contribute to the clinical features of MGS, particularly the bone development abnormalities, and could provide a new dimension for considering developmental impacts of licensing deficiency

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

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

Meier-Gorlin syndrome patients LBLs display impaired origin licensing capacity; some but not all lines show impaired S phase progression.

<p>(a) EBV uses virally encoded EBNA-1, oriP and the host cell origin licensing complex for replication. ORC activity was assessed by the replicative capacity of plasmid-294, which encodes OriP and EBNA-1 in a control LBL (C) and LBLs derived from MGS patients with mutations in <i>ORC1, ORC4, ORC6, CDC6</i> and <i>CDT1</i>. Following transfection of the EBV episome into LBLs and incubation to allow replication, episomal DNA was extracted and examined after <i>BamH1</i> or <i>BamH1+Dpn1</i> digestion using plasmid-294 as a probe. <i>Dpn1</i> degrades unreplicated plasmids that retain bacterial Dam-dependent methylation. The EBV episome has a single <i>BamH1</i> site causing linearization after digestion. Although replication of EBV is less efficient in LBLs compared to hTERT immortalised fibroblasts, ∼5% of the EBV plasmids underwent replication in control cells as shown by the presence of full length episomes (band 1) after <i>Dpn1+BamH1</i> digestion. The level in MGS patient LBLs is substantially reduced. For quantification, the level of the full length plasmid band (1) was plotted relative to one of the <i>Dpn1</i> digestion products (2) and normalised to that obtained in the control (C). Efficient episomal transfection was shown by the similar level of digestion products in all samples. Results represent the mean of two experiments. The reduction was highly significant (t-test, 1-tailed equal variance. Nomenclature used throughout: no significance (ns) P>0.05, * P<0.05, ** P<0.01). (b) Control (C) and ORC1 LBLs were BrdU labelled for 30 min and incubated for the indicated times before fluorescence-activated cell sorting (FACS). The percentage of early S phase cells was assessed. The rate of loss of BrdU⁺ early S phase cells represents the speed of S-phase progression. LBLs with mutations in <i>ORC1, ORC4</i> and <i>ORC6</i> show an impaired rate of S phase progression; CDT1-deficient LBLs were similar to control LBLs and the CDC6-deficient LBLs progressed more rapidly through S phase.</p