Abstract
Ataxia telangiectasia mutated (ATM), the deficiency of which causes a severe neurodegenerative disease, is a crucial mediator for the DNA damage response (DDR). As neurons have high rates of transcription that require topoisomerase I (TOP1), we investigated whether TOP1 cleavage complexes (TOP1cc)-which are potent transcription-blocking lesions-also produce transcription-dependent DNA double-strand breaks (DSBs) with ATM activation. We show the induction of DSBs and DDR activation in post-mitotic primary neurons and lymphocytes treated with camptothecin, with the induction of nuclear DDR foci containing activated ATM, gamma-H2AX (phosphorylated histone H2AX), activated CHK2 (checkpoint kinase 2), MDC1 (mediator of DNA damage checkpoint 1) and 53BP1 (p53 binding protein 1). The DSB-ATM-DDR pathway was suppressed by inhibiting transcription and gamma-H2AX signals were reduced by RNase H1 transfection, which removes transcription-mediated R-loops. Thus, we propose that Top1cc produce transcription arrests with R-loop formation and generate DSBs that activate ATM in post-mitotic cells.
Highlights
DNA double-strand breaks (DSBs) are among the most severe genomic lesions and their repair requires the recruitment of DNAReceived 6 January 2009; revised 30 March 2009; accepted 9 April 2009; published online 26 June 2009 damage response (DDR) proteins (Bakkenist & Kastan, 2004; Shiloh, 2006)
The loss of function of DNA damage response (DDR) proteins leads to Genomic instability and human hereditary diseases such as ataxia telangiectasia syndrome (AT caused by ataxia telangiectasia mutated (ATM) deficiency; Rass et al, 2007)
To determine whether TOP1 cleavage complexes (TOP1cc) can induce DDR in post-mitotic cells, we examined the phosphorylated histone H2AX at Ser 139 (g-H2AX) and its accumulation in nuclear foci
Summary
DNA double-strand breaks (DSBs) are among the most severe genomic lesions and their repair requires the recruitment of DNA. As AT is primarily a neurodegenerative disease and as neurons have high transcription rates, we examined whether transcription-blocking DNA lesions could induce DSBs and activate the ATM-associated DDR. Owing to the high rate of oxygen consumption, neurons produce reactive oxygen species that can damage DNA and trap TOP1cc (Pourquier & Pommier, 2001; Daroui et al, 2004; Pommier, 2006). In agreement with this possibility, defective repair of TOP1cc (by inactivating mutation of tyrosyl-DNA-phosphodiesterase 1 (TDP1)) leads to the hereditary spinocerebellar ataxia with axonal neuropathy (SCAN) syndrome (Rass et al, 2007). We used CPT to induce TOP1cc in non-replicating cells to determine whether transcriptionblocking TOP1cc induce DSBs and activate the ATM-associated DDR
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