Abstract
Although defective repair of DNA double-strand breaks (DSBs) leads to neurodegenerative diseases, the processes underlying their production and signaling in non-replicating cells are largely unknown. Stabilized topoisomerase I cleavage complexes (Top1cc) by natural compounds or common DNA alterations are transcription-blocking lesions whose repair depends primarily on Top1 proteolysis and excision by tyrosyl–DNA phosphodiesterase-1 (TDP1). We previously reported that stabilized Top1cc produce transcription-dependent DSBs that activate ATM in neurons. Here, we use camptothecin (CPT)-treated serum-starved quiescent cells to induce transcription-blocking Top1cc and show that those DSBs are generated during Top1cc repair from Top1 peptide-linked DNA single-strand breaks generated after Top1 proteolysis and before excision by TDP1. Following DSB induction, ATM activates DNA-PK whose inhibition suppresses H2AX and H2A ubiquitination and the later assembly of activated ATM into nuclear foci. Inhibition of DNA-PK also reduces Top1 ubiquitination and proteolysis as well as resumption of RNA synthesis suggesting that DSB signaling further enhances Top1cc repair. Finally, we show that co-transcriptional DSBs kill quiescent cells. Together, these new findings reveal that DSB production and signaling by transcription-blocking Top1 lesions impact on non-replicating cell fate and provide insights on the molecular pathogenesis of neurodegenerative diseases such as SCAN1 and AT syndromes, which are caused by TDP1 and ATM deficiency, respectively.
Highlights
Topoisomerase I (Top1) is required to remove DNA supercoiling generated during transcription
We use serum-starved quiescent cells treated with CPT as a model to induce transcriptionblocking Top1 cleavage complexes (Top1cc) and get molecular insights into the processes underlying both the production and signaling of double-strand breaks (DSBs). We found that those DSBs are produced during Top1cc repair from Top1 peptide-linked DNA single-strand breaks (SSB) generated after Top1 proteolysis and before excision by tyrosyl–DNA phosphodiesterase-1 (TDP1)
These ␥ H2AX foci colocalized with 53BP1, another DNA damage response (DDR) protein (Supplementary Figure S3A), and a neutral Comet assay provided direct evidence for the presence of DSBs (Supplementary Figure S3C and D)
Summary
Topoisomerase I (Top1) is required to remove DNA supercoiling generated during transcription. It relaxes DNA by producing transient Top cleavage complexes (Top1cc), which are Top1-linked DNA single-strand breaks (SSB) [1]. Stabilized Top1cc are potent transcription-blocking DNA lesions [6,7] and their repair (removal) depends primarily on the tyrosyl–DNA phosphodiesterase-1 (TDP1) excision pathway. These co-transcriptional DSBs have been detected in post-mitotic neurons and lymphocytes as well as in replicating cells out of the S-phase [17,18,19] Their production involves the formation of Rloops, a three-strand nucleic acid structure consisting of an RNA:DNA hybrid and displaced single-stranded DNA [20,21].
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.