DNA-PK triggers histone ubiquitination and signaling in response to DNA double-strand breaks produced during the repair of transcription-blocking topoisomerase I lesions

Nucleic Acids Res. 2016 Feb 18;44(3):1161-78. doi: 10.1093/nar/gkv1196. Epub 2015 Nov 17.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Ataxia Telangiectasia Mutated Proteins / genetics
  • Ataxia Telangiectasia Mutated Proteins / metabolism
  • Camptothecin / pharmacology
  • Cell Line
  • Cell Survival / drug effects
  • Cell Survival / genetics
  • Culture Media, Serum-Free / pharmacology
  • DNA Breaks, Double-Stranded*
  • DNA Breaks, Single-Stranded
  • DNA Repair*
  • DNA Topoisomerases, Type I / genetics
  • DNA Topoisomerases, Type I / metabolism*
  • DNA-Activated Protein Kinase / genetics
  • DNA-Activated Protein Kinase / metabolism*
  • Fibroblasts / drug effects
  • Fibroblasts / metabolism
  • Histones / metabolism*
  • Humans
  • Immunoblotting
  • Microscopy, Fluorescence
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism*
  • RNA Interference
  • Signal Transduction
  • Topoisomerase I Inhibitors / pharmacology
  • Ubiquitination / drug effects

Substances

  • Culture Media, Serum-Free
  • Histones
  • Nuclear Proteins
  • Topoisomerase I Inhibitors
  • ATM protein, human
  • Ataxia Telangiectasia Mutated Proteins
  • DNA-Activated Protein Kinase
  • PRKDC protein, human
  • DNA Topoisomerases, Type I
  • TOP1 protein, human
  • Camptothecin