MRE11-RAD50-NBS1 Complex Is Sufficient to Promote Transcription by RNA Polymerase II at Double-Strand Breaks by Melting DNA Ends

Cell Rep. 2021 Jan 5;34(1):108565. doi: 10.1016/j.celrep.2020.108565.

Abstract

The MRE11-RAD50-NBS1 (MRN) complex supports the synthesis of damage-induced long non-coding RNA (dilncRNA) by RNA polymerase II (RNAPII) from DNA double-strand breaks (DSBs) by an unknown mechanism. Here, we show that recombinant human MRN and native RNAPII are sufficient to reconstitute a minimal functional transcriptional apparatus at DSBs. MRN recruits and stabilizes RNAPII at DSBs. Unexpectedly, transcription is promoted independently from MRN nuclease activities. Rather, transcription depends on the ability of MRN to melt DNA ends, as shown by the use of MRN mutants and specific allosteric inhibitors. Single-molecule FRET assays with wild-type and mutant MRN show a tight correlation between the ability to melt DNA ends and to promote transcription. The addition of RPA enhances MRN-mediated transcription, and unpaired DNA ends allow MRN-independent transcription by RNAPII. These results support a model in which MRN generates single-strand DNA ends that favor the initiation of transcription by RNAPII.

Keywords: DNA damage; DNA double-strand breaks; DNA melting; DNA-damage induced transcription; MRE11-RAD50-NBS1 complex; RNA polymerase II; damage-induced long non-coding RNA; dilncRNA; in vitro transcription; single-molecule FRET.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acid Anhydride Hydrolases / genetics
  • Acid Anhydride Hydrolases / metabolism*
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism*
  • DNA Breaks, Double-Stranded
  • DNA Damage
  • DNA Repair
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • HeLa Cells
  • Humans
  • MRE11 Homologue Protein / genetics
  • MRE11 Homologue Protein / metabolism*
  • Mutation
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism*
  • Nucleic Acid Denaturation*
  • RNA Polymerase II / genetics
  • RNA Polymerase II / metabolism*
  • RNA, Long Noncoding / biosynthesis*
  • RNA, Long Noncoding / genetics
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Transcription, Genetic*

Substances

  • Cell Cycle Proteins
  • DNA-Binding Proteins
  • MRE11 protein, human
  • NBN protein, human
  • Nuclear Proteins
  • RNA, Long Noncoding
  • Recombinant Proteins
  • RNA Polymerase II
  • MRE11 Homologue Protein
  • Acid Anhydride Hydrolases
  • RAD50 protein, human