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Transcribing RNA polymerase alone can sense DNA double-strand breaks (DSBs) and form R-loops, crucial for DNA repair. R-loop formation depends on DNA end structures, impacting transcription.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • R-loops at DNA double-strand breaks (DSBs) are critical for DNA repair within transcribed genes.
  • Mechanisms of R-loop formation at DSBs are poorly understood, with several models proposed.
  • Existing models involve RNA polymerase (RNAP) or pre-existing transcripts.

Purpose of the Study:

  • To elucidate the mechanisms of R-loop formation at DSBs.
  • To investigate the role of transcribing RNAP in R-loop generation.
  • To determine the influence of DNA end structures on R-loop formation.

Main Methods:

  • Single-molecule study using Escherichia coli RNAP.
  • Analysis of R-loop formation efficiency across various DNA end structures (blunt, sticky ends with overhangs).

Main Results:

  • Transcribing RNAP alone effectively senses DSBs and generates R-loops without additional factors.
  • R-loop formation efficiency varies significantly (2.8% to 73%) based on DNA end structures, with higher rates for sticky ends.
  • R-loops extend upstream from DSBs, interfere with transcription, and can block subsequent transcription rounds.
  • Results support the bubble extension model over other proposed models.

Conclusions:

  • Transcribing RNAP is a key sensor for DSBs, initiating R-loop formation.
  • DNA end structure is a critical determinant of R-loop formation efficiency.
  • These findings offer insights into DSB repair initiation on transcription templates in diverse organisms.