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Sae2/CtIP prevents R-loop accumulation in eukaryotic cells.

Nodar Makharashvili1,2, Sucheta Arora1,2, Yizhi Yin1,2

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The Sae2/CtIP protein safeguards genome stability by processing DNA lesions. Its depletion causes stalled RNA polymerase and R-loops, highlighting its role in DNA repair and transcription regulation.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Cell Biology

Background:

  • Sae2/CtIP protein is crucial for DNA double-strand break repair via homologous recombination.
  • It also plays a role in surviving single-stranded DNA lesions induced by Top1.
  • CtIP associates with transcription complexes in mammalian cells.

Purpose of the Study:

  • Investigate the function of Sae2/CtIP at single-strand DNA lesions in yeast and human cells.
  • Determine the role of Sae2/CtIP in R-loop formation and processing.
  • Elucidate the mechanism by which Sae2/CtIP contributes to genome stability.

Main Methods:

  • Depletion of Sae2/CtIP in budding yeast and human cells.
  • Analysis of stalled RNA polymerase and RNA-DNA hybrid accumulation.
  • Overexpression of Senataxin (RNA-DNA helicase) to assess its effect.
  • Complementation studies using a catalytic mutant of CtIP.

Main Results:

  • Depletion of Sae2/CtIP leads to accumulation of stalled RNA polymerase and RNA-DNA hybrids at highly expressed genes.
  • Overexpression of Senataxin rescues DNA damage sensitivity and R-loop accumulation in Sae2/CtIP-deficient cells.
  • A catalytically inactive CtIP mutant cannot restore DNA damage resistance, indicating a requirement for CtIP nuclease activity.

Conclusions:

  • Sae2/CtIP plays a critical role in processing single-strand DNA lesions and preventing R-loop accumulation.
  • R-loop processing by 5' flap endonucleases is essential for stabilizing and removing nascent R-loop structures.
  • This highlights a conserved mechanism for maintaining genome integrity during transcription and DNA repair.