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RNA-processing proteins regulate Mec1/ATR activation by promoting generation of RPA-coated ssDNA

Nicola Manfrini1, Camilla Trovesi1, Maxime Wery2

  • 1Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Milan, Italy.

EMBO Reports
|December 21, 2014
PubMed

Insights

RNA decay factors Xrn1, Rrp6, and Trf4 are crucial for activating the Mec1/ATR checkpoint by promoting RPA-coated single-stranded DNA (ssDNA) generation following DNA double-strand breaks (DSBs). Their roles highlight a novel link between RNA processing and maintaining genomic stability.

Area of Science:

  • Molecular Biology
  • Genetics
  • Cell Biology

Background:

  • Eukaryotic cells activate checkpoints involving Tel1/ATM and Mec1/ATR kinases in response to DNA double-strand breaks (DSBs).
  • Mec1/ATR activation relies on RPA-coated single-stranded DNA (ssDNA), generated by DSB resection.
  • RNA-processing factors are increasingly recognized for their roles in genomic stability.

Purpose of the Study:

  • To investigate the role of Saccharomyces cerevisiae RNA decay factors (Xrn1, Rrp6, Trf4) in Mec1/ATR activation and DSB response.
  • To elucidate the mechanisms by which these RNA factors influence ssDNA generation and checkpoint activation.

Main Methods:

  • Analysis of DNA double-strand break (DSB) resection and RPA-coated ssDNA generation in yeast mutants lacking specific RNA decay factors.
  • Assessment of Mec1/ATR checkpoint activation and recruitment to DSBs.
  • Evaluation of homologous recombination (HR) repair, including Rad51/Rad52 association.
  • Deep transcriptome analyses to identify gene expression changes.

Main Results:

  • Xrn1 deficiency inhibits ssDNA generation by preventing MRX complex loading at DSBs.
  • Rrp6 and Trf4 are not essential for DSB resection but are required for efficient RPA and Mec1 recruitment to DSBs.
  • Rrp6 and Trf4 inactivation do not impair Rad51/Rad52 association or homologous recombination repair.
  • Transcriptome analyses revealed no common misregulated gene expression patterns explaining the observed phenotypes.

Conclusions:

  • Saccharomyces cerevisiae RNA decay factors Xrn1, Rrp6, and Trf4 play distinct roles in promoting Mec1/ATR activation by regulating RPA-coated ssDNA generation.
  • Full Mec1 activation may require higher levels of RPA-coated ssDNA than are needed for homologous recombination repair.
  • These findings establish a novel connection between RNA processing pathways and the maintenance of genome stability.
Keywords:
DNA damage checkpointDNA double‐strand breaksRrp6Trf4Xrn1

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