The S-phase checkpoint is required to respond to R-loops accumulated in THO mutants

Mol Cell Biol. 2009 Oct;29(19):5203-13. doi: 10.1128/MCB.00402-09. Epub 2009 Aug 3.

Abstract

Cotranscriptional R-loops are formed in yeast mutants of the THO complex, which functions at the interface between transcription and mRNA export. Despite the relevance of R-loops in transcription-associated recombination, the mechanisms by which they trigger recombination are still elusive. In order to understand how R-loops compromise genome stability, we have analyzed the genetic interaction of THO with 26 genes involved in replication, S-phase checkpoint, DNA repair, and chromatin remodeling. We found a synthetic growth defect in double null mutants of THO and S-phase checkpoint factors, such as the replication factor C- and PCNA-like complexes. Under replicative stress, R-loop-forming THO null mutants require functional S-phase checkpoint functions but not double-strand-break repair functions for survival. Furthermore, R-loop-forming hpr1Delta mutants display replication fork progression impairment at actively transcribed chromosomal regions and trigger Rad53 phosphorylation. We conclude that R-loop-mediated DNA damage activates the S-phase checkpoint, which is required for the cell survival of THO mutants under replicative stress. In light of these results, we propose a model in which R-loop-mediated recombination is explained by template switching.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism
  • Checkpoint Kinase 2
  • Chromosomes
  • DNA Damage
  • DNA Repair*
  • DNA Replication
  • DNA, Fungal / genetics
  • Mad2 Proteins
  • Mutation
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism
  • Phosphorylation
  • Protein Binding
  • Protein Serine-Threonine Kinases / genetics
  • Protein Serine-Threonine Kinases / metabolism
  • S Phase*
  • Saccharomyces cerevisiae / cytology
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae / physiology
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism
  • Transcription Factors / genetics*
  • Transcription Factors / metabolism

Substances

  • Cell Cycle Proteins
  • DNA, Fungal
  • HPR1 protein, S cerevisiae
  • MAD2 protein, S cerevisiae
  • Mad2 Proteins
  • Nuclear Proteins
  • Saccharomyces cerevisiae Proteins
  • Transcription Factors
  • Checkpoint Kinase 2
  • Protein Serine-Threonine Kinases
  • RAD53 protein, S cerevisiae