The S-phase checkpoint is required to respond to R-loops accumulated in THO mutants.
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.
Gómez-González
Belén
B
Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla-CSIC, Av. Américo Vespucio s/n, 41092 Seville, Spain.
Felipe-Abrio
Irene
I
Aguilera
Andrés
A
D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D064447:Checkpoint Kinase 2; D002875:Chromosomes; D004249:DNA Damage; D004260:DNA Repair; D004261:DNA Replication; D004271:DNA, Fungal / Q000235:genetics; D064246:Mad2 Proteins; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D016196:S Phase; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics* / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism
D018797:Cell Cycle Proteins; D004271:DNA, Fungal; C063494:HPR1 protein, S cerevisiae; C577695:MAD2 protein, S cerevisiae; D064246:Mad2 Proteins; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D064447:Checkpoint Kinase 2; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae