title: Checkpoint-dependent RNR induction promotes fork restart after replicative stress.
abstract: The checkpoint kinase Rad53 is crucial to regulate DNA replication in the presence of replicative stress. Under conditions that interfere with the progression of replication forks, Rad53 prevents Exo1-dependent fork degradation. However, although EXO1 deletion avoids fork degradation in rad53 mutants, it does not suppress their sensitivity to the ribonucleotide reductase (RNR) inhibitor hydroxyurea (HU). In this case, the inability to restart stalled forks is likely to account for the lethality of rad53 mutant cells after replication blocks. Here we show that Rad53 regulates replication restart through the checkpoint-dependent transcriptional response, and more specifically, through RNR induction. Thus, in addition to preventing fork degradation, Rad53 prevents cell death in the presence of HU by regulating RNR-expression and localization. When RNR is induced in the absence of Exo1 and RNR negative regulators, cell viability of rad53 mutants treated with HU is increased and the ability of replication forks to restart after replicative stress is restored.
authors.affiliation: Instituto de Biología Funcional y Genómica and Departamento de Microbiología y Genética, (CSIC/USAL), Campus Miguel de Unamuno, Salamanca 37007, Spain.
authors.forename: Esther C
authors.initials: EC
authors.lastname: Morafraile
authors.affiliation: Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Herts. EN6 3LD, United Kingdom.
authors.forename: John F X
authors.initials: JF
authors.lastname: Diffley
authors.affiliation: Centro de Biología Molecular Severo Ochoa (CSIC/UAM), Cantoblanco, 28049 Madrid, Spain.
authors.forename: José Antonio
authors.initials: JA
authors.lastname: Tercero
authors.affiliation: 1] Instituto de Biología Funcional y Genómica and Departamento de Microbiología y Genética, (CSIC/USAL), Campus Miguel de Unamuno, Salamanca 37007, Spain [2] Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Herts. EN6 3LD, United Kingdom [3] Centro de Biología Molecular Severo Ochoa (CSIC/UAM), Cantoblanco, 28049 Madrid, Spain.
authors.forename: Mónica
authors.initials: M
authors.lastname: Segurado
mesh_terms: D018797:Cell Cycle Proteins / Q000096:biosynthesis* / Q000235:genetics; D002470:Cell Survival / Q000187:drug effects; D064447:Checkpoint Kinase 2 / Q000096:biosynthesis* / Q000235:genetics; D004260:DNA Repair / Q000235:genetics; D004261:DNA Replication / Q000187:drug effects / Q000235:genetics*; D005090:Exodeoxyribonucleases / Q000235:genetics* / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects; D006918:Hydroxyurea / Q000494:pharmacology; D009154:Mutation; D012264:Ribonucleotide Reductases / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D018797:Cell Cycle Proteins; D029701:Saccharomyces cerevisiae Proteins; D012264:Ribonucleotide Reductases; D064447:Checkpoint Kinase 2; C067022:RAD53 protein, S cerevisiae; D005090:Exodeoxyribonucleases; C028450:exodeoxyribonuclease I; D006918:Hydroxyurea