title: The Mre11-Rad50-Nbs1 complex acts both upstream and downstream of ataxia telangiectasia mutated and Rad3-related protein (ATR) to regulate the S-phase checkpoint following UV treatment.
abstract: The Mre11-Rad50-Nbs1 (MRN) complex is required for mediating the S-phase checkpoint following UV treatment, but the underlying mechanism is not clear. Here we demonstrate that at least two mechanisms are involved in regulating the S-phase checkpoint in an MRN-dependent manner following UV treatment. First, when replication forks are stalled, MRN is required upstream of ataxia telangiectasia mutated and Rad3-related protein (ATR) to facilitate ATR activation in a substrate and dosage-dependent manner. In particular, MRN is required for ATR-directed phosphorylation of RPA2, a critical event in mediating the S-phase checkpoint following UV treatment. Second, MRN is a downstream substrate of ATR. Nbs1 is phosphorylated by ATR at Ser-343 when replication forks are stalled, and this phosphorylation event is also important for down-regulating DNA replication following UV treatment. Moreover, we demonstrate that MRN and ATR/ATR-interacting protein (TRIP) interact with each other, and the forkhead-associated/breast cancer C-terminal domains (FHA/BRCT) of Nbs1 play a significant role in mediating this interaction. Mutations in the FHA/BRCT domains do not prevent ATR activation but specifically impair ATR-mediated Nbs1 phosphorylation at Ser-343, which results in a defect in the S-phase checkpoint. These data suggest that MRN plays critical roles both upstream and downstream of ATR to regulate the S-phase checkpoint when replication forks are stalled.
authors.affiliation: Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: Erin
authors.initials: E
authors.lastname: Olson
authors.forename: Christian J
authors.initials: CJ
authors.lastname: Nievera
authors.forename: Alan Yueh-Luen
authors.initials: AY
authors.lastname: Lee
authors.forename: Longchuan
authors.initials: L
authors.lastname: Chen
authors.forename: Xiaohua
authors.initials: X
authors.lastname: Wu
mesh_terms: D017766:Acid Anhydride Hydrolases; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000502:physiology*; D002460:Cell Line; D045744:Cell Line, Tumor; D004249:DNA Damage; D045643:DNA Repair Enzymes / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000502:physiology*; D015972:Gene Expression Regulation, Neoplastic; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D000076228:MRE11 Homologue Protein; D009687:Nuclear Proteins / Q000502:physiology*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D017434:Protein Structure, Tertiary; D016196:S Phase; D012694:Serine / Q000737:chemistry; D014466:Ultraviolet Rays
chemical_list: D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C121766:MRE11 protein, human; C112156:NBN protein, human; D009687:Nuclear Proteins; D012694:Serine; D005982:Glutathione Transferase; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; D000076228:MRE11 Homologue Protein; D017766:Acid Anhydride Hydrolases; C112158:RAD50 protein, human; D045643:DNA Repair Enzymes