title: Replication independent ATR signalling leads to G2/M arrest requiring Nbs1, 53BP1 and MDC1.
abstract: Ataxia telangiectasia and Rad3-related (ATR) is a phosphoinositol-3-kinase like kinase (PIKK) that initiates a signal transduction response to replication fork stalling. Defects in ATR signalling have been reported in several disorders characterized by microcephaly and growth delay. Here, we gain insight into factors influencing the ATR signalling pathway and consider how they can be exploited for diagnostic purposes. Activation of ATR at stalled replication forks leads to intra-S and G2/M phase checkpoint arrest. ATR also phosphorylates gamma-H2AX at single-stranded (ss) DNA regions generated during nucleotide excision repair (NER) in non-replicating cells, but the critical analysis of any functional consequence has not been reported. Here, we show that UV irradiation of G2 phase cells causes ATR-dependent but replication-independent G2/M checkpoint arrest. This process requires the Nbs1 N-terminus encompassing the FHA and BRCT domains but not the Nbs1 C-terminus in contrast to ATM-dependent activation of G2/M arrest in response to ionizing radiation. Thus, Nbs1 has a function in ATR signalling in a manner distinct to any role at stalled replication forks. Replication-independent ATR signalling also requires the mediator proteins, 53BP1 and MDC1, providing direct evidence for their role in ATR signalling, but not H2AX. Finally, the process is activated in Cockayne's syndrome but not Xeroderma pigmentosum group A cells providing evidence that ssDNA regions generated during NER are the ATR-pathway-specific activating lesion. Replication-independent G2/M checkpoint arrest represents a suitable assay to specifically identify patients with defective ATR signalling, including Seckel syndrome, Nijmegen breakage syndrome and MCPH-1-dependent primary microcephaly.
authors.affiliation: Genome Damage and Stability Centre, University of Sussex, Brighton, East Sussex BN1 9RQ, UK.
authors.forename: Tom
authors.initials: T
authors.lastname: Stiff
authors.forename: Karen
authors.initials: K
authors.lastname: Cerosaletti
authors.forename: Patrick
authors.initials: P
authors.lastname: Concannon
authors.forename: Mark
authors.initials: M
authors.lastname: O'Driscoll
authors.forename: Penny A
authors.initials: PA
authors.lastname: Jeggo
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D001260:Ataxia Telangiectasia / Q000235:genetics / Q000378:metabolism; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002455:Cell Division; D002460:Cell Line; D004249:DNA Damage; D004260:DNA Repair; D004261:DNA Replication; D016195:G2 Phase; D006657:Histones / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D009154:Mutation; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D015534:Trans-Activators / Q000378:metabolism*; D000071857:Tumor Suppressor p53-Binding Protein 1; D014466:Ultraviolet Rays
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; C468783:H2AX protein, human; D006657:Histones; D047908:Intracellular Signaling Peptides and Proteins; C413163:MDC1 protein, human; C112156:NBN protein, human; D009687:Nuclear Proteins; D011994:Recombinant Proteins; C498301:TP53BP1 protein, human; D015534:Trans-Activators; D000071857:Tumor Suppressor p53-Binding Protein 1; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases