title: The Rad4(TopBP1) ATR-activation domain functions in G1/S phase in a chromatin-dependent manner.
abstract: DNA damage checkpoint activation can be subdivided in two steps: initial activation and signal amplification. The events distinguishing these two phases and their genetic determinants remain obscure. TopBP1, a mediator protein containing multiple BRCT domains, binds to and activates the ATR/ATRIP complex through its ATR-Activation Domain (AAD). We show that Schizosaccharomyces pombe Rad4(TopBP1) AAD-defective strains are DNA damage sensitive during G1/S-phase, but not during G2. Using lacO-LacI tethering, we developed a DNA damage-independent assay for checkpoint activation that is Rad4(TopBP1) AAD-dependent. In this assay, checkpoint activation requires histone H2A phosphorylation, the interaction between TopBP1 and the 9-1-1 complex, and is mediated by the phospho-binding activity of Crb2(53BP1). Consistent with a model where Rad4(TopBP1) AAD-dependent checkpoint activation is ssDNA/RPA-independent and functions to amplify otherwise weak checkpoint signals, we demonstrate that the Rad4(TopBP1) AAD is important for Chk1 phosphorylation when resection is limited in G2 by ablation of the resecting nuclease, Exo1. We also show that the Rad4(TopBP1) AAD acts additively with a Rad9 AAD in G1/S phase but not G2. We propose that AAD-dependent Rad3(ATR) checkpoint amplification is particularly important when DNA resection is limiting. In S. pombe, this manifests in G1/S phase and relies on protein-chromatin interactions.
authors: Genome Damage and Stability Centre, University of Sussex, Brighton, Sussex, United Kingdom.
authors: Su-Jiun
authors: SJ
authors: Lin
authors: Christopher P
authors: CP
authors: Wardlaw
authors: Takashi
authors: T
authors: Morishita
authors: Izumi
authors: I
authors: Miyabe
authors: Charly
authors: C
authors: Chahwan
authors: Thomas
authors: T
authors: Caspari
authors: Ulrike
authors: U
authors: Schmidt
authors: Antony M
authors: AM
authors: Carr
authors: Valerie
authors: V
authors: Garcia
mesh_terms: D002352:Carrier Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D002843:Chromatin / Q000235:genetics*; D004249:DNA Damage / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D059585:G1 Phase Cell Cycle Checkpoints / Q000235:genetics*; D015966:Gene Expression Regulation, Fungal; D006657:Histones / Q000235:genetics; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D016196:S Phase / Q000235:genetics; D012568:Schizosaccharomyces / Q000235:genetics*; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D011503:Transglutaminases / Q000235:genetics / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002843:Chromatin; C109867:Crb2 protein, S pombe; D004268:DNA-Binding Proteins; D006657:Histones; D009687:Nuclear Proteins; C082463:RAD4 protein, S pombe; D029702:Schizosaccharomyces pombe Proteins; C069930:rad9 protein; D011503:Transglutaminases; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; D000071877:Checkpoint Kinase 1; C000605873:Chk1 protein, S pombe; C456840:rad3 protein, S pombe