The Rad4TopBP1 ATR-Activation Domain Functions in G1/S Phase in a Chromatin-Dependent Manner

et al. (2012) The Rad4TopBP1 ATR-Activation Domain Functions in G1/S Phase in a Chromatin- Dependent Manner. PLoS Genet 8(6): e1002801. doi:10.1371/journal.pgen.1002801 TopBP1 The Rad4 ATR-Activation Domain Functions in G1/S Phase in a Chromatin-Dependent Manner Su-Jiun Lin 0 Christopher P. Wardlaw 0 Takashi Morishita 0 Izumi Miyabe 0 Charly Chahwan 0 Thomas Caspari 0 Ulrike Schmidt 0 Antony M. Carr 0 Valerie Garcia 0 Sue Jinks-Robertson, Duke University, United States of America 0 1 Genome Damage and Stability Centre, University of Sussex , Brighton, Sussex , United Kingdom , 2 Department of Molecular Genetics, University of Toronto , Toronto, Ontario , Canada , 3 School of Biological Sciences, Bangor University , Bangor, Gwynedd , United Kingdom 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 Rad4TopBP1 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 Rad4TopBP1 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 Crb253BP1. Consistent with a model where Rad4TopBP1 AAD-dependent checkpoint activation is ssDNA/RPA-independent and functions to amplify otherwise weak checkpoint signals, we demonstrate that the Rad4TopBP1 AAD is important for Chk1 phosphorylation when resection is limited in G2 by ablation of the resecting nuclease, Exo1. We also show that the Rad4TopBP1 AAD acts additively with a Rad9 AAD in G1/S phase but not G2. We propose that AAD-dependent Rad3ATR checkpoint amplification is particularly important when DNA resection is limiting. In S. pombe, this manifests in G1/S phase and relies on proteinchromatin interactions. - Funding: This work was supported by CRUK grant C5514/A10722 (www.cancerresearchuk.org/) and MRC grant G0600233 (www.mrc.ac.uk/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. The DNA damage checkpoint is an elaborate signal transduction pathway that monitors the integrity of the DNA, prevents cell cycle progression and promotes appropriate DNA metabolism [1] reviewed in [2]. The DNA damage sensors associated with checkpoint activation define two separate DNA structure-dependent signal transduction cascades. Each pathway engages a phospho-inositol-3 kinase-like protein kinase (PIKK); either the Ataxia Telangiectasia Mutated (ATM) or the Ataxia Telangiectasia and Rad3 related (ATR) kinase [3]. ATM detects DNA double strand breaks (DSBs) by interaction with the Mre11Rad50-Nbs1 repair complex, while ATR primarily senses single stranded-DNA (ss-DNA) through interactions with RPA. Both ATM and ATR are conserved in the model organisms S. pombe and S. cerevisiae. For ATR to recognise a DNA lesion, single-stranded DNA (ssDNA) needs to be formed - for example by DNA repairdependent DNA processing [4] or following the replication machinery encountering the unrepaired lesion [5]. Once ssDNA is generated, it is immediately coated by replication protein A (RPA) (Reviewed in: [6]). Multiple ATR molecules are initially recruited to ssDNA regions via ATRs obligate binding partner, ATRIP, which binds directly to RPA [7,8]. ATR-ATRIP recruitment to ssDNA-RPA is necessary for basal ATR activation, but is insufficient for full checkpoint activation: corecruitment of a second complex consisting of three PCNA-like proteins, Rad9, Hus1 and Rad1 (known as the 9-1-1 clamp) is also necessary. 9-1-1 is loaded in parallel to ATR recruitment at 59 ssDNA/dsDNA junctions by the checkpoint clamp loader Rad17RFC[25] [9,10,11]. (Figure 1A). When ATR-ATRIP is first loaded at the site of ssDNA, its basal kinase activity promotes phosphorylation of its immediate neighbours, including ATRIP [12,13], an in trans phosphorylation of a residue within ATR itself, T1989 [14], and the subunits of the 9-1-1 clamp [15,16]. Dependent on the concomitant recruitment of 9-1-1, a further protein, TopBP1, is recruited [17]. TopBP1 is recruited via an interaction between its BRCT (1+2) domains and a constitutive phosphosphorylation on the C-terminus of Rad9 [18,19]. Similarly in both yeast systems, Saccharomyces cerevisiae and Schizosaccharomyces pombe, the TopBP1 homologs, Dpb11 and Rad4 respectively, are recruited by the phosphorylation of the Cterminus of Rad9Ddc1 creating a binding site for a pair of BRCT domains (Figure 1A). In S. pombe, the C-terminal phosphorylations occur on Rad9 at residues T412 and S423 [20]. This subsequently recruits Rad4TopBP1 via interaction with BRCT pair (3+4). However, unlike in mammalian cells, T412 and S423 in S. pombe are directly targeted by Rad3ATR in response to its ssDNA/RPA DNA structuredependent checkpoint activation and the amplification of checkpoint signals are carefully modulated to allow the checkpoint kinases to delay mitosis and regulate DNA metabolism. While much work has gone into understanding how this checkpoint functions, the mechanism by which the checkpoint signal is amplified is less clear. We have characterised a conserved domain in the Schizosaccharomyces pombe TopBP1 homolog, Rad4TopBP1 (also known as Cut5) that is capable of activating the ATR homolog Rad3ATR. We demonstrate that this domain is not required for initial checkpoint activation, but functions to amplify the checkpoint signal, likely when the presence of single-stranded DNA is limiting. Our data suggest that the function of the Rad4TopBP1 ATR-Activation Domain (AAD) is mediated by interactions between checkpoint proteins and phosphorylated histone H2A, which is itself promoted by Rad3ATR. We propose that the resulting amplification of the checkpoint signal is particularly important in G1-S phase, when resection is limited. binding and concomitant 9-1-1 loading [16,20,21]. Despite these differences, in both S. pombe [20] and mammalian cells [14], Rad4TopBP1 recruitment promotes the formation of a Rad3ATR/91-1/Rad4TopBP1 complex (Figure 1A). However, the mode of interaction of Rad3ATR and Rad4TopBP1 within this complex has not been defined in S. pombe. Mammalian TopBP1 can directly activate ATR-ATRIP both in vitro in the absence of ssDNA/RPA and when over-expressed in cells. TopBP1-dependent ATR activation requires an ATR activation domain (AAD) situated between the 6th and 7th BRCT domains [17] and mutation of a conserved aromatic residue wi (...truncated)