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      An Essential Function for the ATR-Activation-Domain (AAD) of TopBP1 in Mouse Development and Cellular Senescence

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          Abstract

          ATR activation is dependent on temporal and spatial interactions with partner proteins. In the budding yeast model, three proteins – Dpb11 TopBP1, Ddc1 Rad9 and Dna2 - all interact with and activate Mec1 ATR. Each contains an ATR activation domain (ADD) that interacts directly with the Mec1 ATR:Ddc2 ATRIP complex. Any of the Dpb11 TopBP1, Ddc1 Rad9 or Dna2 ADDs is sufficient to activate Mec1 ATR in vitro. All three can also independently activate Mec1 ATR in vivo: the checkpoint is lost only when all three AADs are absent. In metazoans, only TopBP1 has been identified as a direct ATR activator. Depletion-replacement approaches suggest the TopBP1-AAD is both sufficient and necessary for ATR activation. The physiological function of the TopBP1 AAD is, however, unknown. We created a knock-in point mutation (W1147R) that ablates mouse TopBP1-AAD function. TopBP1-W1147R is early embryonic lethal. To analyse TopBP1-W1147R cellular function in vivo, we silenced the wild type TopBP1 allele in heterozygous MEFs. AAD inactivation impaired cell proliferation, promoted premature senescence and compromised Chk1 signalling following UV irradiation. We also show enforced TopBP1 dimerization promotes ATR-dependent Chk1 phosphorylation. Our data suggest that, unlike the yeast models, the TopBP1-AAD is the major activator of ATR, sustaining cell proliferation and embryonic development.

          Author Summary

          DNA damage checkpoint signalling is an essential component of the DNA damage response. Many of the key proteins initiating the checkpoint signal have been identified and characterised in yeast. Here we explore the role of the ATR activating domain (AAD) of TopBP1 in embryonic development, cell growth and checkpoint activation using a mouse model. In contrast to yeasts, where the TopBP1 AAD plays a redundant, and thus phenotypically minor, role in ATR activation, our data demonstrate that the mouse TopBP1 AAD is essential for cellular proliferation. Interestingly, this suggests evolution has provided a simpler ATR activation mechanism in metazoans than it has in yeasts.

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          Most cited references45

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          ATR and ATRIP: partners in checkpoint signaling.

          The checkpoint kinases ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3 related) transduce genomic stress signals to halt cell cycle progression and promote DNA repair. We report the identification of an ATR-interacting protein (ATRIP) that is phosphorylated by ATR, regulates ATR expression, and is an essential component of the DNA damage checkpoint pathway. ATR and ATRIP both localize to intranuclear foci after DNA damage or inhibition of replication. Deletion of ATR mediated by the Cre recombinase caused the loss of ATR and ATRIP expression, loss of DNA damage checkpoint responses, and cell death. Therefore, ATR is essential for the viability of human somatic cells. Small interfering RNA directed against ATRIP caused the loss of both ATRIP and ATR expression and the loss of checkpoint responses to DNA damage. Thus, ATRIP and ATR are mutually dependent partners in cell cycle checkpoint signaling pathways.
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            ATR disruption leads to chromosomal fragmentation and early embryonic lethality.

            Although a small decrease in survival and increase in tumor incidence was observed in ATR(+/-) mice, ATR(-/-) embryos die early in development, subsequent to the blastocyst stage and prior to 7.5 days p.c. In culture, ATR(-/-) blastocysts cells continue to cycle into mitosis for 2 days but subsequently fail to expand and die of caspase-dependent apoptosis. Importantly, caspase-independent chromosome breaks are observed in ATR(-/-) cells prior to widespread apoptosis, implying that apoptosis is caused by a loss of genomic integrity. These data show that ATR is essential for early embryonic development and must function in processes other than regulation of p53.
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              Phosphorylation of Sld2 and Sld3 by cyclin-dependent kinases promotes DNA replication in budding yeast.

              Cyclin-dependent kinases (CDKs) drive major cell cycle events including the initiation of chromosomal DNA replication. We identified two S phase CDK (S-CDK) phosphorylation sites in the budding yeast Sld3 protein that, together, are essential for DNA replication. Here we show that, when phosphorylated, these sites bind to the amino-terminal BRCT repeats of Dpb11. An Sld3-Dpb11 fusion construct bypasses the requirement for both Sld3 phosphorylation and the N-terminal BRCT repeats of Dpb11. Co-expression of this fusion with a phospho-mimicking mutant in a second essential CDK substrate, Sld2, promotes DNA replication in the absence of S-CDK. Therefore, Sld2 and Sld3 are the minimal set of S-CDK targets required for DNA replication. DNA replication in cells lacking G1 phase CDK (G1-CDK) required expression of the Cdc7 kinase regulatory subunit, Dbf4, as well as Sld2 and Sld3 bypass. Our results help to explain how G1- and S-CDKs promote DNA replication in yeast.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Genet
                PLoS Genet
                plos
                plosgen
                PLoS Genetics
                Public Library of Science (San Francisco, USA )
                1553-7390
                1553-7404
                August 2013
                August 2013
                8 August 2013
                : 9
                : 8
                : e1003702
                Affiliations
                [1 ]Leibniz Institute for Age Research – Fritz Lipmann Institute (FLI), Jena, Germany
                [2 ]Sussex for Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton, Sussex, United Kingdom
                [3 ]Faculty of Biology and Pharmacy, Friedrich-Schiller-University Jena, Jena, Germany
                University of Washington, United States of America
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: AMC ZQW. Performed the experiments: CL TLL ZWZ CB AK WM. Analyzed the data: ZQW AMC CL ZWZ. Wrote the paper: ZQW AMC ZWZ.

                Article
                PGENETICS-D-13-01218
                10.1371/journal.pgen.1003702
                3738440
                23950734
                a5d4b22a-448d-4016-aa79-4280f039811e
                Copyright @ 2013

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 9 May 2013
                : 20 June 2013
                Page count
                Pages: 11
                Funding
                This work was supported in part by the Deutsche Forschungsgemeinschaft (DFG) to ZQW and MRC grants G0600233 and G1100074 to AMC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology

                Genetics
                Genetics

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