An essential function for the ATR-Activation-Domain (AAD) of TopBP1 in mouse development and cellular senescence

ATR activation is dependent on temporal and spatial interactions with partner proteins. In the budding yeast model, three proteins – Dpb11TopBP1, Ddc1Rad9 and Dna2 - all interact with and activate Mec1ATR. Each contains an ATR activation domain (ADD) that interacts directly with the Mec1ATR:Ddc2ATRIP complex. Any of the Dpb11TopBP1, Ddc1Rad9 or Dna2 ADDs is sufficient to activate Mec1ATR in vitro. All three can also independently activate Mec1ATR 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

Kinetics of poly(ADP-ribosyl)ation, but not PARP1 itself, determines the cell fate in response to DNA damage in vitro and in vivo

One of the fastest cellular responses to genotoxic stress is the formation of poly(ADP-ribose) polymers (PAR) by poly(ADP-ribose)polymerase 1 (PARP1, or ARTD1). PARP1 and its enzymatic product PAR regulate diverse biological processes, such as DNA repair, chromatin remodeling, transcription and cell death. However, the inter-dependent function of the PARP1 protein and its enzymatic activity clouds the mechanism underlying the biological response. We generated a PARP1 knock-in mouse model carrying a point mutation in the catalytic domain of PARP1 (D993A), which impairs the kinetics of the PARP1 activity and the PAR chain complexity in vitro and in vivo, designated as hypo-PARylation. PARP1D993A/D993A mice and cells are viable and show no obvious abnormalities. Despite a mild defect in base excision repair (BER), this hypo-PARylation compromises the DNA damage response during DNA replication, leading to cell death or senescence. Strikingly, PARP1D993A/D993A mice are hypersensitive to alkylation in vivo, phenocopying the phenotype of PARP1 knockout mice. Our study thus unravels a novel regulatory mechanism, which could not be revealed by classical loss-of-function studies, on how PAR homeostasis, but not the PARP1 protein, protects cells and organisms from acute DNA damage.publishe

Genotype distribution of offspring from TopBP1^ki/+ breeding.

<p>Genotyping analysis of offspring derived from AAD-mutant heterozygote backcrosses (ki/+ x +/+) and intercrosses (ki/+ x ki/+). P0: postnatal day 0; +/+, wild type; ki/+, heterozygotes mutant; ki/ki, homozygotes knock-in mutant; f, female; m, male.</p>*<p>: These embryos were too small to reliably define their genotype.</p

Generation of TopBP1 AAD mutant transgenic mice.

<p>(<b>A</b>) Schematic of the C-terminus of the <i>TopBP1</i> locus: wild type (wt), targeted (tg) and knock-in (ki) alleles. The red line marks the targeting vector. Exons are numbered in the boxes, Southern blot probes (p8 and p5), sizes of DNA fragments after indicated enzyme digestion and the location of primers for PCR genotyping are shown. The targeting vector contained a neomycin resistance gene (Neo) flanked by two <i>frt</i> sites (grey triangles). Exon 20 is flanked by two <i>loxP</i> sites (black triangle). Tryptophan 1147 (W1147) was mutated into arginine in AAD by replacing T3439 with C in exon 20. (<b>B–C</b>) Southern blot analyses of gene targeted ES cell clones: Homologous integration was verified by digestion with <i>Ase</i>I and hybridization with a 5′ external probe (p8) and by <i>Ppu</i>MI digestion and hybridization with a 3′ external probe (p5). (<b>D</b>) PCR genotyping analysis of the wild type, targeted and the knock-in allele following Neo excission. (<b>E</b>). Sequencing of genomic DNA from <i>TopBP1^ki/+</i> heterozygous MEFs confirms the mutation of TTT (tryptophan) to TTC (Arginine) (W1147R).</p

Inducible dimerization of TopBP1 activates ATR-Chk1.

<p>(<b>A</b>) Schematic showing the inducible dimerization. Addition of AP20187 induces dimerization (interaction) of FKBP-containing proteins. (<b>B</b>) Immunoprecipitation (IP) coupled immunoblot (IB) analysis of HEK293T cells that were transfected with the indicated vectors (Flag-tagged and HA-tagged). Cells were treated with 100 nm of AP20187 for 1 hr and analyzed by IP-IB using indicated antibodies. (<b>C</b>) IP-IB analysis of HEK293T cells after transfection with empty vector (HA-FKBP) or wild type TopBP1 (HA-FKBP-wtTopBP1). Cells at 40 hr after transfection were incubated with 100 nm of AP20187 for 1 hr without or with 1.6 mM of ATR inhibitor (ATRi) or were treated with 100 J/m-2 UV. Immunoblot analysis was performed 1 hr after respective treatment. (<b>D</b>) IP-IB analysis of HEK293T cells after transfection with empty vector (HA-FKBP), or FKBP-fused wild type TopBP1 (HA-FKBP-wtTopBP1), or FKBP-fused AAD mutant TopBP1 (HA-FKBP-mtTopBP1), or wild type TopBP1 without FKBP (HA-wtTopBP1). Cells were treated with 100 nm of AP20187 for 1 hour then analysed by immunoblotting with indicated antibodies.</p

Poly(ADP-ribose) binding to Chk1 at stalled replication forks is required for S-phase checkpoint activation

Damaged replication forks activate poly(ADP-ribose) polymerase 1 (PARP1), which catalyses poly(ADP-ribose) (PAR) formation; however, how PARP1 or poly(ADP-ribosyl)ation is involved in the S-phase checkpoint is unknown. Here we show that PAR, supplied by PARP1, interacts with Chk1 via a novel PAR-binding regulatory (PbR) motif in Chk1, independent of ATR and its activity. iPOND studies reveal that Chk1 associates readily with the unperturbed replication fork and that PAR is required for efficient retention of Chk1 and phosphorylated Chk1 at the fork. A PbR mutation, which disrupts PAR binding, but not the interaction with its partners Claspin or BRCA1, impairs Chk1 and the S-phase checkpoint activation, and mirrors Chk1 knockdown-induced hypersensitivity to fork poisoning. We find that long chains, but not short chains, of PAR stimulate Chk1 kinase activity. Collectively, we disclose a previously unrecognized mechanism of the S-phase checkpoint by PAR metabolism that modulates Chk1 activity at the replication fork

AAD mutation induces premature cellular senescence.

<p>(<b>A</b>) GFP+ <i>TopBP1^ki/+</i> cells were sorted 24 hr after transfection and cultured. Images show the cell density and morphology at D1 and D5. Enlargement shows a representative area of <i>TopBP1</i>^ki/− cells from D5. (<b>B</b>) SA-<i>β</i>-<i>galactosidase staining</i> of cells 6 days after shRNA transfection shown in blue. (<b>C</b>) Quantification of SA-β-<i>galactosidase positive cells</i> from B. The data represent the mean ± SD of at least 500 cells from 2 independent experiments. <i>P</i> value: Student's <i>t</i>-test. (<b>D</b>) Semi-quantitative RT-PCR analysis of RNA isolated from D5 cultures from A. The expression level (indicated on top of each sample) was estimated by quantification normalized to the level of GAPDH and then correlated with GFP-shLuc transfected cells. Two independent experiments were performed which showed equivalent results.</p

Inactivation of the AAD results in early embryonic developmental defects.

<p>(<b>A</b>) Decidua at E11.5 from intercrossing of <i>TopBP1^ki/+</i>mice. Decidua in (I) was genotype ki/+. Decidua in (II) was empty, thus with unclear genotype. Bar = 1 mm. (<b>B</b>) Cultures of E3.5 blastocysts from <i>TopBP1^ki/+</i> intercrosses. D1: 1 day after culture, D4: 4 days after culture. Arrows indicate inner cell mass (ICM). Bar = 50 micrometers. Genotypes are indicated on the top of respective images. (<b>C</b>) Example of PCR genotyping from ICM of blastocyst outgrowth. Expected product size for alleles labeled. ki = knock-in; wt = wild type.</p

Mutation of AAD impairs ATR-Chk1 pathway <i>in vivo</i>.

<p>(<b>A</b>) Immunostaining of RPA (red and upper panel) in <i>TopBP1^ki/+</i> cells 36 hr after transfection with GFP-shLuc or GFP-shTop2 without or with the indicated treatment. ATRi, ATR inhibitor. GFP-shLuc or GFP-shTop2 transfection is visualized by GFP (green). DNA was stained with DAPI (blue). Inset shows the enlargement of selected areas. (<b>B</b>) Immunostaining of phosphorylation of Chk1-S317 (pChk1, red, upper panel) in <i>TopBP1^ki/+</i> cells 36 hr after transfection with GFP-shLuc or GFP-shTop2 without or with the indicated treatment. ATRi, ATR inhibitor. GFP-shLuc or GFP-shTop2 transfection is visualized by GFP (green). DNA was stained with DAPI (blue). GFP-shChk1 transfection (right panel, arrows) served as a negative control for pChk1 staining. (<b>C</b>) Quantification of fluorescent density of phosphor Chk1-S317 staining (pChk1) of indicated samples from B. The data represent the mean ± SD of at least 200 cells (or GFP positive cells) and were repeated three times. One-way ANOVA pair-test was performed for the statistical analysis. ***<i>P</i><0.001; <i>n.s.</i>, not significant.</p