The Rad9-Hus1-Rad1 checkpoint clamp regulates interaction of TopBP1 with ATR

TopBP1 serves as an activator of the ATR-ATRIP complex in response to the presence of incompletely replicated or damaged DNA. This process involves binding of ATR to the ATR-activating domain of TopBP1, which is located between BRCT domains VI and VII. TopBP1 displays increased binding to ATR-ATRIP in Xenopus egg extracts containing checkpoint-inducing DNA templates. We show that an N-terminal region of TopBP1 containing BRCT repeats I-II is essential for this checkpoint-stimulated binding of TopBP1 to ATR-ATRIP. The BRCT I-II region of TopBP1 also binds specifically to the Rad9-Hus1-Rad1 (9-1-1) complex in Xenopus egg extracts. This binding occurs via the C-terminal domain of Rad9 and depends upon phosphorylation of its Ser-373 residue. Egg extracts containing either a mutant of TopBP1 lacking the BRCT I-II repeats or a mutant of Rad9 with an alanine substitution at Ser-373 are defective in checkpoint regulation. Furthermore, an isolated C-terminal fragment from Rad9 is an effective inhibitor of checkpoint signaling in egg extracts. These findings suggest that interaction of the 9-1-1 complex with the BRCT I-II region of TopBP1 is necessary for binding of ATR-ATRIP to the ATR-activating domain of TopBP1 and the ensuing activation of ATR.     

Claspin operates downstream of TopBP1 to direct ATR signaling towards Chk1 activation

TopBP1 and Claspin are adaptor proteins that facilitate phosphorylation of Chk1 by the ATR kinase in response to genotoxic stress. Despite their established requirement for Chk1 activation, the exact way in which TopBP1 and Claspin control Chk1 phosphorylation remains unclear. We show that TopBP1 tightly colocalizes with ATR in distinct nuclear subcompartments generated by DNA damage. Although depletion of TopBP1 by RNA interference (RNAi) strongly impaired phosphorylation of multiple ATR targets, including Chk1, Nbs1, Smc1, and H2AX, it did not interfere with ATR assembly at the sites of DNA damage. These findings challenge the current concept of ATR activation by recruitment to damaged DNA. In contrast, Claspin, like Chk1, remained distributed throughout the nucleus both before and after DNA damage. Consistently, the RNAi-mediated ablation of Claspin selectively abrogated ATR's ability to phosphorylate Chk1 but not other ATR targets. In addition, downregulation of Claspin mimicked Chk1 inactivation by inducing spontaneous DNA damage. Finally, we show that TopBP1 is required for the DNA damage-induced interaction between Claspin and Chk1. Together, these results suggest that while TopBP1 is a general regulator of ATR, Claspin operates downstream of TopBP1 to selectively regulate the Chk1-controlled branch of the genotoxic stress response.     

Claspin,a Chk1-regulatory protein,monitors DNA replication on chromatin independently of RPA,ATR, and Rad17

Claspin is required for the ATR-dependent activation of Chk1 in Xenopus egg extracts containing incompletely replicated DNA. We show here that Claspin associates with chromatin in a regulated manner during S phase. Binding of Claspin to chromatin depends on the pre-replication complex (pre-RC) and Cdc45 but not on replication protein A (RPA). These dependencies suggest that binding of Claspin occurs around the time of initial DNA unwinding at replication origins. By contrast, both ATR and Rad17 require RPA for association with DNA. Claspin, ATR, and Rad17 all bind to chromatin independently. These findings suggest that Claspin plays a role in monitoring DNA replication during S phase. Claspin, ATR, and Rad17 may collaborate in checkpoint regulation by detecting different aspects of a DNA replication fork.     

Two Distinct Modes of ATR Activation Orchestrated by Rad17 and Nbs1

1. Download : Download full-size image

     

Phosphorylation of Xenopus Rad1 and Hus1 defines a readout for ATR activation that is independent of Claspin and the Rad9 carboxy terminus

The DNA damage checkpoint pathways sense and respond to DNA damage to ensure genomic stability. The ATR kinase is a central regulator of one such pathway and phosphorylates a number of proteins that have roles in cell cycle progression and DNA repair. Using the Xenopus egg extract system, we have investigated regulation of the Rad1/Hus1/Rad9 complex. We show here that phosphorylation of Rad1 and Hus1 occurs in an ATR- and TopBP1-dependent manner on T5 of Rad1 and S219 and T223 of Hus1. Mutation of these sites has no effect on the phosphorylation of Chk1 by ATR. Interestingly, phosphorylation of Rad1 is independent of Claspin and the Rad9 carboxy terminus, both of which are required for Chk1 phosphorylation. These data suggest that an active ATR signaling complex exists in the absence of the carboxy terminus of Rad9 and that this carboxy-terminal domain may be a specific requirement for Chk1 phosphorylation and not necessary for all ATR-mediated signaling events. Thus, Rad1 phosphorylation provides an alternate and early readout for the study of ATR activation.     

ATR,Claspin and the Rad9-Rad1-Hus1 Complex Regulate Chk1 and Cdc25A in the Absence of DNA Damage

The ATR and Chk1 kinases are essential to maintain genomicintegrity. ATR, with Claspin and the Rad9-Rad1-Hus1 complex,activates Chk1 after DNA damage. Chk1-mediated phosphorylation ofthe Cdc25A phosphatase is required for the mammalian S-phasecheckpoint. Here, we show that during physiological S phase theregulation of the Chk1-Cdc25A pathway depends on ATR, Claspin,Rad9, and Hus1. Human cells with chemically or genetically ablatedATR showed inhibition of Chk1-dependent phosphorylation of Cdc25A,and they accumulated Cdc25A without external DNA damage.Furthermore, siRNA-mediated depletion of Claspin, Rad9 and Hus1also stabilized Cdc25A. ATR ablation also inhibited the activatoryphosphorylation of Chk1 on serine 345. Thus, the ATR-Chk1-Cdc25Apathway represents an integral part of physiological S-phaseprogression, and interference with this mechanism underminesviability of somatic mammalian cells. DNA damage further activatesand switches this pathway from its constitutively operating“surveillance mode” compatible with DNA replication into an“emergency” checkpoint response.     

Structure-based predictions of Rad1, Rad9, Hus1 and Rad17 participation in sliding clamp and clamp-loading complexes

The repair of damaged DNA is coupled to the completion of DNA replication by several cell cycle checkpoint proteins, including, for example, in fission yeast Rad1^Sp, Hus1^Sp, Rad9^Sp and Rad17^Sp. We have found that these four proteins are conserved with protein sequences throughout eukaryotic evolution. Using computational techniques, including fold recognition, comparative modeling and generalized sequence profiles, we have made high confidence structure predictions for the each of the Rad1, Hus1 and Rad9 protein families (Rad17^Sc, Mec3^Sc and Ddc1^Sc in budding yeast, respectively). Each of these families was found to share a common protein fold with that of PCNA, the sliding clamp protein that tethers DNA polymerase to its template. We used previously reported genetic and biochemical data for these proteins from yeast and human cells to predict a heterotrimeric PCNA-like ring structure for the functional Rad1/Rad9/Hus1 complex and to determine their exact order within it. In addition, for each individual protein family, contact regions with neighbors within the PCNA-like ring were identified. Based on a molecular model for Rad17^Sp, we concluded that members of this family, similar to the subunits of the RFC clamp-loading complex, are capable of coupling ATP binding with conformational changes required to load a sliding clamp onto DNA. This model substantiates previous findings regarding the behavior of Rad17 family proteins upon DNA damage and within the RFC complex of clamp-loading proteins.     

Structure and function of the Rad9-binding region of the DNA-damage checkpoint adaptor TopBP1

TopBP1 is a scaffold protein that coordinates activation of the DNA-damage-checkpoint response by coupling binding of the 9-1-1 checkpoint clamp at sites of ssDNA, to activation of the ATR–ATRIP checkpoint kinase complex. We have now determined the crystal structure of the N-terminal region of human TopBP1, revealing an unexpected triple-BRCT domain structure. The arrangement of the BRCT domains differs significantly from previously described tandem BRCT domain structures, and presents two distinct sites for binding phosphopeptides in the second and third BRCT domains. We show that the site in the second but not third BRCT domain in the N-terminus of TopBP1, provides specific interaction with a phosphorylated motif at pSer387 in Rad9, which can be generated by CK2.     

A Highlights from MBoC Selection: Rad17 Plays a Central Role in Establishment of the Interaction between TopBP1 and the Rad9-Hus1-Rad1 Complex at Stalled Replication Forks

Rad17 is critical for the ATR-dependent activation of Chk1 during checkpoint responses. It is known that Rad17 loads the Rad9-Hus1-Rad1 (9-1-1) complex onto DNA. We show that Rad17 also mediates the interaction of 9-1-1 with the ATR-activating protein TopBP1 in Xenopus egg extracts. Studies with Rad17 mutants indicate that binding of ATP to Rad17 is essential for the association of 9-1-1 and TopBP1. Furthermore, hydrolysis of ATP by Rad17 is necessary for the loading of 9-1-1 onto DNA and the elevated, checkpoint-dependent accumulation of TopBP1 on chromatin. Significantly, a mutant 9-1-1 complex that cannot bind TopBP1 has a normal capacity to promote elevated accumulation of TopBP1 on chromatin. Taken together, we propose the following mechanism. First, Rad17 loads 9-1-1 onto DNA. Second, TopBP1 accumulates on chromatin in a manner that depends on both Rad17 and 9-1-1. Finally, 9-1-1 and TopBP1 dock in a Rad17-dependent manner before activation of Chk1.     

Pannexin 1 Channels Play Essential Roles in Urothelial Mechanotransduction and Intercellular Signaling

Urothelial cells respond to bladder distension with ATP release, and ATP signaling within the bladder and from the bladder to the CNS is essential for proper bladder function. In other cell types, pannexin 1 (Panx1) channels provide a pathway for mechanically-induced ATP efflux and for ATP-induced ATP release through interaction with P2X₇ receptors (P2X₇Rs). We report that Panx1 and P2X₇R are functionally expressed in the bladder mucosa and in immortalized human urothelial cells (TRT-HU1), and participate in urothelial ATP release and signaling. ATP release from isolated rat bladders induced by distention was reduced by the Panx1 channel blocker mefloquine (MFQ) and was blunted in mice lacking Panx1 or P2X₇R expression. Hypoosmotic shock induced YoPro dye uptake was inhibited by MFQ and the P2X₇R blocker A438079 in TRT-HU1 cells, and was also blunted in primary urothelial cells derived from mice lacking Panx1 or P2X₇R expression. Rinsing-induced mechanical stimulation of TRT-HU1 cells triggered ATP release, which was reduced by MFQ and potentiated in low divalent cation solution (LDPBS), a condition known to enhance P2X₇R activation. ATP signaling evaluated as intercellular Ca²⁺ wave radius was significantly larger in LDPBS, reduced by MFQ and by apyrase (ATP scavenger). These findings indicate that Panx1 participates in urothelial mechanotransduction and signaling by providing a direct pathway for mechanically-induced ATP release and by functionally interacting with P2X₇Rs.     

TopBP1 and ATR colocalization at meiotic chromosomes: role of TopBP1/Cut5 in the meiotic recombination checkpoint

Mammalian TopBP1 is a BRCT domain-containing protein whose function in mitotic cells is linked to replication and DNA damage checkpoint. Here, we study its possible role during meiosis in mice. TopBP1 foci are abundant during early prophase I and localize mainly to histone gamma-H2AX-positive domains, where DNA double-strand breaks (required to initiate recombination) occur. Strikingly, TopBP1 showed a pattern almost identical to that of ATR, a PI3K-like kinase involved in mitotic DNA damage checkpoint. In the synapsis-defective Fkbp6(-/-) mouse, TopBP1 heavily stains unsynapsed regions of chromosomes. We also tested whether Schizosaccharomyces pombe Cut5 (the TopBP1 homologue) plays a role in the meiotic recombination checkpoint, like spRad3, the ATR homologue. Indeed, we found that a cut5 mutation suppresses the checkpoint-dependent meiotic delay of a meiotic recombination defective mutant, indicating a direct role of the Cut5 protein in the meiotic checkpoint. Our findings suggest that ATR and TopBP1 monitor meiotic recombination and are required for activation of the meiotic recombination checkpoint.     

Site-Specific Phosphorylation of the DNA Damage Response Mediator Rad9 by Cyclin-Dependent Kinases Regulates Activation of Checkpoint Kinase 1

The mediators of the DNA damage response (DDR) are highly phosphorylated by kinases that control cell proliferation, but little is known about the role of this regulation. Here we show that cell cycle phosphorylation of the prototypical DDR mediator Saccharomyces cerevisiae Rad9 depends on cyclin-dependent kinase (CDK) complexes. We find that a specific G2/M form of Cdc28 can phosphorylate in vitro the N-terminal region of Rad9 on nine consensus CDK phosphorylation sites. We show that the integrity of CDK consensus sites and the activity of Cdc28 are required for both the activation of the Chk1 checkpoint kinase and its interaction with Rad9. We have identified T125 and T143 as important residues in Rad9 for this Rad9/Chk1 interaction. Phosphorylation of T143 is the most important feature promoting Rad9/Chk1 interaction, while the much more abundant phosphorylation of the neighbouring T125 residue impedes the Rad9/Chk1 interaction. We suggest a novel model for Chk1 activation where Cdc28 regulates the constitutive interaction of Rad9 and Chk1. The Rad9/Chk1 complex is then recruited at sites of DNA damage where activation of Chk1 requires additional DDR–specific protein kinases.     

A tale of two tails: Activation of DNA damage checkpoint kinase Mec1/ATR by the 9-1-1 clamp and by Dpb11/TopBP1

The DNA damage and replication checkpoint kinase Mec1/ATR is a member of the PI3-kinase related kinases that function in response to various genotoxic stresses. The checkpoint clamp 9-1-1 (Rad9-Rad1-Hus1 in S. pombe and mammals; Ddc1-Rad17-Mec3 in S. cerevisiae) executes two distinct checkpoint functions. In S. cerevisiae, DNA-bound 9-1-1 directly activates Mec1 kinase activity, a function that has not been demonstrated in other organisms. A second, conserved activity of 9-1-1 is that of TopBP1/Cut5/Dpb11 recruitment to stalled replication sites; subsequent activation of Mec1/ATR is carried out by TopBP1/Cut5/Dpb11. Biochemical studies indicate that the mode of Mec1/ATR activation by S. cerevisiae 9-1-1 is analogous to activation by S. cerevisiae Dpb11 or by vertebrate TopBP1: activation is mediated by the intrinsically disordered C-terminal tail of each activator. The relative contributions made by multiple activators of Mec1/ATR are discussed.     

Cooperative activation of the ATR checkpoint kinase by TopBP1 and damaged DNA

TopBP1, acting in concert with DNA containing bulky base lesions, stimulates ATR kinase activity under physiologically relevant reaction conditions. Here, we analyze the roles of the three components in ATR activation: DNA, base damage and TopBP1. We show that base adducts caused by a potent carcinogen, benzo[a]pyrene diol epoxide (BPDE), constitute a strong signal for TopBP1-dependent ATR kinase activity on Chk1 and p53. We find that the C-terminus of TopBP1 binds preferentially to damaged DNA and is sufficient to mediate damaged DNA-dependent ATR activation in a manner similar to full-length TopBP1. Significantly, we find that stimulation of ATR by BPDE-damaged DNA exhibits strong dependence on the length of DNA, with essentially no stimulation with fragments of 0.2 kb and reaching maximum stimulation with 2 kb fragments. Moreover, TopBP1 shows preferential binding to longer DNA fragments and, in contrast to previous biochemical studies, TopBP1 binding is completely independent of DNA ends. We find that TopBP1 binds to circular and linear DNAs with comparable affinities and that these DNA forms elicit the same level of TopBP1-dependent ATR activation. Taken together, these findings suggest a cooperative activation mechanism for the ATR checkpoint kinase by TopBP1 and damaged DNA.     

Rad9, Rad17, and Rad24 Are Required for S Phase Regulation in Saccharomyces Cerevisiae in Response to DNA Damage

We have previously shown that a checkpoint dependent on MEC1 and RAD53 slows the rate of S phase progression in Saccharomyces cerevisiae in response to alkylation damage. Whereas wild-type cells exhibit a slow S phase in response to damage, mec1-1 and rad53 mutants replicate rapidly in the presence or absence of DNA damage. In this report, we show that other genes (RAD9, RAD17, RAD24) involved in the DNA damage checkpoint pathway also play a role in regulating S phase in response to DNA damage. Furthermore, RAD9, RAD17, and RAD24 fall into two groups with respect to both sensitivity to alkylation and regulation of S phase. We also demonstrate that the more dramatic defect in S phase regulation in the mec1-1 and rad53 mutants is epistatic to a less severe defect seen in rad9Δ, rad17Δ, and rad24Δ. Furthermore, the triple rad9Δ rad17Δ rad24Δ mutant also has a less severe defect than mec1-1 or rad53 mutants. Finally, we demonstrate the specificity of this phenotype by showing that the DNA repair and/or checkpoint mutants mgt1Δ, mag1Δ, apn1Δ, rev3Δ, rad18Δ, rad16Δ, dun1-Δ100, sad4-1, tel1Δ, rad26Δ, rad51Δ, rad52-1, rad54Δ, rad14Δ, rad1Δ, pol30-46, pol30-52, mad3Δ, pds1Δ/esp2Δ, pms1Δ, mlh1Δ, and msh2Δ are all proficient at S phase regulation, even though some of these mutations confer sensitivity to alkylation.     

Ubiquitin-specific Peptidase 20 Regulates Rad17 Stability,Checkpoint Kinase 1 Phosphorylation and DNA Repair by Homologous Recombination

Rad17 is a subunit of the Rad9-Hus1-Rad1 clamp loader complex, which is required for Chk1 activation after DNA damage. Rad17 has been shown to be regulated by the ubiquitin-proteasome system. We have identified a deubiquitylase, USP20 that is required for Rad17 protein stability in the steady-state and post DNA damage. We demonstrate that USP20 and Rad17 interact, and that this interaction is enhanced by UV exposure. We show that USP20 regulation of Rad17 is at the protein level in a proteasome-dependent manner. USP20 depletion results in poor activation of Chk1 protein by phosphorylation, consistent with Rad17 role in ATR-mediated phosphorylation of Chk1. Similar to other DNA repair proteins, USP20 is phosphorylated post DNA damage, and its depletion sensitizes cancer cells to damaging agents that form blocks ahead of the replication forks. Similar to Chk1 and Rad17, which enhance recombinational repair of collapsed replication forks, we demonstrate that USP20 depletion impairs DNA double strand break repair by homologous recombination. Together, our data establish a new function of USP20 in genome maintenance and DNA repair.