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.     

Involvement of Hus1 in the chain elongation step of DNA replication after exposure to camptothecin or ionizing radiation

DNA damage-induced S phase (S) checkpoint includes inhibition of both replicon initiation and chain elongation. The precise mechanism for controlling the two processes remains unclear. In this study, we showed that Hus1-deficient mouse cells had an impaired S checkpoint after exposure to DNA strand break-inducing agents such as camptothecin (CPT) (≥1.0 µM), or ionizing radiation (IR) (≥15 Gy). The Hus1-dependent S checkpoint contributes to cell resistance to CPT. This impaired S checkpoint induced by CPT or IR in Hus1-deficient cells reflected mainly the chain elongation step of DNA replication and was correlated with the reduction of dissociation of PCNA from DNA replication foci. Although Hus1 is required for Rad9 phosphorylation following exposure of cells to CPT or IR, Hus1-deficient cells showed normal activation of ATR/CHK1 and ATM kinases at doses where the checkpoint defects were manifested, suggesting that Hus1 is not a component of the sensor system for activating these pathways in S checkpoint induced by CPT or IR.     

Viral transport of DNA damage that mimics a stalled replication fork

Adeno-associated virus type 2 (AAV2) infection incites cells to arrest with 4N DNA content or die if the p53 pathway is defective. This arrest depends on AAV2 DNA, which is single stranded with inverted terminal repeats that serve as primers during viral DNA replication. Here, we show that AAV2 DNA triggers damage signaling that resembles the response to an aberrant cellular DNA replication fork. UV treatment of AAV2 enhances the G2 arrest by generating intrastrand DNA cross-links which persist in infected cells, disrupting viral DNA replication and maintaining the viral DNA in the single-stranded form. In cells, such DNA accumulates into nuclear foci with a signaling apparatus that involves DNA polymerase delta, ATR, TopBP1, RPA, and the Rad9/Rad1/Hus1 complex but not ATM or NBS1. Focus formation and damage signaling strictly depend on ATR and Chk1 functions. Activation of the Chk1 effector kinase leads to the virus-induced G2 arrest. AAV2 provides a novel way to study the cellular response to abnormal DNA replication without damaging cellular DNA. By using the AAV2 system, we show that in human cells activation of phosphorylation of Chk1 depends on TopBP1 and that it is a prerequisite for the appearance of DNA damage foci.     

Analysis of Rad3 and Chk1 protein kinases defines different checkpoint responses.

Eukaryotic cells respond to DNA damage and S phase replication blocks by arresting cell-cycle progression through the DNA structure checkpoint pathways. In Schizosaccharomyces pombe, the Chk1 kinase is essential for mitotic arrest and is phosphorylated after DNA damage. During S phase, the Cds1 kinase is activated in response to DNA damage and DNA replication blocks. The response of both Chk1 and Cds1 requires the six 'checkpoint Rad' proteins (Rad1, Rad3, Rad9, Rad17, Rad26 and Hus1). We demonstrate that DNA damage-dependent phosphorylation of Chk1 is also cell-cycle specific, occurring primarily in late S phase and G2, but not during M/G1 or early S phase. We have also isolated and characterized a temperature-sensitive allele of rad3. Rad3 functions differently depending on which checkpoint pathway is activated. Following DNA damage, rad3 is required to initiate but not maintain the Chk1 response. When DNA replication is inhibited, rad3 is required for both initiation and maintenance of the Cds1 response. We have identified a strong genetic interaction between rad3 and cds1, and biochemical evidence shows a physical interaction is possible between Rad3 and Cds1, and between Rad3 and Chk1 in vitro. Together, our results highlight the cell-cycle specificity of the DNA structure-dependent checkpoint response and identify distinct roles for Rad3 in the different checkpoint responses. Keywords: ATM/ATR/cell-cycle checkpoints/Chk1/Rad3     

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.     

NER initiation factors,DDB2 and XPC,regulate UV radiation response by recruiting ATR and ATM kinases to DNA damage sites

ATR and ATM kinases are central to the checkpoint activation in response to DNA damage and replication stress. However, the nature of the signal, which initially activates these kinases in response to UV damage, is unclear. Here, we have shown that DDB2 and XPC, two early UV damage recognition factors, are required for the damage-specific ATR and ATM recruitment and phosphorylation. ATR and ATM physically interacted with XPC and promptly localized to the UV damage sites. ATR and ATM recruitment and their phosphorylation were negatively affected in cells defective in DDB2 or XPC functions. Consequently, the phosphorylation of ATR and ATM substrates, Chk1, Chk2, H2AX, and BRCA1 was significantly reduced or abrogated in mutant cells. Furthermore, UV exposure of cells defective in DDB2 or XPC resulted in a marked decrease in BRCA1 and Rad51 recruitment to the damage site. Conversely, ATR- and ATM-deficiency failed to affect the recruitment of DDB2 and XPC to the damage site, and therefore did not influence the NER efficiency. These findings demonstrate a novel function of DDB2 and XPC in maintaining a vital cross-talk with checkpoint proteins, and thereby coordinating subsequent repair and checkpoint activation.     

Genotoxin-induced Rad9-Hus1-Rad1 (9-1-1) chromatin association is an early checkpoint signaling event

Rad17, Rad1, Hus1, and Rad9 are key participants in checkpoint signaling pathways that block cell cycle progression in response to genotoxins. Biochemical and molecular modeling data predict that Rad9, Hus1, and Rad1 form a heterotrimeric complex, dubbed 9-1-1, which is loaded onto chromatin by a complex of Rad17 and the four small replication factor C (RFC) subunits (Rad17-RFC) in response to DNA damage. It is unclear what checkpoint proteins or checkpoint signaling events regulate the association of the 9-1-1 complex with DNA. Here we show that genotoxin-induced chromatin binding of 9-1-1 does not require the Rad9-inducible phosphorylation site (Ser-272). Although we found that Rad9 undergoes an additional phosphatidylinositol 3-kinase-related kinase (PIKK)-dependent posttranslational modification, we also show that genotoxin-triggered 9-1-1 chromatin binding does not depend on the catalytic activity of the PIKKs ataxia telangiectasia-mutated (ATM), ataxia telangiectasia and Rad3-related (ATR), or DNA-PK. Additionally, 9-1-1 chromatin binding does not require DNA replication, suggesting that the complex can be loaded onto DNA in response to DNA structures other than stalled DNA replication forks. Collectively, these studies demonstrate that 9-1-1 chromatin binding is a proximal event in the checkpoint signaling cascade.     

Cdc2 phosphorylation of Crb2 is required for reestablishing cell cycle progression after the damage checkpoint.

DNA damage induces cell cycle arrest (called the damage checkpoint), during which cells carry out actions for repair. A fission yeast protein, Crb2/Rhp9, which resembles budding yeast Rad9p and human BRCA1, promotes checkpoint by activating Chk1 kinase, which restrains Cdc2 activation. We show here that phosphorylation of the T215 Cdc2 site of Crb2 is required for reentering the cell cycle after the damage-induced checkpoint arrest. If this site is nonphosphorylatable, irradiated cells remain arrested, though damage is repaired, and maintain the phosphorylated state of Chk1 kinase. The T215 site is in vitro phosphorylated by purified Cdc2 kinase. Phosphorylation of T215 occurs intensely in response to DNA damage at a late stage, suggesting an antagonistic role of Cdc2 phosphorylation toward checkpoint.     

Protein phosphatase 5 is necessary for ATR-mediated DNA repair

Several recent studies have shown that protein phosphatase 5 (PP5) participates in cell cycle arrest after DNA damage, but its roles in DNA repair have not yet been fully characterized. We investigated the roles of PP5 in the repair of ultraviolet (UV)- and neocarzinostatin (NCS)-induced DNA damage. The results of comet assays revealed different repair patterns in UV- and NCS-exposed U2OS-PS cells. PP5 is only essential for Rad3-related (ATR)-mediated DNA repair. Furthermore, the phosphorylation of 53BP1 and BRCA1, important mediators of DNA damage repair, and substrates of ATR and ATM decreased in U2OS-PS cells exposed to UV radiation. In contrast, the cell cycle arrest proteins p53, CHK1, and CHK2 were normally phosphorylated in U2OS and U2OS-PS cells exposed to UV radiation or treated with NCS. In view of these results, we suggest that PP5 plays a crucial role in ATR-mediated repair of UV-induced DNA damage.     

BRCA1 is required for hMLH1 stabilization following doxorubicin-induced DNA damage

Human DNA mismatch repair (MMR) is involved in the removal of DNA base mismatches that arise either during DNA replication or are caused by DNA damage. In this study, we show that the activation of the MMR component hMLH1 in response to doxorubicin (DOX) treatment requires the presence of BRCA1 and that this phenomenon is mediated by an ATM/ATR dependent phosphorylation of the hMLH1 Ser-406 residue. BRCA1 is an oncosuppressor protein with a central role in the DNA damage response and it is a critical component of the ATM/ATR mediated checkpoint signaling. Starting from a previous finding in which we demonstrated that hMLH1 is able to bind to BRCA1, in this study we asked whether BRCA1 might be the bridge for ATM/ATR dependent phosphorylation of the hMLH1 molecular partner. We found that: (i) the negative modulation of BRCA1 expression is able to produce a remarkable reversal of hMLH1 stabilization, (ii) BRCA1 is required for post-translational modification produced by DOX treatment on hMLH1 which is, in turn, attributed to the ATM/ATR activity, (iii) the serine 406 phosphorylatable residue is critical for hMLH1 activation by ATM/ATR via BRCA1. Taken together, our data lend support to the hypothesis suggesting an important role of this oncosuppressor as a scaffold or bridging protein in DNA-damage response signaling via downstream phosphorylation of the ATM/ATR substrate hMLH1.     

Activation of the S phase DNA damage checkpoint by mitomycin C

We have studied the rate of DNA synthesis, cell cycle distribution, formation of gamma-H2AX, and Rad51 nuclear foci and association of Rad51 with the nuclear matrix after treatment of HeLa cells with the interstrand crosslinking agent mitomycin C (MMC) in the presence of the kinase inhibitors caffeine and wortmannin. The results showed that MMC treatment arrested the cells in S-phase and induced the appearance of gamma-H2AX and Rad51 nuclear foci, accompanied with a sequestering of Rad51 to the nuclear matrix. These effects were abrogated by caffeine, which inhibits the Ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR) kinases. However, wortmannin at a concentration that inhibits ATM, but not ATR did not affect cell cycle progression, damage-induced phosphorylation of H2AX and Rad51 foci formation, and association with the nuclear matrix, suggesting that the S-phase arrest induced by MMC is ATR-dependent. These findings were confirmed by experiments with ATR-deficient and AT cells. They indicate that the DNA damage ATR-dependent S-phase checkpoint pathway may regulate the spatiotemporal organization of the process of repair of interstrand crosslinks.     

Differential roles of ATR and ATM in p53, Chk1, and histone H2AX phosphorylation in response to hyperoxia: ATR-dependent ATM activation

Elevated level of oxygen (hyperoxia) is widely used in critical care units and in respiratory insufficiencies. In addition, hyperoxia has been implicated in many diseases such as bronchopulmonary dysplasia or acute respiratory distress syndrome. Although hyperoxia is known to cause DNA base modifications and strand breaks, the DNA damage response has not been adequately investigated. We have investigated the effect of hyperoxia on DNA damage signaling and show that hyperoxia is a unique stress that activates the ataxia telangiectasia mutant (ATM)- and Rad3-related protein kinase (ATR)-dependent p53 phosphorylations (Ser6, -15, -37, and -392), phosphorylation of histone H2AX (Ser139), and phosphorylation of checkpoint kinase 1 (Chk1). In addition, we show that phosphorylation of p53 (Ser6) and histone H2AX (Ser139) depend on both ATM and ATR. We demonstrate that ATR activation precedes ATM activation in hyperoxia. Finally, we show that ATR is required for ATM activation in hyperoxia. Taken together, we report that ATR is the major DNA damage signal transducer in hyperoxia that activates ATM.     

Spatial organization of the mammalian genome surveillance machinery in response to DNA strand breaks

We show that DNA double-strand breaks (DSBs) induce complex subcompartmentalization of genome surveillance regulators. Chromatin marked by gamma-H2AX is occupied by ataxia telangiectasia-mutated (ATM) kinase, Mdc1, and 53BP1. In contrast, repair factors (Rad51, Rad52, BRCA2, and FANCD2), ATM and Rad-3-related (ATR) cascade (ATR, ATR interacting protein, and replication protein A), and the DNA clamp (Rad17 and -9) accumulate in subchromatin microcompartments delineated by single-stranded DNA (ssDNA). BRCA1 and the Mre11-Rad50-Nbs1 complex interact with both of these compartments. Importantly, some core DSB regulators do not form cytologically discernible foci. These are further subclassified to proteins that connect DSBs with the rest of the nucleus (Chk1 and -2), that assemble at unprocessed DSBs (DNA-PK/Ku70), and that exist on chromatin as preassembled complexes but become locally modified after DNA damage (Smc1/Smc3). Finally, checkpoint effectors such as p53 and Cdc25A do not accumulate at DSBs at all. We propose that subclassification of DSB regulators according to their residence sites provides a useful framework for understanding their involvement in diverse processes of genome surveillance.     

Characterization of Schizosaccharomyces pombe Hus1: a PCNA-related protein that associates with Rad1 and Rad9

Hus1 is one of six checkpoint Rad proteins required for all Schizosaccharomyces pombe DNA integrity checkpoints. MYC-tagged Hus1 reveals four discrete forms. The main form, Hus1-B, participates in a protein complex with Rad9 and Rad1, consistent with reports that Rad1-Hus1 immunoprecipitation is dependent on the rad9(+) locus. A small proportion of Hus1-B is intrinsically phosphorylated in undamaged cells and more becomes phosphorylated after irradiation. Hus1-B phosphorylation is not increased in cells blocked in early S phase with hydroxyurea unless exposure is prolonged. The Rad1-Rad9-Hus1-B complex is readily detectable, but upon cofractionation of soluble extracts, the majority of each protein is not present in this complex. Indirect immunofluorescence demonstrates that Hus1 is nuclear and that this localization depends on Rad17. We show that Rad17 defines a distinct protein complex in soluble extracts that is separate from Rad1, Rad9, and Hus1. However, two-hybrid interaction, in vitro association and in vivo overexpression experiments suggest a transient interaction between Rad1 and Rad17.     

BRCA1-dependent Chk1 phosphorylation triggers partial chromatin disassociation of phosphorylated Chk1 and facilitates S-phase cell cycle arrest

Chk1 phosphorylation by the PI3-like kinases ATR and ATM is critical for its activation and its role in prevention of premature mitotic entry in response to DNA damage or stalled replication. The breast and ovarian tumor suppressor, BRCA1, is among several checkpoint mediators that are required for Chk1 activation by ATM and ATR. Previously we showed that BRCA1 is necessary for Chk1 phosphorylation and activation following ionizing radiation. BRCA1 has been implicated in S-phase checkpoint control yet its mechanism of action is not well characterized. Here we report that BRCA1 is critical for Chk1 phosphorylation in response to inhibition of replication by either cisplatin or hydroxyurea. While Chk1 phosphorylation of S317 is fully dependent on BRCA1, additional proteins may mediate S345 phosphorylation at later time points. In addition, we show that a subset of phosphorylated Chk1 is released from the chromatin in a BRCA1-dependent manner which may lead to the phosphorylation of Chk1 substrate, Cdc25C, on S216 and to S-phase checkpoint activation. Inhibition of Chk1 kinase by UCN-01 or expression of Chk1 phosphorylation mutants in which the serine residues were substituted with alanine residues abrogates BRCA1-dependent cell cycle arrest in response replication inhibition. These data reveal that BRCA1 facilitates Chk1 phosphorylation and its partial chromatin dissociation following replication inhibition that is likely to be required for S-phase checkpoint signaling.     

Tid1/Rdh54 translocase is phosphorylated through a Mec1- and Rad53-dependent manner in the presence of DSB lesions in budding yeast

Saccharomyces cerevisiae cells with a single double-strand break (DSB) activate the ATR/Mec1-dependent checkpoint response as a consequence of extensive ssDNA accumulation. The recombination factor Tid1/Rdh54, a member of the Swi2-like family proteins, has an ATPase activity and may contribute to the remodelling of nucleosomes on DNA. Tid1 dislocates Rad51 recombinase from dsDNA, can unwind and supercoil DNA filaments, and has been implicated in checkpoint adaptation from a G2/M arrest induced by an unrepaired DSB.Here we show that both ATR/Mec1 and Chk2/Rad53 kinases are implicated in the phosphorylation of Tid1 in the presence of DNA damage, indicating that the protein is regulated during the DNA damage response. We show that Tid1 ATPase activity is dispensable for its phosphorylation and for its recruitment near a DSB, but it is required to switch off Rad53 activation and for checkpoint adaptation. Mec1 and Rad53 kinases, together with Rad51 recombinase, are also implicated in the hyper-phosphorylation of the ATPase defective Tid1-K318R variant and in the efficient binding of the protein to the DSB site.In summary, Tid1 is a novel target of the DNA damage checkpoint pathway that is also involved in checkpoint adaptation.