Tipin-Replication Protein A Interaction Mediates Chk1 Phosphorylation by ATR in Response to Genotoxic Stress

Mammalian Timeless is a multifunctional protein that performs essential roles in the circadian clock, chromosome cohesion, DNA replication fork protection, and DNA replication/DNA damage checkpoint pathways. The human Timeless exists in a tight complex with a smaller protein called Tipin (Timeless-interacting protein). Here we investigated the mechanism by which the Timeless-Tipin complex functions as a mediator in the ATR-Chk1 DNA damage checkpoint pathway. We find that the Timeless-Tipin complex specifically mediates Chk1 phosphorylation by ATR in response to DNA damage and replication stress through interaction of Tipin with the 34-kDa subunit of replication protein A (RPA). The Tipin-RPA interaction stabilizes Timeless-Tipin and Tipin-Claspin complexes on RPA-coated ssDNA and in doing so promotes Claspin-mediated phosphorylation of Chk1 by ATR. Our results therefore indicate that RPA-covered ssDNA not only supports recruitment and activation of ATR but also, through Tipin and Claspin, it plays an important role in the action of ATR on its critical downstream target Chk1.     

miR-107 Activates ATR/Chk1 Pathway and Suppress Cervical Cancer Invasion by Targeting MCL1

MicroRNAs (miRNAs) are a class of single-stranded, non-coding RNAs of about 22 nucleotides in length. Increasing evidence implicates miRNAs may function as oncogenes or tumor suppressors. Here we showed that miR-107 directly targeted MCL1 and activated ATR/Chk1 pathway to inhibit proliferation, migration and invasiveness of cervical cancer cells. Moreover, we found that MCL1 was frequently up-regulated in cervical cancer, and knockdown of MCL1 markedly inhibited cancer cell proliferation, migration and invasion, whereas ectopic expression of MCL1 significantly enhances these properties. The restoration of MCL1 expression can counteract the effect of miR-107 on the cancer cells. Together, miR-107 is a new regulator of MCL1, and both miR-107 and MCL1 play important roles in the pathogenesis of cervical cancer. We have therefore identified a mechanism for ATR/Chk1 pathway which involves an increase in miR-107 leading to a decrease in MCL1. Correspondingly, our results revealed that miR-107 affected ATR/Chk1 signalling and gene expression, and implicated miR-107 as a therapeutic target in human cervical cancer. We also demonstrated that taxol attenuated migration and invasion in cervical cancer cells by activating the miR-107, in which miR-107 play an important role in regulating the expression of MCL1. Elucidation of this discovered MCL1 was directly regulated by miR-107 will greatly enhance our understanding of the mechanisms responsible for cervical cancer and will provide an additional arm for the development of anticancer therapies.     

Replication of single-stranded porcine circovirus DNA by DNA polymerases α and δ

Porcine circovirus is the only mammalian DNA virus so far known to contain a single-stranded circular genome (Tischer et al. (1982) Nature 295, 64–66). Replication of its small viral DNA (1.76 kb) appears to be dependent on cellular enzymes expressed during S-phase of the cell cycle (Tischer et al. (1987) Arch. Virol. 96, 39–57). In this paper we have exploited the porcine circovirus genome to probe for in vitro initiation and elongation of DNA replication by different preparations of calf thymus DNA polymerase α and δ as well as by a partially purified preparation from pig thymus. The results indicated that three different purification fractions of calf thymus DNA polymerase α and one from pig thymus initiate DNA synthesis at several sites on the porcine circovirus DNA. It appears that the sites at which DNA primase synthesizes primers are not entirely random. Subsequent DNA elongation by a highly purified DNA polymerase α holoenzyme which had been isolated by the criterion of replicating single-stranded M13 DNA (Ottiger et al. (1987) Nucleic Acids Res. 15, 4789–4807) is very efficient. Complete conversion to the double-stranded form is obtained in less than 1 min. When the DNA synthesis by DNA polymerase α is blocked with the DNA polymerase α specific monoclonal antibody SJK 132-20 after initiation by DNA primase, DNA polymerase δ can efficiently replicate from the primers. This in vitro DNA replication system may be used in analogy to the bacteriophage systems in E. coli to study initiation and elongation of DNA replication.     

Chk1 Inhibits E2F6 Repressor Function in Response to Replication Stress to Maintain Cell-Cycle Transcription

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Reversal of DDK-Mediated MCM Phosphorylation by Rif1-PP1 Regulates Replication Initiation and Replisome Stability Independently of ATR/Chk1

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Inhibition of human Chk1 causes increased initiation of DNA replication, phosphorylation of ATR targets, and DNA breakage

Human checkpoint kinase 1 (Chk1) is an essential kinase required to preserve genome stability. Here, we show that Chk1 inhibition by two distinct drugs, UCN-01 and CEP-3891, or by Chk1 small interfering RNA (siRNA) leads to phosphorylation of ATR targets. Chk1-inhibition triggered rapid, pan-nuclear phosphorylation of histone H2AX, p53, Smc1, replication protein A, and Chk1 itself in human S-phase cells. These phosphorylations were inhibited by ATR siRNA and caffeine, but they occurred independently of ATM. Chk1 inhibition also caused an increased initiation of DNA replication, which was accompanied by increased amounts of nonextractable RPA protein, formation of single-stranded DNA, and induction of DNA strand breaks. Moreover, these responses were prevented by siRNA-mediated downregulation of Cdk2 or the replication initiation protein Cdc45, or by addition of the CDK inhibitor roscovitine. We propose that Chk1 is required during normal S phase to avoid aberrantly increased initiation of DNA replication, thereby protecting against DNA breakage. These results may help explain why Chk1 is an essential kinase and should be taken into account when drugs to inhibit this kinase are considered for use in cancer treatment.     

Phosphorylation of Minichromosome Maintenance 3 (MCM3) by Checkpoint Kinase 1 (Chk1) Negatively Regulates DNA Replication and Checkpoint Activation

Mechanisms controlling DNA replication and replication checkpoint are critical for the maintenance of genome stability and the prevention or treatment of human cancers. Checkpoint kinase 1 (Chk1) is a key effector protein kinase that regulates the DNA damage response and replication checkpoint. The heterohexameric minichromosome maintenance (MCM) complex is the core component of mammalian DNA helicase and has been implicated in replication checkpoint activation. Here we report that Chk1 phosphorylates the MCM3 subunit of the MCM complex at Ser-205 under normal growth conditions. Mutating the Ser-205 of MCM3 to Ala increased the length of DNA replication track and shortened the S phase duration, indicating that Ser-205 phosphorylation negatively controls normal DNA replication. Upon replicative stress treatment, the inhibitory phosphorylation of MCM3 at Ser-205 was reduced, and this reduction was accompanied with the generation of single strand DNA, the key platform for ataxia telangiectasia mutated and Rad3-related (ATR) activation. As a result, the replication checkpoint is activated. Together, these data provide significant insights into the regulation of both normal DNA replication and replication checkpoint activation through the novel phosphorylation of MCM3 by Chk1.     

DNA Mismatch Repair and Chk1-Dependent Centrosome Amplification in Response to DNA Alkylation Damage

Centrosome amplification is frequently observed in tumour cells exposed to genotoxic stress, however the underlying mechanisms and biological consequences are poorly understood. Here, we show that the anti-metabolite and alkylating agent 6-thioguanine (6-TG) induces centrosome amplification resulting in the formation of multi-polar spindles when damaged cells subsequently enter mitosis. These aberrant, multi-polar mitoses are frequently resolved by asymmetric cell divisions causing unequal segregation of genetic material and cell death in one or both daughter products. We show that this phenomenon is associated with transient cell cycle delay in S- and G2-phase and is dependent on DNA mismatch repair (DNA MMR) proficiency and Chk1 protein kinase activity. Although Chk1-deficient cells do not exhibit cell cycle delay, centrosome amplification, or multi-polar spindle formation, continued cell cycle progression in the presence of 6-TG eventually results in increased levels of mitotic catastrophe, most probably due to mitosis with incompletely replicated DNA. Taken together, these results reveal novel mechanisms of cell killing by 6-TG and underscore the importance of interactions between cell cycle checkpoints and DNA MMR in determining the fate of cells bearing DNA damage.     

α-, β-, and γ-Tubulin Polymerization in Response to DNA Damage

Microtubules provide structural support for a cell and play key roles in cell motility, mitosis, and meiosis. They are also the targets of several anticancer agents, indicating their importance in maintaining cell viability. We have investigated the possibility that alterations in microtubule structure and tubulin polymerization may be part of the cellular response to DNA damage. In this report, we find that γ-radiation stimulates the production and polymerization of α-, β-, and γ- tubulin in hematopoeitic cell lines (Ramos, DP16), leading to visible changes in microtubule structures. We have found that this microtubule reorganization can be prevented by caffeine, a drug that concomitantly inhibits DNA damage-induced cell cycle arrest and apoptosis. Our results support the idea that microtubule polymerization is an important facet of the mammalian response to DNA damage.     

Structure of the human RAD17–RFC clamp loader and 9–1–1 checkpoint clamp bound to a dsDNA–ssDNA junction

The RAD9–RAD1–HUS1 (9–1–1) clamp forms one half of the DNA damage checkpoint system that signals the presence of substantial regions of single-stranded DNA arising from replication fork collapse or resection of DNA double strand breaks. Loaded at the 5′-recessed end of a dsDNA–ssDNA junction by the RAD17–RFC clamp loader complex, the phosphorylated C-terminal tail of the RAD9 subunit of 9–1–1 engages with the mediator scaffold TOPBP1 which in turn activates the ATR kinase, localised through the interaction of its constitutive partner ATRIP with RPA-coated ssDNA. Using cryogenic electron microscopy (cryoEM) we have determined the structure of a complex of the human RAD17–RFC clamp loader bound to human 9–1–1, engaged with a dsDNA–ssDNA junction. The structure answers the key questions of how RAD17 confers specificity for 9–1–1 over PCNA, and how the clamp loader specifically recognises the recessed 5′ DNA end and fixes the orientation of 9–1–1 on the ssDNA.     

ATR and a Chk1-Aurora B pathway coordinate postmitotic genome surveillance with cytokinetic abscission

Aurora B regulates cytokinesis timing and plays a central role in the abscission checkpoint. Cellular events monitored by this checkpoint are beginning to be elucidated, yet signaling pathways upstream of Aurora B in this context remain poorly understood. Here we reveal a new connection between postmitotic genome surveillance and cytokinetic abscission. Underreplicated DNA lesions are known to be transmitted through mitosis and protected in newly formed nuclei by recruitment of 53BP1 and other proteins until repair takes place. We find that this genome surveillance initiates before completion of cytokinesis. Elevating replication stress increases this postmitotic process and delays cytokinetic abscission by keeping the abscission checkpoint active. We further find that ATR activity in midbody-stage cells links postmitotic genome surveillance to abscission timing and that Chk1 integrates this and other signals upstream of Aurora B to regulate when the final step in the physical separation of daughter cells occurs.     

The Caenorhabditis elegans Werner Syndrome Protein Functions Upstream of ATR and ATM in Response to DNA Replication Inhibition and Double-Strand DNA Breaks

WRN-1 is the Caenorhabditis elegans homolog of the human Werner syndrome protein, a RecQ helicase, mutations of which are associated with premature aging and increased genome instability. Relatively little is known as to how WRN-1 functions in DNA repair and DNA damage signaling. Here, we take advantage of the genetic and cytological approaches in C. elegans to dissect the epistatic relationship of WRN-1 in various DNA damage checkpoint pathways. We found that WRN-1 is required for CHK1 phosphorylation induced by DNA replication inhibition, but not by UV radiation. Furthermore, WRN-1 influences the RPA-1 focus formation, suggesting that WRN-1 functions in the same step or upstream of RPA-1 in the DNA replication checkpoint pathway. In response to ionizing radiation, RPA-1 focus formation and nuclear localization of ATM depend on WRN-1 and MRE-11. We conclude that C. elegans WRN-1 participates in the initial stages of checkpoint activation induced by DNA replication inhibition and ionizing radiation. These functions of WRN-1 in upstream DNA damage signaling are likely to be conserved, but might be cryptic in human systems due to functional redundancy.     

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.     

Replication-dependent cytotoxicity and Spartan-mediated repair of trapped PARP1–DNA complexes

The antitumor activity of poly(ADP-ribose) polymerase inhibitors (PARPis) has been ascribed to PARP trapping, which consists in tight DNA–protein complexes. Here we demonstrate that the cytotoxicity of talazoparib and olaparib results from DNA replication. To elucidate the repair of PARP1–DNA complexes associated with replication in human TK6 and chicken DT40 lymphoblastoid cells, we explored the role of Spartan (SPRTN), a metalloprotease associated with DNA replication, which removes proteins forming DPCs. We find that SPRTN-deficient cells are hypersensitive to talazoparib and olaparib, but not to veliparib, a weak PARP trapper. SPRTN-deficient cells exhibit delayed clearance of trapped PARP1 and increased replication fork stalling upon talazoparib and olaparib treatment. We also show that SPRTN interacts with PARP1 and forms nuclear foci that colocalize with the replicative cell division cycle 45 protein (CDC45) in response to talazoparib. Additionally, SPRTN is deubiquitinated and epistatic with translesion synthesis (TLS) in response to talazoparib. Our results demonstrate that SPRTN is recruited to trapped PARP1 in S-phase to assist in the excision and replication bypass of PARP1–DNA complexes.     

Multiple hPOT1–TPP1 cooperatively unfold contiguous telomeric G-quadruplexes proceeding from 3′ toward 5′, a feature due to a 3′-end binding preference and to structuring of telomeric DNA

Telomeres are DNA repeated sequences that associate with shelterin proteins and protect the ends of eukaryotic chromosomes. Human telomeres are composed of 5′TTAGGG repeats and ends with a 3′ single-stranded tail, called G-overhang, that can be specifically bound by the shelterin protein hPOT1 (human Protection of Telomeres 1). In vitro studies have shown that the telomeric G-strand can fold into stable contiguous G-quadruplexes (G4). In the present study we investigated how hPOT1, in complex with its shelterin partner TPP1, binds to telomeric sequences structured into contiguous G4 in potassium solutions. We observed that binding of multiple hPOT1–TPP1 preferentially proceeds from 3′ toward 5′. We explain this directionality in terms of two factors: (i) the preference of hPOT1–TPP1 for the binding site situated at the 3′ end of a telomeric sequence and (ii) the cooperative binding displayed by hPOT1–TPP1 in potassium. By comparing binding in K⁺ and in Li⁺, we demonstrate that this cooperative behaviour does not stem from protein-protein interactions, but from structuring of the telomeric DNA substrate into contiguous G4 in potassium. Our study suggests that POT1-TPP1, in physiological conditions, might preferentially cover the telomeric G-overhang starting from the 3′-end and proceeding toward 5′.