TopBP1 comes into focus.

Frattini et al. (2021) demonstrate that TopBP1 forms phase-separated nuclear condensates to promote activation of ATR in cells experiencing genomic stress.

Treslin collaborates with TopBP1 in triggering the initiation of DNA replication.

TopBP1 has important roles in both DNA replication and checkpoint regulation in vertebrates. We have identified a protein called Treslin that associates with TopBP1 in Xenopus egg extracts. Depletion of Treslin from egg extracts strongly inhibits chromosomal DNA replication. Binding of Treslin to chromatin in egg extracts occurs independently of TopBP1. However, loading of the initiator protein Cdc45 onto chromatin cannot take place in the absence of Treslin. Prior to the initiation of DNA replication, Treslin associates with TopBP1 in a Cdk2-dependent manner. Ablation of Treslin from human cells also strongly inhibits DNA replication. Taken together, these results indicate that Treslin and TopBP1 collaborate in the Cdk2-mediated loading of Cdc45 onto replication origins. Thus, Treslin regulates a pivotal step in the initiation of DNA replication in vertebrates.

Interaction of Chk1 with Treslin negatively regulates the initiation of chromosomal DNA replication.

Treslin helps to trigger the initiation of DNA replication by promoting integration of Cdc45 into the replicative helicase. Treslin is a key positive-regulatory target of cell-cycle control mechanisms; activation of Treslin by cyclin-dependent kinase is essential for the initiation of replication. Here we demonstrate that Treslin is also a critical locus for negative regulatory mechanisms that suppress initiation. We found that the checkpoint-regulatory kinase Chk1 associates specifically with a C-terminal domain of Treslin (designated TRCT). Mutations in the TRCT domain abolish binding of Chk1 to Treslin and thereby eliminate Chk1-catalyzed phosphorylation of Treslin. Significantly, abolition of the Treslin-Chk1 interaction results in elevated initiation of chromosomal DNA replication during an unperturbed cell cycle, which reveals a function for Chk1 during a normal S phase. This increase is due to enhanced loading of Cdc45 onto potential replication origins. These studies provide important insights into how vertebrate cells orchestrate proper initiation of replication.

Rif1 restrains the rate of replication origin firing in Xenopus laevis.

Metazoan genomes are duplicated by the coordinated activation of clusters of replication origins at different times during S phase, but the underlying mechanisms of this temporal program remain unclear during early development. Rif1, a key replication timing factor, inhibits origin firing by recruiting protein phosphatase 1 (PP1) to chromatin counteracting S phase kinases. We have previously described that Rif1 depletion accelerates early Xenopus laevis embryonic cell cycles. Here, we find that in the absence of Rif1, patterns of replication foci change along with the acceleration of replication cluster activation. However, initiations increase only moderately inside active clusters. Our numerical simulations suggest that the absence of Rif1 compresses the temporal program towards more homogeneity and increases the availability of limiting initiation factors. We experimentally demonstrate that Rif1 depletion increases the chromatin-binding of the S phase kinase Cdc7/Drf1, the firing factors Treslin, MTBP, Cdc45, RecQL4, and the phosphorylation of both Treslin and MTBP. We show that Rif1 globally, but not locally, restrains the replication program in early embryos, possibly by inhibiting or excluding replication factors from chromatin.

Ultrafast and hypersensitive phase imaging of propagating internodal current flows in myelinated axons and electromagnetic pulses in dielectrics.

Many ultrafast phenomena in biology and physics are fundamental to our scientific understanding but have not yet been visualized owing to the extreme speed and sensitivity requirements in imaging modalities. Two examples are the propagation of passive current flows through myelinated axons and electromagnetic pulses through dielectrics, which are both key to information processing in living organisms and electronic devices. Here, we demonstrate differentially enhanced compressed ultrafast photography (Diff-CUP) to directly visualize propagations of passive current flows at approximately 100 m/s along internodes, i.e., continuous myelinated axons between nodes of Ranvier, from Xenopus laevis sciatic nerves and of electromagnetic pulses at approximately 5 × 107 m/s through lithium niobate. The spatiotemporal dynamics of both propagation processes are consistent with the results from computational models, demonstrating that Diff-CUP can span these two extreme timescales while maintaining high phase sensitivity. With its ultrahigh speed (picosecond resolution), high sensitivity, and noninvasiveness, Diff-CUP provides a powerful tool for investigating ultrafast biological and physical phenomena.

Drf1-dependent kinase interacts with Claspin through a conserved protein motif.

The Dbf4/Drf1-dependent kinase (DDK) is required for the initiation of DNA replication in eukaryotes. Another protein, Claspin, mediates the activation of a cellular checkpoint response to stalled replication forks and is also a regulator of replication. In this study, we found that DDK phosphorylates Claspin in vitro and forms a nuclear complex containing Cdc7, Drf1, and Claspin in Xenopus egg extracts. In addition, purified Claspin and DDK are capable of a direct in vitro interaction. We identified a conserved binding site on Claspin required for its interaction with DDK. This site corresponds to the first of two sequence repeats in the Chk1-binding domain of Claspin. Furthermore, we have established that two amino acids in this motif, Asp(861) and Gln(866), are essential for the interaction between Claspin and DDK. We found that mutant forms of Claspin incapable of interacting with DDK are still able to associate with and activate Chk1 in response to DNA replication blockages. However, Claspin-depleted egg extracts that have been reconstituted with these mutants of Claspin undergo DNA replication more slowly. These findings suggest that the interaction of DDK with Claspin mediates a checkpoint-independent function of Claspin related to DNA replication.

Aven-dependent activation of ATM following DNA damage.

BACKGROUND: In response to DNA damage, cells undergo either cell-cycle arrest or apoptosis, depending on the extent of damage and the cell's capacity for DNA repair. Cell-cycle arrest induced by double-stranded DNA breaks depends on activation of the ataxia-telangiectasia (ATM) protein kinase, which phosphorylates cell-cycle effectors such as Chk2 and p53 to inhibit cell-cycle progression. ATM is recruited to double-stranded DNA breaks by a complex of sensor proteins, including Mre11/Rad50/Nbs1, resulting in autophosphorylation, monomerization, and activation of ATM kinase. RESULTS: In characterizing Aven protein, a previously reported apoptotic inhibitor, we have found that Aven can function as an ATM activator to inhibit G2/M progression. Aven bound to ATM and Aven overexpressed in cycling Xenopus egg extracts prevented mitotic entry and induced phosphorylation of ATM and its substrates. Immunodepletion of endogenous Aven allowed mitotic entry even in the presence of damaged DNA, and RNAi-mediated knockdown of Aven in human cells prevented autophosphorylation of ATM at an activating site (S1981) in response to DNA damage. Interestingly, Aven is also a substrate of the ATM kinase. Mutation of ATM-mediated phosphorylation sites on Aven reduced its ability to activate ATM, suggesting that Aven activation of ATM after DNA damage is enhanced by ATM-mediated Aven phosphorylation. CONCLUSIONS: These results identify Aven as a new ATM activator and describe a positive feedback loop operating between Aven and ATM. In aggregate, these findings place Aven, a known apoptotic inhibitor, as a critical transducer of the DNA-damage signal.

Repeated phosphopeptide motifs in Claspin mediate the regulated binding of Chk1.

In vertebrates, the checkpoint-regulatory kinase Chk1 mediates cell-cycle arrest in response to a block in DNA replication or to DNA damaged by ultraviolet radiation. The activation of Chk1 depends on both Claspin and the upstream regulatory kinase ATR. Claspin is a large acidic protein that becomes phosphorylated and binds to Chk1 in the presence of checkpoint-inducing DNA templates in Xenopus egg extracts. Here we identify, by means of deletion analysis, a region of Claspin of 57 amino acids that is both necessary and sufficient for binding to Xenopus Chk1. This Chk1-binding domain contains two highly conserved repeats of approximately ten amino acids. A serine residue in each repeat (serine 864 and serine 895) undergoes phosphorylation during a checkpoint response. A mutant of Claspin containing non-phosphorylatable amino acids at positions 864 and 895 cannot bind to Chk1 and is unable to mediate its activation. Our results indicate that two phosphopeptide motifs in Claspin are essential for checkpoint signalling.

Binding of the Treslin-MTBP Complex to Specific Regions of the Human Genome Promotes the Initiation of DNA Replication.

The processes that control where higher eukaryotic cells initiate DNA replication throughout the genome are not understood clearly. In metazoans, the Treslin-MTBP complex mediates critical final steps in formation of the activated replicative helicase prior to initiation of replication. Here, we map the genome-wide distribution of the MTBP subunit of this complex in human cells. Our results indicate that MTBP binds to at least 30,000 sites in the genome. A majority of these sites reside in regions of open chromatin that contain transcriptional-regulatory elements (e.g., promoters, enhancers, and super-enhancers), which are known to be preferred areas for initiation of replication. Furthermore, many binding sites encompass two genomic features: a nucleosome-free DNA sequence (e.g., G-quadruplex DNA or AP-1 motif) and a nucleosome bearing histone marks characteristic of open chromatin, such as H3K4me2. Taken together, these findings indicate that Treslin-MTBP associates coordinately with multiple genomic signals to promote initiation of replication.

Redox priming promotes Aurora A activation during mitosis.

Cell cycle-dependent redox changes can mediate transient covalent modifications of cysteine thiols to modulate the activities of regulatory kinases and phosphatases. Our previously reported finding that protein cysteine oxidation is increased during mitosis relative to other cell cycle phases suggests that redox modifications could play prominent roles in regulating mitotic processes. The Aurora family of kinases and their downstream targets are key components of the cellular machinery that ensures the proper execution of mitosis and the accurate segregation of chromosomes to daughter cells. In this study, x-ray crystal structures of the Aurora A kinase domain delineate redox-sensitive cysteine residues that, upon covalent modification, can allosterically regulate kinase activity and oligomerization state. We showed in both Xenopus laevis egg extracts and mammalian cells that a conserved cysteine residue within the Aurora A activation loop is crucial for Aurora A activation by autophosphorylation. We further showed that covalent disulfide adducts of this residue promote autophosphorylation of the Aurora A kinase domain. These findings reveal a potential mechanistic link between Aurora A activation and changes in the intracellular redox state during mitosis and provide insights into how novel small-molecule inhibitors may be developed to target specific subpopulations of Aurora A.

Absence of BLM leads to accumulation of chromosomal DNA breaks during both unperturbed and disrupted S phases.

Bloom's syndrome (BS), a disorder associated with genomic instability and cancer predisposition, results from defects in the Bloom's helicase (BLM) protein. In BS cells, chromosomal abnormalities such as sister chromatid exchanges occur at highly elevated rates. Using Xenopus egg extracts, we have studied Xenopus BLM (Xblm) during both unperturbed and disrupted DNA replication cycles. Xblm binds to replicating chromatin and becomes highly phosphorylated in the presence of DNA replication blocks. This phosphorylation depends on Xenopus ATR (Xatr) and Xenopus Rad17 (Xrad17), but not Claspin. Xblm and Xenopus topoisomerase IIIalpha (Xtop3alpha) interact in a regulated manner and associate with replicating chromatin interdependently. Immunodepletion of Xblm from egg extracts results in accumulation of chromosomal DNA breaks during both normal and perturbed DNA replication cycles. Disruption of the interaction between Xblm and Xtop3alpha has similar effects. The occurrence of DNA damage in the absence of Xblm, even without any exogenous insult to the DNA, may help to explain the genesis of chromosomal defects in BS cells.

RPA-coated single-stranded DNA promotes the ETAA1-dependent activation of ATR.

Besides TopBP1, ETAA1 has been identified more recently as an activator of the ATR-ATRIP complex in human cells. We have examined the role of ETAA1 in the Xenopus egg-extract system, which has been instrumental in the study of ATR-ATRIP. Depletion of ETAA1 from egg extracts did not noticeably reduce the activation of ATR-ATRIP in response to replication stress, as monitored by the ATR-dependent phosphorylation of Chk1 and RPA. Moreover, lack of ETAA1 did not appear to affect DNA replication during an unperturbed S-phase. Significantly, we find that TopBP1 is considerably more abundant than ETAA1 in egg extracts. We proceeded to show that ETAA1 could support the activation of ATR-ATRIP in response to replication stress if we increased its concentration in egg extracts by adding extra full-length recombinant ETAA1. Thus, TopBP1 appears to be the predominant activator of ATR-ATRIP in response to replication stress in this system. We have also explored the biochemical mechanism by which ETAA1 activates ATR-ATRIP. We have developed an in vitro system in which full-length recombinant ETAA1 supports activation of ATR-ATRIP in the presence of defined components. We find that binding of ETAA1 to RPA associated with single-stranded DNA (ssDNA) greatly stimulates its ability to activate ATR-ATRIP. Thus, RPA-coated ssDNA serves as a direct positive effector in the ETAA1-mediated activation of ATR-ATRIP.

Roles of replication fork-interacting and Chk1-activating domains from Claspin in a DNA replication checkpoint response.

Claspin is essential for the ATR-dependent activation of Chk1 in Xenopus egg extracts containing incompletely replicated DNA. Claspin associates with replication forks upon origin unwinding. We show that Claspin contains a replication fork-interacting domain (RFID, residues 265-605) that associates with Cdc45, DNA polymerase epsilon, replication protein A, and two replication factor C complexes on chromatin. The RFID contains two basic patches (BP1 and BP2) at amino acids 265-331 and 470-600, respectively. Deletion of either BP1 or BP2 compromises optimal binding of Claspin to chromatin. Absence of BP1 has no effect on the ability of Claspin to mediate activation of Chk1. By contrast, removal of BP2 causes a large reduction in the Chk1-activating potency of Claspin. We also find that Claspin contains a small Chk1-activating domain (residues 776-905) that does not bind stably to chromatin, but it is fully effective at high concentrations for mediating activation of Chk1. These results indicate that stable retention of Claspin on chromatin is not necessary for activation of Chk1. Instead, our findings suggest that only transient interaction of Claspin with replication forks potentiates its Chk1-activating function. Another implication of this work is that stable binding of Claspin to chromatin may play a role in other functions besides the activation of Chk1.

MTBP, the partner of Treslin, contains a novel DNA-binding domain that is essential for proper initiation of DNA replication.

Treslin, which is essential for incorporation of Cdc45 into the replicative helicase, possesses a partner called MTBP (Mdm2-binding protein). We have analyzed Xenopus and human MTBP to assess its role in DNA replication. Depletion of MTBP from Xenopus egg extracts, which also removes Treslin, abolishes DNA replication. These extracts be can rescued with recombinant Treslin-MTBP but not Treslin or MTBP alone. Thus, Treslin-MTBP is collectively necessary for replication. We have identified a C-terminal region of MTBP (the CTM domain) that binds efficiently to both double-stranded DNA and G-quadruplex (G4) DNA. This domain also exhibits homology with budding yeast Sld7. Mutants of MTBP without a functional CTM domain are defective for DNA replication in Xenopus egg extracts. These mutants display an impaired localization to chromatin and the inability to support loading of Cdc45. Human cells harboring such a mutant also display severe S-phase defects. Thus, the CTM domain of MTBP plays a critical role in localizing Treslin-MTBP to the replication apparatus for initiation.

CKS Proteins Promote Checkpoint Recovery by Stimulating Phosphorylation of Treslin.

CKS proteins are small (9-kDa) polypeptides that bind to a subset of the cyclin-dependent kinases. The two paralogs expressed in mammals, Cks1 and Cks2, share an overlapping function that is essential for early development. However, both proteins are frequently overexpressed in human malignancy. It has been shown that CKS protein overexpression overrides the replication stress checkpoint, promoting continued origin firing. This finding has led to the proposal that CKS protein-dependent checkpoint override allows premalignant cells to evade oncogene stress barriers, providing a causal link to oncogenesis. Here, we provide mechanistic insight into how overexpression of CKS proteins promotes override of the replication stress checkpoint. We show that CKS proteins greatly enhance the ability of Cdk2 to phosphorylate the key replication initiation protein treslin in vitro Furthermore, stimulation of treslin phosphorylation does not occur by the canonical adapter mechanism demonstrated for other substrates, as cyclin-dependent kinase (CDK) binding-defective mutants are capable of stimulating treslin phosphorylation. This effect is recapitulated in vivo, where silencing of Cks1 and Cks2 decreases treslin phosphorylation, and overexpression of wild-type or CDK binding-defective Cks2 prevents checkpoint-dependent dephosphorylation of treslin. Finally, we provide evidence that the role of CKS protein-dependent checkpoint override involves recovery from checkpoint-mediated arrest of DNA replication.

SUMOylation of the C-terminal domain of DNA topoisomerase IIα regulates the centromeric localization of Claspin.

DNA topoisomerase II (TopoII) regulates DNA topology by its strand passaging reaction, which is required for genome maintenance by resolving tangled genomic DNA. In addition, TopoII contributes to the structural integrity of mitotic chromosomes and to the activation of cell cycle checkpoints in mitosis. Post-translational modification of TopoII is one of the key mechanisms by which its broad functions are regulated during mitosis. SUMOylation of TopoII is conserved in eukaryotes and plays a critical role in chromosome segregation. Using Xenopus laevis egg extract, we demonstrated previously that TopoIIα is modified by SUMO on mitotic chromosomes and that its activity is modulated via SUMOylation of its lysine at 660. However, both biochemical and genetic analyses indicated that TopoII has multiple SUMOylation sites in addition to Lys660, and the functions of the other SUMOylation sites were not clearly determined. In this study, we identified the SUMOylation sites on the C-terminal domain (CTD) of TopoIIα. CTD SUMOylation did not affect TopoIIα activity, indicating that its function is distinct from that of Lys660 SUMOylation. We found that CTD SUMOylation promotes protein binding and that Claspin, a well-established cell cycle checkpoint mediator, is one of the SUMOylation-dependent binding proteins. Claspin harbors 2 SUMO-interacting motifs (SIMs), and its robust association to mitotic chromosomes requires both the SIMs and TopoIIα-CTD SUMOylation. Claspin localizes to the mitotic centromeres depending on mitotic SUMOylation, suggesting that TopoIIα-CTD SUMOylation regulates the centromeric localization of Claspin. Our findings provide a novel mechanistic insight regarding how TopoIIα-CTD SUMOylation contributes to mitotic centromere activity.

The Mre11-Rad50-Nbs1 (MRN) complex has a specific role in the activation of Chk1 in response to stalled replication forks.

The activation of Chk1 in response to stalled replication forks in Xenopus egg extracts involves a complex pathway containing ATM and Rad3-related (ATR), topoisomerase IIß-binding protein 1 (TopBP1), Rad17, the Rad9-Hus1-Rad1 (9-1-1) complex, and Claspin. We have observed that egg extracts lacking the Mre11-Rad50-Nbs1 (MRN) complex show greatly, although not completely, reduced activation of Chk1 in response to replication blockages. Depletion of both Rad17 and MRN leads to a further, essentially complete, reduction in the activation of Chk1. Thus, Rad17 and MRN act in at least a partially additive manner in promoting activation of Chk1. There was not an obvious change in the binding of RPA, ATR, Rad17, or the 9-1-1 complex to chromatin in aphidicolin (APH)-treated, MRN-depleted extracts. However, there was a substantial reduction in the binding of TopBP1. In structure-function studies of the MRN complex, we found that the Mre11 subunit is necessary for the APH-induced activation of Chk1. Moreover, a nuclease-deficient mutant of Mre11 cannot substitute for wild-type Mre11 in this process. These results indicate that the MRN complex, in particular the nuclease activity of Mre11, plays an important role in the activation of Chk1 in response to stalled replication forks. These studies reveal a previously unknown property of the MRN complex in genomic stability.

Role for Rif1 in the checkpoint response to damaged DNA in Xenopus egg extracts.

TopBP1 is critical for both DNA replication and checkpoint regulation in vertebrate cells. In this study, we have identified Rif1 as a binding partner of TopBP1 in Xenopus egg extracts. In addition, Rif1 also interacts with both ATM and the Mre11-Rad50-Nbs1 (MRN) complex, which are key regulators of checkpoint responses to double-stranded DNA breaks (DSBs). Depletion of Rif1 from egg extracts compromises the activation of Chk1 in response to DSBs but not stalled replication forks. Removal of Rif1 also has a significant impact on the chromatin-binding behavior of key checkpoint proteins. In particular, binding of TopBP1, ATR and the MRN complex to chromatin containing DSBs is reduced in the absence of Rif1. Rif1 interacts with chromatin in a highly regulated and dynamic manner. In unperturbed egg extracts, the association of Rif1 with chromatin depends upon formation of replication forks. In the presence of DSBs, there is elevated accumulation of Rif1 on chromatin under conditions where the activation of ATM is suppressed. Taken together, these results suggest that Rif1 plays a dynamic role in the early steps of a checkpoint response to DSBs in the egg-extract system by promoting the correct accumulation of key regulators on the DNA.

Direct regulation of Treslin by cyclin-dependent kinase is essential for the onset of DNA replication.

Treslin, a TopBP1-interacting protein, is necessary for deoxyribonucleic acid (DNA) replication in vertebrates. Association between Treslin and TopBP1 requires cyclin-dependent kinase (Cdk) activity in Xenopus laevis egg extracts. We investigated the mechanism and functional importance of Cdk for this interaction using both X. laevis egg extracts and human cells. We found that Treslin also associated with TopBP1 in a Cdk-regulated manner in human cells and that Treslin was phosphorylated within a conserved Cdk consensus target sequence (on S976 in X. laevis and S1000 in humans). Recombinant human Cdk2-cyclin E also phosphorylated this residue of Treslin in vitro very effectively. Moreover, a mutant of Treslin that cannot undergo phosphorylation on this site showed significantly diminished binding to TopBP1. Finally, human cells harboring this mutant were severely deficient in DNA replication. Collectively, these results indicate that Cdk-mediated phosphorylation of Treslin during S phase is necessary for both its effective association with TopBP1 and its ability to promote DNA replication in human cells.

Role for casein kinase 1 in the phosphorylation of Claspin on critical residues necessary for the activation of Chk1.

The mediator protein Claspin is critical for the activation of the checkpoint kinase Chk1 during checkpoint responses to stalled replication forks. This function involves the Chk1-activating domain (CKAD) of Claspin, which undergoes phosphorylation on multiple conserved sites. These phosphorylations promote binding of Chk1 to Claspin and ensuing activation of Chk1 by ATR. However, despite the importance of this regulatory process, the kinase responsible for these phosphorylations has remained unknown. By using a multifaceted approach, we have found that casein kinase 1 gamma 1 (CK1γ1) carries out this function. CK1γ1 phosphorylates the CKAD of Claspin efficiently in vitro, and depletion of CK1γ1 from human cells by small interfering RNA (siRNA) results in dramatically diminished phosphorylation of Claspin. Consequently, the siRNA-treated cells display impaired activation of Chk1 and resultant checkpoint defects. These results indicate that CK1γ1 is a novel component of checkpoint responses that controls the interaction of a key checkpoint effector kinase with its cognate mediator protein.