Biology subjects

Day, M.

Publications and source records attributed to Day, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Mechanistic basis for the spatiotemporal assembly of the Bloom Syndrome complex on ultrafine DNA bridges

The BTRR (BLM/TOP3A/RMI1/RMI2) complex resolves DNA replication and recombination intermediates, as well as mitotic DNA intertwinements, yet how its diverse activities are regulated remains unclear. Here, we identify key determinants of stable assembly of the BTRR complex on ultrafine DNA bridges (UFBs). We reveal that the RMI1-RMI2 subcomplex acts as a molecular reader of hydrophobic-rich short linear motifs (SLiMs) embedded within PICH and FANCM, thereby providing a conserved mechanism for engaging distinct Superfamily2 DNA translocases during chromosome segregation and DNA repair. During UFB resolution, SLiM binding stabilises the PICH-BTRR complex assembly and supports essential interactions among BLM, PICH, and DNA. Persistent PICH-BTRR complex association post-mitosis would compromise interphase DNA damage responses and is prevented by active PICH nuclear exclusion. Our findings define the molecular basis of UFB-binding complex assembly and highlight its spatiotemporal control required to specialise BTRR complex functions.

cell biology↗

bioRxiv2026

Human coronavirus nucleocapsid proteins have disparate innate immune evasion abilities

During infection, coronaviruses produce abundant double-stranded RNA (dsRNA) which can induce antiviral innate immune responses such as the interferon, 2-5-oligoadenylate synthetase (OAS)/RNase L, and protein kinase R (PKR) pathways. Coronaviruses must antagonize these dsRNA responses for successful replication. The SARS-CoV-2 nucleocapsid (N) protein plays a central role in evasion of dsRNA responses, interacting with dsRNA to block interferon-{beta} production and the activation of OAS/RNase L and PKR. Despite intensive study of SARS-CoV-2 N, our understanding of the innate immune evasion abilities of N proteins produced by other human coronaviruses (HCoVs) remains incomplete. Here, we provide a comprehensive comparison of HCoV N proteins expressed in a human lung cell line and show that their abilities to block dsRNA-induced innate immune responses differ. Highly pathogenic HCoV N proteins inhibited the production of interferon-{beta} mRNA and activation of OAS/RNase L, while common cold HCoV N proteins did not. While most HCoV N proteins inhibited PKR phosphorylation, HCoV-OC43 N did not, an observation that correlated with high levels of PKR activation observed during HCoV-OC43 infection. The ability of HCoV N proteins to antagonize PKR required colocalization with dsRNA, yet the overall decrease in PKR phosphorylation mediated by N was not due to sequestration of dsRNA away from PKR. Rather, N colocalized with dsRNA and PKR at dsRNA-induced foci (dRIFs) and inhibited PKR phosphorylation within dRIFs. Sarbecovirus N proteins also relocalized PKR to G3BP1 foci, suggesting that these N proteins can inhibit PKR using two distinct mechanisms. Collectively, our work reveals an unexpected level of functional and mechanistic diversity among the innate immune evasion abilities of human coronavirus N proteins. These findings challenge existing presumptions that observations made in one coronavirus can be extrapolated to others, because even conserved essential proteins such as N can exhibit considerable functional heterogeneity.

microbiology↗

bioRxiv2026

Evolutionary Recoding of Olfactory Sensory Neurons

Odorant receptors (Ors) are the interface between an animals nervous system and its olfactory environment. In insect genomes, the Ors are often the largest gene family, and their rapid duplications and deletions result in extensive copy-number differences between species. Because olfactory sensory neurons (OSNs) typically express only one Or (the so-called one-receptor one-neuron rule), this dynamism at the level of Or genes raises fundamental questions regarding their cellular regulation: How do new Or duplicates gain their own neuron-specific expression? Such Or-OSN changes are thought to provide a key evolutionary path for modifying olfactory perception and related behaviours, but the absence of examples of these transitions has prevented an understanding of how they occur. Using a highly duplicated Drosophila Or subfamily (the Or67a subfamily) as a model system, we discovered parallel instances of Or67a duplicates gaining new OSN expression and reconstructed their evolutionary histories. Functional work in D. suzukii, a species with two novel Or67a-expressing OSN populations, revealed that their Or67a expression has arisen in preexisting OSNs, which have lost their ancestral Ors. As a result, these neurons were recoded and acquired new olfactory identities, thereby demonstrating the diversification of an OSN repertoire without the invention of developmentally new OSN lineages.

evolutionary biology↗

bioRxiv2025

Microtubule end stabilisation by cooperative oligomers of Ska and Ndc80 complexes

During mitosis, properly aligned chromosomes stabilise microtubule ends with the help of kinetochores to ensure timely segregation of chromosomes. Microtubule-binding components of the human outer kinetochore, such as Ndc80 and Ska complexes, are present in multiple copies and together bind several microtubule ends, creating a highly multivalent binding interface. Whereas Ndc80:Ndc80 and Ndc80:microtubule binding is crucial for interface stability, Ndc80 alone in absence of Ska is unable to support stable kinetochore-attachments. Using cryoET, we demonstrate that oligomeric Ndc80:Ska assemblies stabilise microtubule ends against shortening by strengthening lateral contacts between tubulin protofilaments at microtubule plus-ends. We further identify a point mutation within the SKA1 microtubule-binding domain that does not affect microtubule-binding of individual Ska molecules, but does abolish Ska:Ska interactions. Finally, we report that oligomerisation of Ska, in a cooperative fashion together with the Ndc80, is necessary to maintain stable microtubule attachments both in vivo and in vitro.

biochemistry↗

bioRxiv2025

Assessing impacts of mitochondrial dysfunction on axonal microtubule bundles as potential mechanism in neurodegeneration

Mitochondrial dysfunction is an important cause for neurodegeneration, often associated with dyshomeostasis of reactive oxygen species, i.e. oxidative stress. However, apart from ATP production, mitochondria have many other functions the aberration of which may impact neurons in very different ways. Oxidative stress can cause the deterioration of axonal microtubule bundles, thus critically affecting the highways for life-sustaining transport and providing a potential path to neurodegeneration. We recently found that aberrant transport of mitochondria can have this effect by causing oxidative stress. We therefore asked which aberrations of mitochondrial physiology might impact microtubules, which of these might explain the observed consequences of aberrant mitochondrial transport, and whether mitochondria-induced microtubule phenotypes are always mediated by oxidative stress. Using one consistent Drosophila primary neuron system, we deleted 13 different mitochondrial factors known to be detrimental for neurons in vivo. Losses of five factors caused MT damage, all involving oxidative stress, hence supporting the path from mitochondria via oxidative stress to microtubule deterioration; we discuss Sod2 as potential candidate explaining effects of mitochondrial transport aberration. However, the loss of eight factors - seven of them important mitochondrial morphogenesis regulators - caused no microtubule damage, suggesting potential oxidative stress-independent pathways. Summary StatementAssessing mutant effects of 13 mitochondrial factors on axonal microtubule organisation to unravel potential mechanisms underpinning neurodegeneration

cell biology↗

bioRxiv2025

The human RIF1-Long isoform interacts with BRCA1 to promote recombinational fork repair under DNA replication stress

RIF1 is a multifunctional protein that regulates DNA replication and repair. RIF1-deficient cells are hypersensitive to DNA replication stress. Of the two alternatively spliced RIF1 isoforms, called RIF1-Short and RIF1-Long, the RIF1-Long isoform is more capable than RIF1-Short in supporting cell recovery from replication stress. Examining replication stress resistance mechanisms specific to RIF1-Long, we find that prolonged replication stress unexpectedly induces interaction of RIF1-Long with BRCA1. Mechanistically, a phosphorylated SPKF motif unique to the RIF1-Long isoform binds the tandem BRCT domain of BRCA1. BRCA1-RIF1-Long interaction is strongly down-regulated through dephosphorylation by RIF1-associated Protein Phosphatase 1. BRCA1-RIF1-Long interaction requires ATR signaling, and occurs predominantly during S phase. Loss of RIF1-Long impairs the formation of RAD51 foci, and reduces the efficiency of homology-mediated repair at broken replication forks. In summary, our investigation establishes RIF1-Long as a new functional binding partner of the BRCA1-BRCT domain, crucial to protect cells from extended DNA replication stress by enabling RAD51-dependent repair of broken replication forks.

molecular biology↗

bioRxiv2025

Different mechanisms link gain and loss of kinesin functions to axonal degeneration

Axons are the slender, often meter-long projections of neurons that form the biological cables wiring our bodies. Most of these delicate structures must survive for an organisms lifetime, meaning up to a century in humans. Long-term maintenance and sustained functionality of axons requires motor protein-driven transport distributing life-sustaining materials and organelles to places of need. It seems therefore plausible that loss of motor function would cause axon degeneration; however, also gain-of-function conditions were linked to disorders including motor neuron disease or spastic paraplegia. To understand this phenomenon, we studied [~]40 genetic manipulations of motor proteins, cargo linkers and regulators of reactive oxygen species in one standardised Drosophila primary neuron system. Using axonal microtubule bundle organisation as a relevant readout reflecting the state of axon integrity, we found that losses of Dynein heavy chain, KIF1A/Unc-104 and KIF5/Kinesin heavy chain (Khc) all cause bundle disintegration in the form of chaotically curled microtubules. Detailed functional studies of Khc and its adaptor proteins revealed that losses of mitochondrial or lysosomal transport cause ROS dyshomeostasis, which is a microtubule-curl-inducing condition in fly and mouse neurons alike. We find that hyper-activated Khc induces the same microtubule curling phenotype, not through ROS but likely more directly through enhanced mechanical forces. Studies with loss of Unc-104 or KIFBP and expression of an ALS-linked mutant form of the human Khc orthologue KIF5A suggest that loss or hyperactivation of different types of transport motors cause MT curling as a shared feature. We discuss a model which can explain our findings and their relevance for understanding motor-linked neurodegeneration.

neuroscience↗

bioRxiv2024

The mitotic CIP2A-TOPBP1 axis facilitates mitotic pathway choice between MiDAS and MMEJ

Mitotic DNA double-strand breaks (DSBs) accumulate in response to replication stress or BRCA1/2 deficiency posing a significant threat to genome stability as repair by non-homologous end-joining (NHEJ) and homologous recombination (HR) is inactivated in mitosis. Mitotic cells instead rely on the mechanisms of microhomology mediated end-joining (MMEJ) and mitotic DNA synthesis (MiDAS). However, how these pathways are regulated in mitosis remains unknown. Here we reveal the CIP2A-TOPBP1 complex facilitates recruitment of SMX complex components to mitotic chromatin marked by CIP2A, through a CDK1-dependent interaction between TOPBP1 BRCT 1/2 and SLX4 phospho-threonine1260, that drives MiDAS. Furthermore, CIP2A promotes the recruitment of Pol{theta} to facilitate mitotic MMEJ. This defines a mechanistic framework for mitotic DSB repair, where simultaneous disruption of MiDAS and MMEJ pathways underpins the synthetic lethality observed in BRCA1/2-deficient cells following CIP2A depletion. These findings provide critical insights into mitotic DNA repair and highlights therapeutic opportunities in HR deficient tumours.

molecular biology↗

bioRxiv2024

Centromere protection requires strict mitotic inactivation of the Bloom syndrome helicase complex

The BTRR (BLM/TOP3A/RMI1/RMI2) complex resolves various DNA replication and recombination intermediates to suppress genome instability. Alongside PICH, they target mitotic DNA intertwinements, known as ultrafine DNA bridges, facilitating chromosome segregation. Both BLM and PICH undergo transient mitotic hyper-phosphorylation, but the biological significance of this remains elusive. Here, we uncover that during early mitosis, multiple protein kinases act together to strictly constrain the BTRR complex for the protection of centromeres. Mechanistically, CDK1 destabilises the complex and suppresses its association with PICH at the chromatin underneath kinetochores. Inactivating the BLM and TOP3A interaction compromises the UFB-binding complex mitotic functions and can prevent centromere destruction. We further unravel how different clusters of mitotic phosphorylation on BLM affect its interaction with the TOP3A/RMI1/RMI2 subcomplex and illegitimate centromere unwinding. Furthermore, we identify specific phosphorylation sites targeted by the MPS1-PLK1 axis functioning to prevent BLM hyper-activation at centromeres. Notably, unleashing such activity after sister-chromatid cohesion loss facilitates separation of entangled chromosomes. Together, our study defines a centromere protection pathway in human mitotic cells, heavily reliant on a tight spatiotemporal control of the BTRR complex.

cell biology↗

bioRxiv2024

Mating proximity blinds threat perception.

Romantic engagement can bias sensory perception. This love blindness reflects a common behavioral principle across organisms: favoring pursuit of a coveted reward over potential risks. In the case of animal courtship, such sensory biases may support reproductive success but can also expose individuals to danger, such as predation. How do neural networks balance the trade-off between risk and reward? Here, we discover a dopamine-governed filter mechanism in male Drosophila that reduces threat perception as courtship progresses. We show that during early courtship stages, threat-activated visual neurons inhibit central courtship nodes via specific serotonergic neurons. This serotonergic inhibition prompts flies to abort courtship when they see imminent danger. However, as flies advance in the courtship process, the dopaminergic filter system reduces visual threat responses, shifting the balance from survival to mating. By recording neural activity from males as they approach mating, we demonstrate that progress in courtship is registered as dopaminergic activity levels ramping up. This dopamine signaling inhibits the visual threat detection pathway via Dop2R receptors, allowing male flies to focus on courtship when they are close to copulation. Thus, dopamine signaling biases sensory perception based on perceived goal proximity, in order to prioritize between competing behaviors.

neuroscience↗

bioRxiv2023

Inhibitory G proteins play multiple roles to polarize sensory hair cell morphogenesis

Inhibitory G alpha (GNAI or Gi) proteins are critical for the polarized morphogenesis of sensory hair cells and for hearing. The extent and nature of their actual contributions remains unclear, however, as previous studies did not investigate all GNAI proteins and included non-physiological approaches. Pertussis toxin can downregulate functionally redundant GNAI1, GNAI2, GNAI3 and GNAO proteins, but may also induce unrelated defects. Here we directly and systematically determine the role(s) of each individual GNAI protein in mouse auditory hair cells. GNAI2 and GNAI3 are similarly polarized at the hair cell apex with their binding partner GPSM2, whereas GNAI1 and GNAO are not detected. In Gnai3 mutants, GNAI2 progressively fails to fully occupy the subcellular compartments where GNAI3 is missing. In contrast, GNAI3 can fully compensate for the loss of GNAI2 and is essential for hair bundle morphogenesis and auditory function. Simultaneous inactivation of Gnai2 and Gnai3 recapitulates for the first time two distinct types of defects only observed so far with pertussis toxin: 1) a delay or failure of the basal body to migrate off-center in prospective hair cells, and 2) a reversal in the orientation of some hair cell types. We conclude that GNAI proteins are critical for hair cells to break planar symmetry and to orient properly before GNAI2/3 regulate hair bundle morphogenesis with GPSM2.

developmental biology↗

bioRxiv2023

TopBP1 utilises a bipartite GINS binding mode to activate the replicative helicase

Activation of the replicative Mcm2-7 helicase by loading GINS and Cdc45 is crucial for replication origin firing, and as such for faithful genetic inheritance. Our biochemical and structural studies demonstrate that the helicase activator GINS interacts with TopBP1 through two separate binding surfaces, the first involving a stretch of highly conserved amino acids in the TopBP1-GINI region, the second a surface on TopBP1-BRCT4. The two surfaces bind to opposite ends of the A domain of the GINS subunit Psf1. Mutation analysis reveals that either surface is individually able to support TopBP1-GINS interaction, albeit with reduced affinity. Consistently, either surface is sufficient for replication origin firing in Xenopus egg extracts and becomes essential in the absence of the other. The TopBP1-GINS interaction appears sterically incompatible with simultaneous binding of DNA polymerase epsilon (Pol{varepsilon}) to GINS when bound to Mcm2-7-Cdc45, although TopBP1-BRCT4 and the Pol{varepsilon} subunit PolE2 show only partial competitivity in binding to Psf1. Our TopBP1-GINS model improves the understanding of the recently characterised metazoan pre-loading complex. It further predicts the coordination of three molecular origin firing processes, DNA polymerase epsilon arrival, TopBP1 ejection and GINS integration into Mcm2-7-Cdc45.

molecular biology↗

bioRxiv2023

State-dependent GABAergic regulation of striatal spiny projection neuron excitability

Synaptic transmission mediated by GABAA receptors (GABAARs) in adult, principal striatal spiny projection neurons (SPNs) can suppress ongoing spiking, but its effect on synaptic integration at sub-threshold membrane potentials is less well characterized, particularly those near the resting down-state. To fill this gap, a combination of molecular, optogenetic, optical and electrophysiological approaches were used to study SPNs in mouse ex vivo brain slices, and computational tools were used to model somatodendritic synaptic integration. Activation of GABAARs, either by uncaging of GABA or by optogenetic stimulation of GABAergic synapses, evoked currents with a reversal potential near -60 mV in perforated patch recordings from both juvenile and adult SPNs. Molecular profiling of SPNs suggested that this relatively positive reversal potential was not attributable to NKCC1 expression, but rather to a dynamic equilibrium between KCC2 and Cl-/HCO3-cotransporters. Regardless, from down-state potentials, optogenetic activation of dendritic GABAergic synapses depolarized SPNs. This GABAAR-mediated depolarization summed with trailing ionotropic glutamate receptor (iGluR) stimulation, promoting dendritic spikes and increasing somatic depolarization. Simulations revealed that a diffuse dendritic GABAergic input to SPNs effectively enhanced the response to coincident glutamatergic input. Taken together, our results demonstrate that GABAARs can work in concert with iGluRs to excite adult SPNs when they are in the resting down-state, suggesting that their inhibitory role is limited to brief periods near spike threshold. This state-dependence calls for a reformulation of the role intrastriatal GABAergic circuits.

neuroscience↗

bioRxiv2023

Signalling pathway crosstalk stimulated by L-proline drives differentiation of mouse embryonic stem cells to primitive-ectoderm-like cells

The amino acid L-proline exhibits novel growth factor-like properties during development - from improving blastocyst development to driving neurogenesis in vitro. Addition of 400 M L-proline to self-renewal medium drives mouse embryonic stem cells (ESCs) to a transcriptionally distinct pluripotent cell population - early primitive ectoderm-like (EPL) cells - which lies between the naive and primed states. EPL cells retain expression of pluripotency genes, upregulate primitive ectoderm markers, undergo a morphological change and have increased cell number. These changes are facilitated by a complex signalling network hinging on the Mapk, Fgfr, Pi3k and mTor pathways. We use a factorial experimental design coupled with linear modelling and Bayesian regularised neural networks to understand which signalling pathways are involved in the transition between ESCs and EPL cells, and how they underpin changes in morphology, cell number, apoptosis, proliferation and gene expression. This approach allows for consideration of where pathways work antagonistically or synergistically. Modelling showed that most properties were affected by more than one inhibitor, and each inhibitor blocked specific aspects of differentiation. These mechanisms underpin both progression of stem cells across the in vitro pluripotency continuum and serve as a model for pre-, peri- and post-implantation embryogenesis. Summary StatementL-proline acts as growth factor to modulate phosphorylation of the Mapk, Pi3k, Fgf and mTor signalling pathways to drive embryonic stem cells to primitive ectoderm-like cells.

developmental biology↗

bioRxiv2022

Metabolomics and transcriptomics reveal the response mechanisms of Mikania micrantha to Puccinia spegazzinii infection

Mikania micrantha is one of the 100 worst invasive species globally and can cause significant negative impacts to agricultural and forestry economics, particularly in Asia and the Pacific region. The rust Puccinia spegazzinii has been used successfully as a biological control agent in several countries to help manage M. micrantha. However, the response mechanisms of M. micrantha to P. spegazzinii infection have never been studied. To investigate the response of M. micrantha to infection by P. spegazzinii, an integrated analysis of metabolomics and transcriptomics was performed. The levels of 101 metabolites, including organic acids, amino acids and secondary metabolites in M. micrantha infected with P. spegazzinii, were significantly different compared to those in plants that were not infected. The relative amounts of L-leucine, L-ioleucine, L-tryptophan, histidine, L-phenylalanine and L-citrulline were increased under P. spegazzinii infection. These may convert to intermediates within the TCA cycle, participating in energy biosynthesis. In addition, phytoalexins, such as maackiain, nobiletin, vasicin, arachidonic acid and JA-Ile, were synthesized and accumulated in M. micrantha in response to P. spegazzinii infection. A total of 4,978 differentially expressed genes were identified in M. micrantha infected by P. spegazzinii. Many key genes of the Mitogen-activated protein kinase (MAPK) signalling pathway, showed significantly higher expression under P. spegazzinii infection. These results not only help us to understand the response of metabolites and gene expression in M. micrantha following infection by P. spegazzinii, but also help us understand the potential for the continuous biological control of M. micrantha using P. spegazzinii.

pathology↗

bioRxiv2022

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.

biochemistry↗

bioRxiv2021

CK2 phosphorylation of human papillomavirus 16 E2 on serine 23 promotes interaction with TopBP1 and is critical for E2 plasmid retention function

During the human papillomavirus 16 (HPV16) life cycle, the E2 protein interacts with host factors to regulate viral transcription, replication and genome segregation/retention. Our understanding of host partner proteins and their roles in E2 functions remains incomplete. Here, we demonstrate that CK2 phosphorylation of E2 on serine 23 promotes interaction with TopBP1 in vitro and in vivo, and that E2 is phosphorylated on this residue during the HPV16 life cycle. We investigated the consequences of mutating serine 23 on E2 functions. E2-S23A activates and represses transcription identically to E2-WT (wild-type), and E2-S23A is as efficient as E2-WT in transient replication assays. However, E2-S23A has compromised interaction with mitotic chromatin when compared with E2-WT. In E2-WT cells, both E2 and TopBP1 levels increase during mitosis when compared with vector control cells. In E2-S23A cells, neither E2 nor TopBP1 levels increase during mitosis. We next tested whether this difference in E2-S23A levels during mitosis disrupts E2 plasmid retention function. We developed a novel plasmid retention assay and demonstrate that E2-S23A is deficient in plasmid retention when compared with E2-WT. siRNA targeted knockdown of TopBP1 abrogates E2-WT plasmid retention function. Introduction of the S23A mutation into the HPV16 genome resulted in delayed immortalization of human foreskin keratinocytes (HFK) and higher episomal viral genome copy number in resulting established HFK. Overall, our results demonstrate that CK2 phosphorylation of E2 on serine 23 promotes interaction with TopBP1, which is critical for E2 plasmid retention function and in HPV16 immortalization of keratinocytes. ImportanceHuman papillomaviruses are causative agents in around 5% of all cancers, with no specific anti-viral therapeutics available for treating infections or resultant cancers. In this report, we demonstrate that phosphorylation of HPV16 E2 by CK2 promotes formation of a complex formation with the cellular protein TopBP1 in vitro and in vivo. This complex results in stabilization of E2 during mitosis and mediates plasmid retention by E2. This function promotes the partitioning of viral genomes into the nuclei of daughter cells following mitosis. We demonstrate that CK2 phosphorylates E2 on serine 23 in vivo, and that CK2 inhibitors disrupt the E2-TopBP1 complex. Mutation of E2 serine 23 to alanine disrupts the HPV16 life cycle, demonstrating a critical function for this residue. Together, our results suggest that CK2 inhibitors may disrupt the E2-TopBP1 dependent HPV16 life cycle and potentially kill HPV16 positive cancers, which lays a molecular foundation to develop novel therapeutic approaches for combating HPV16 disease.

molecular biology↗

bioRxiv2020

Structural basis for recruitment of the CHK1 DNA damage kinase by the CLASPIN scaffold protein

CHK1 is a protein kinase that functions downstream of activated ATR to phosphorylate multiple targets as part of intra-S and G2/M DNA damage checkpoints. Its role in allowing cells to survive replicative stress has made it an important target for anti-cancer drug discovery. Activation of CHK1 by ATR depends on their mutual interaction with CLASPIN - a natively unstructured protein that interacts with CHK1 through a cluster of phosphorylation sites in its C-terminal half. We have now determined the crystal structure of the kinase domain of CHK1 bound to a high-affinity motif from CLASPIN. Our data show that CLASPIN engages a conserved site on CHK1 adjacent to the substrate-binding cleft, involved in phosphate sensing in other kinases. The CLASPIN motif is not phosphorylated by CHK1, nor does it affect phosphorylation of a CDC25 substrate peptide, suggesting that it functions purely as a scaffold for CHK1 activation by ATR.

biochemistry↗