Spatial organization and functions of Chk1 activation by TopBP1 biomolecular condensates.

Assembly of TopBP1 biomolecular condensates triggers activation of the ataxia telangiectasia-mutated and Rad3-related (ATR)/Chk1 signaling pathway, which coordinates cell responses to impaired DNA replication. Here, we used optogenetics and reverse genetics to investigate the role of sequence-specific motifs in the formation and functions of TopBP1 condensates. We propose that BACH1/FANCJ is involved in the partitioning of BRCA1 within TopBP1 compartments. We show that Chk1 is activated at the interface of TopBP1 condensates and provide evidence that these structures arise at sites of DNA damage and in primary human fibroblasts. Chk1 phosphorylation depends on the integrity of a conserved arginine motif within TopBP1's ATR activation domain (AAD). Its mutation uncouples Chk1 activation from TopBP1 condensation, revealing that optogenetically induced Chk1 phosphorylation triggers cell cycle checkpoints and slows down replication forks in the absence of DNA damage. Together with previous work, these data suggest that the intrinsically disordered AAD encodes distinct molecular steps in the ATR/Chk1 pathway.

The nucleolar protein GNL3 prevents resection of stalled replication forks.

Faithful DNA replication requires specific proteins that protect replication forks and so prevent the formation of DNA lesions that may damage the genome. Identification of new proteins involved in this process is essential to understand how DNA lesions accumulate in cancer cells and how they tolerate them. Here, we show that human GNL3/nucleostemin, a GTP-binding protein localized mostly in the nucleolus and highly expressed in cancer cells, prevents nuclease-dependent resection of nascent DNA in response to replication stress. We demonstrate that inhibiting origin firing reduces resection. This suggests that the heightened replication origin activation observed upon GNL3 depletion largely drives the observed DNA resection probably due to the exhaustion of the available RPA pool. We show that GNL3 and DNA replication initiation factor ORC2 interact in the nucleolus and that the concentration of GNL3 in the nucleolus is required to limit DNA resection. We propose that the control of origin firing by GNL3 through the sequestration of ORC2 in the nucleolus is critical to prevent nascent DNA resection in response to replication stress.

Compartmentalization of the DNA damage response: Mechanisms and functions.

Cells have evolved an arsenal of molecular mechanisms to respond to continuous alterations in the primary structure of DNA. At the cellular level, DNA damage response proteins accumulate at sites of DNA damage and organize into nuclear foci. As recounted by Errol Friedberg, pioneering work on DNA repair in the 1930 s was stimulated by collaborations between physicists and geneticists. In recent years, the introduction of ideas from physics on self-organizing compartments has taken the field of cell biology by storm. Percolation and phase separation theories are increasingly used to model the self-assembly of compartments, called biomolecular condensates, that selectively concentrate molecules without a surrounding membrane. In this review, we discuss these concepts in the context of the DNA damage response. We discuss how studies of DNA repair foci as condensates can link molecular mechanisms with cell physiological functions, provide new insights into regulatory mechanisms, and open new perspectives for targeting DNA damage responses for therapeutic purposes.

Compartmentalization of the SUMO/RNF4 pathway by SLX4 drives DNA repair.

SLX4, disabled in the Fanconi anemia group P, is a scaffolding protein that coordinates the action of structure-specific endonucleases and other proteins involved in the replication-coupled repair of DNA interstrand cross-links. Here, we show that SLX4 dimerization and SUMO-SIM interactions drive the assembly of SLX4 membraneless compartments in the nucleus called condensates. Super-resolution microscopy reveals that SLX4 forms chromatin-bound clusters of nanocondensates. We report that SLX4 compartmentalizes the SUMO-RNF4 signaling pathway. SENP6 and RNF4 regulate the assembly and disassembly of SLX4 condensates, respectively. SLX4 condensation per se triggers the selective modification of proteins by SUMO and ubiquitin. Specifically, SLX4 condensation induces ubiquitylation and chromatin extraction of topoisomerase 1 DNA-protein cross-links. SLX4 condensation also induces the nucleolytic degradation of newly replicated DNA. We propose that the compartmentalization of proteins by SLX4 through site-specific interactions ensures the spatiotemporal control of protein modifications and nucleolytic reactions during DNA repair.

The BLM helicase is a new therapeutic target in multiple myeloma involved in replication stress survival and drug resistance.

Multiple myeloma (MM) is a hematologic cancer characterized by accumulation of malignant plasma cells in the bone marrow. To date, no definitive cure exists for MM and resistance to current treatments is one of the major challenges of this disease. The DNA helicase BLM, whose depletion or mutation causes the cancer-prone Bloom's syndrome (BS), is a central factor of DNA damage repair by homologous recombination (HR) and genomic stability maintenance. Using independent cohorts of MM patients, we identified that high expression of BLM is associated with a poor outcome with a significant enrichment in replication stress signature. We provide evidence that chemical inhibition of BLM by the small molecule ML216 in HMCLs (human myeloma cell lines) leads to cell cycle arrest and increases apoptosis, likely by accumulation of DNA damage. BLM inhibition synergizes with the alkylating agent melphalan to efficiently inhibit growth and promote cell death in HMCLs. Moreover, ML216 treatment re-sensitizes melphalan-resistant cell lines to this conventional therapeutic agent. Altogether, these data suggest that inhibition of BLM in combination with DNA damaging agents could be of therapeutic interest in the treatment of MM, especially in those patients with high BLM expression and/or resistance to melphalan.

An optogenetic proximity labeling approach to probe the composition of inducible biomolecular condensates in cultured cells.

Inducible biomolecular condensates play fundamental roles in cellular responses to intracellular and environmental cues. Knowledge about their composition is crucial to understand the functions that arise specifically from the assembly of condensates. This protocol combines an optogenetic and an efficient proximity labeling approach to analyze protein modifications driven by protein condensation in cultured cells. Low endogenous biotin level ensures sharp signals. For complete details on the use and execution of this protocol, please refer to Frattini et al. (2021).

TopBP1 assembles nuclear condensates to switch on ATR signaling.

ATR checkpoint signaling is crucial for cellular responses to DNA replication impediments. Using an optogenetic platform, we show that TopBP1, the main activator of ATR, self-assembles extensively to yield micrometer-sized condensates. These opto-TopBP1 condensates are functional entities organized in tightly packed clusters of spherical nano-particles. TopBP1 condensates are reversible, occasionally fuse, and co-localize with TopBP1 partner proteins. We provide evidence that TopBP1 condensation is a molecular switch that amplifies ATR activity to phosphorylate checkpoint kinase 1 (Chk1) and slow down replication forks. Single amino acid substitutions of key residues in the intrinsically disordered ATR activation domain disrupt TopBP1 condensation and consequently ATR/Chk1 signaling. In physiologic salt concentration and pH, purified TopBP1 undergoes liquid-liquid phase separation in vitro. We propose that the actuation mechanism of ATR signaling is the assembly of TopBP1 condensates driven by highly regulated multivalent and cooperative interactions.

Dihydropyrimidinase protects from DNA replication stress caused by cytotoxic metabolites.

Imbalance in the level of the pyrimidine degradation products dihydrouracil and dihydrothymine is associated with cellular transformation and cancer progression. Dihydropyrimidines are degraded by dihydropyrimidinase (DHP), a zinc metalloenzyme that is upregulated in solid tumors but not in the corresponding normal tissues. How dihydropyrimidine metabolites affect cellular phenotypes remains elusive. Here we show that the accumulation of dihydropyrimidines induces the formation of DNA-protein crosslinks (DPCs) and causes DNA replication and transcriptional stress. We used Xenopus egg extracts to recapitulate DNA replication invitro. We found that dihydropyrimidines interfere directly with the replication of both plasmid and chromosomal DNA. Furthermore, we show that the plant flavonoid dihydromyricetin inhibits human DHP activity. Cellular exposure to dihydromyricetin triggered DPCs-dependent DNA replication stress in cancer cells. This study defines dihydropyrimidines as potentially cytotoxic metabolites that may offer an opportunity for therapeutic-targeting of DHP activity in solid tumors.

A tumor suppressive DNA translocase named FANCM.

FANCM is named after Fanconi anemia (FA) complement group M. The clinical symptoms of FA include congenital abnormalities, pancytopenia, and cancer proneness. However, recent studies reveal that biallelic inactivation of FANCM does not cause the constellation of FA symptoms, but predisposes patients to cancer and infertility. FANCM is a tumor suppressor gene that encodes a conserved and structure-specific DNA translocase. It controls the outcome of homologous recombination and facilitates DNA replication across a variety of natural and chemically induced obstacles. This review details our current understanding of FANCM as a facilitator of the cellular functions of caretaker proteins, including FA, Bloom syndrome, and Ataxia telangiectasia and RAD3-related proteins, which collectively ensure the maintenance of chromosome stability during DNA replication.

Recruitment of ubiquitin-activating enzyme UBA1 to DNA by poly(ADP-ribose) promotes ATR signalling.

The DNA damage response (DDR) ensures cellular adaptation to genotoxic insults. In the crowded environment of the nucleus, the assembly of productive DDR complexes requires multiple protein modifications. How the apical E1 ubiquitin activation enzyme UBA1 integrates spatially and temporally in the DDR remains elusive. Using a human cell-free system, we show that poly(ADP-ribose) polymerase 1 promotes the recruitment of UBA1 to DNA. We find that the association of UBA1 with poly(ADP-ribosyl)ated protein-DNA complexes is necessary for the phosphorylation replication protein A and checkpoint kinase 1 by the serine/threonine protein kinase ataxia-telangiectasia and RAD3-related, a prototypal response to DNA damage. UBA1 interacts directly with poly(ADP-ribose) via a solvent-accessible and positively charged patch conserved in the Animalia kingdom but not in Fungi. Thus, ubiquitin activation can anchor to poly(ADP-ribose)-seeded protein assemblies, ensuring the formation of functional ataxia-telangiectasia mutated and RAD3-related-signalling complexes.

Premature activation of the SLX4 complex by Vpr promotes G2/M arrest and escape from innate immune sensing.

The HIV auxiliary protein Vpr potently blocks the cell cycle at the G2/M transition. Here, we show that G2/M arrest results from untimely activation of the structure-specific endonuclease (SSE) regulator SLX4 complex (SLX4com) by Vpr, a process that requires VPRBP-DDB1-CUL4 E3-ligase complex. Direct interaction of Vpr with SLX4 induced the recruitment of VPRBP and kinase-active PLK1, enhancing the cleavage of DNA by SLX4-associated MUS81-EME1 endonucleases. G2/M arrest-deficient Vpr alleles failed to interact with SLX4 or to induce recruitment of MUS81 and PLK1. Furthermore, knockdown of SLX4, MUS81, or EME1 inhibited Vpr-induced G2/M arrest. In addition, we show that the SLX4com is involved in suppressing spontaneous and HIV-1-mediated induction of type 1 interferon and establishment of antiviral responses. Thus, our work not only reveals the identity of the cellular factors required for Vpr-mediated G2/M arrest but also identifies the SLX4com as a regulator of innate immunity.

DNA structure-specific priming of ATR activation by DNA-PKcs.

Three phosphatidylinositol-3-kinase-related protein kinases implement cellular responses to DNA damage. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and ataxia-telangiectasia mutated respond primarily to DNA double-strand breaks (DSBs). Ataxia-telangiectasia and RAD3-related (ATR) signals the accumulation of replication protein A (RPA)-covered single-stranded DNA (ssDNA), which is caused by replication obstacles. Stalled replication intermediates can further degenerate and yield replication-associated DSBs. In this paper, we show that the juxtaposition of a double-stranded DNA end and a short ssDNA gap triggered robust activation of endogenous ATR and Chk1 in human cell-free extracts. This DNA damage signal depended on DNA-PKcs and ATR, which congregated onto gapped linear duplex DNA. DNA-PKcs primed ATR/Chk1 activation through DNA structure-specific phosphorylation of RPA32 and TopBP1. The synergistic activation of DNA-PKcs and ATR suggests that the two kinases combine to mount a prompt and specific response to replication-born DSBs.

Induction of ASAP (MAP9) contributes to p53 stabilization in response to DNA damage.

p53 is a key tumor suppressor that controls DNA damage response and genomic integrity. In response to genotoxic stress, p53 is stabilized and activated, resulting in controlled activation of genes involved in cell cycle arrest, DNA repair and/or apoptosis. ASAP is a centrosome- and spindle-associated protein, the deregulation of which induces severe mitotic defects. We show here that following double-strand break DNA formation, ASAP directly interacts with and stabilizes p53 by enhancing its p300-mediated acetylation and blocking its MDM2-mediated ubiquitination and degradation, leading to an increase of p53 transcriptional activity. Upon DNA damage, ASAP is transiently accumulated before being degraded upon persistent damage. This work links the p53 response with the cytoskeleton and confirms that the DNA-damaging signaling pathway is coordinated by centrosomal proteins. We reveal the existence of a new pathway through which ASAP signals the DNA damage response by regulating the p300-MDM2-p53 loop. These results point out ASAP as a possible target for the design of drugs to sensitize radio-resistant tumors.

Fos family protein degradation by the proteasome.

c-Fos proto-oncoprotein defines a family of closely related transcription factors (Fos proteins) also comprising Fra-1, Fra-2, FosB and DeltaFosB, the latter two proteins being generated by alternative splicing. Through the regulation of many genes, most of them still unidentified, they regulate major functions from the cell level up to the whole organism. Thus they are involved in the control of proliferation, differentiation and apoptosis, as well as in the control of responses to stresses, and they play important roles in organogenesis, immune responses and control of cognitive functions, among others. Fos proteins are intrinsically unstable. We have studied how two of them, c-Fos and Fra-1, are degraded. Departing from the classical scenario where unstable key cell regulators are hydrolysed by the proteasome after polyubiquitination, we showed that the bulk of c-Fos and Fra-1 can be broken down independently of any prior ubiquitination. Certain conserved structural domains suggest that similar mechanisms may also apply to Fra-2 and FosB. Computer search indicates that certain motifs shared by the Fos proteins and putatively responsible for instability are found in no other protein, suggesting the existence of degradation mechanisms specific for this protein family. Under particular signalling conditions, others have shown that a part of cytoplasmic c-Fos requires ubiquitination for fast turnover. This poses the question of the multiplicity of degradation pathways that apply to proteins depending on their intracellular localization.

ASAP is a novel substrate of the oncogenic mitotic kinase Aurora-A: phosphorylation on Ser625 is essential to spindle formation and mitosis.

Proper chromosome segregation is required to maintain the appropriate number of chromosomes from one cell generation to another and to prevent aneuploidy, which is mainly found in solid cancers. A correct mitotic spindle is necessary to accomplish such a process. Aurora kinases play critical roles in chromosome segregation and cell division; their deregulation impairs spindle assembly, checkpoint function and cell division causing chromosome mis-segregation. These kinases have been implicated in tumorigenesis. Aurora-A (AurA), in particular has been identified as a cancer-susceptibility gene, is overexpressed in a number of tumors and is required for G2/M transition and spindle assembly. ASAP is a novel spindle-associated protein, the deregulation of which induces severe mitotic defects. We show here that ASAP is a novel substrate of AurA kinase. We have identified serine 625 as the major phosphorylation site for AurA in vivo and localized the phosphorylated form of ASAP to centrosomes from late G2 to telophase, and around the midbody during cytokinesis. AurA depletion induces a proteasome-dependent degradation of ASAP. ASAP depletion induces spindle defects rescued by the expression of the phosphorylation-mimetic mutant ASAP-S625E and not by the non-phosphorylatable mutant ASAP-S625A. Microinjection of mono-specific S625 phospho-antibodies also impaired spindle formation and mitosis. These results strongly indicate that the phosphorylation of ASAP on S625 by AurA is required for bipolar spindle assembly and is essential for a correct mitotic progression. All together, these results suggest that we have identified a novel AurA substrate, pointing out ASAP as a new potential target for antitumoral drugs.

Ubiquitin-independent- versus ubiquitin-dependent proteasomal degradation of the c-Fos and Fra-1 transcription factors: is there a unique answer?

The Fos family of transcription factors comprises c-Fos, Fra-1, Fra-2 and FosB, which are all intrinsically unstable proteins. Fos proteins heterodimerize with a variety of other transcription factors to control genes encoding key cell regulators. Their best known partners are the Jun family proteins (c-Jun, JunB, and JunD). At the cellular level, Fos-involving dimers control proliferation, differentiation, apoptosis and responses to environmental cues. At the organism level, they play paramount parts in organogenesis, immune responses and cognitive functions, among others. fos family genes are subjected to exquisite, complex and intermingled transcriptional and post-transcriptional regulations, which are necessary to avoid pathological effects. In particular, the Fos proteins undergo to numerous post-translational modifications, such as phosphorylations and sumoylation, regulating their transcriptional activity, their subcellular localization and their turnover. The mechanisms whereby c-Fos and Fra-1 are degraded have been studied in detail. Contrasting with the classical scenario, according to which most unstable key cell regulators are hydrolyzed by the proteasome after conjugation of polyubiquitin chains, the bulk of c-Fos and Fra-1 can be hydrolyzed independently of any prior ubiquitylation in different situations. c-Fos and Fra-1 share a common destabilizing domain whose primary sequence is conserved in Fra-2 and FosB, suggesting that similar breakdown mechanisms might be at play in the latter two proteins. However, a database search indicates that this domain is not found in any other protein, suggesting that the mechanisms underlying Fos protein destruction may be specific to this family. Interestingly, under particular conditions, a fraction of cytoplasmic c-Fos is ubiquitylated, leading to faster turnover. This poses the question of the multiplicity of degradation pathways that can target the same substrate depending on its activation state, its protein partnership and/or its intracellular localization. This issue is discussed here together with the, thus far, overlooked roles of the various proteasomal complexes found in all cells.

Ubiquitin-independent proteasomal degradation of Fra-1 is antagonized by Erk1/2 pathway-mediated phosphorylation of a unique C-terminal destabilizer.

Fra-1, a transcription factor that is phylogenetically and functionally related to the proto-oncoprotein c-Fos, controls many essential cell functions. It is expressed in many cell types, albeit with differing kinetics and abundances. In cells reentering the cell cycle, Fra-1 expression is transiently stimulated albeit later than that of c-Fos and for a longer time. Moreover, Fra-1 overexpression is found in cancer cells displaying high Erk1/2 activity and has been linked to tumorigenesis. One crucial point of regulation of Fra-1 levels is controlled protein degradation, the mechanism of which remains poorly characterized. Here, we have combined genetic, pharmacological, and signaling studies to investigate this process in nontransformed cells and to elucidate how it is altered in cancer cells. We report that the intrinsic instability of Fra-1 depends on a single destabilizer contained within the C-terminal 30 to 40 amino acids. Two serines therein, S252 and S265, are phosphorylated by kinases of the Erk1/2 pathway, which compromises protein destruction upon both normal physiological induction and tumorigenic constitutive activation of this cascade. Our data also indicate that Fra-1, like c-Fos, belongs to a small group of proteins that may, under certain circumstances, undergo ubiquitin-independent degradation by the proteasome. Our work reveals both similitudes and differences between Fra-1 and c-Fos degradation mechanisms. In particular, the presence of a single destabilizer within Fra-1, instead of two that are differentially regulated in c-Fos, explains the much faster turnover of the latter when cells traverse the G(0)/G(1)-to-S-phase transition. Finally, our study offers further insights into the signaling-regulated expression of the other Fos family proteins.

The HBZ-SP1 isoform of human T-cell leukemia virus type I represses JunB activity by sequestration into nuclear bodies.

BACKGROUND: The human T-cell leukemia virus type I (HTLV-I) basic leucine-zipper factor (HBZ) has previously been shown to modulate transcriptional activity of Jun family members. The presence of a novel isoform of HBZ, termed HBZ-SP1, has recently been characterized in adult T-cell leukemia (ATL) cells and has been found to be associated with intense nuclear spots. In this study, we investigated the role of these nuclear bodies in the regulation of the transcriptional activity of JunB. RESULTS: Using fluorescence microscopy, we found that the HBZ-SP1 protein localizes to intense dots corresponding to HBZ-NBs and to nucleoli. We analyzed the relative mobility of the EGFP-HBZ-SP1 fusion protein using fluorescence recovery after photobleaching (FRAP) analysis and found that the deletion of the ZIP domain perturbs the association of the HBZ-SP1 protein to the HBZ-NBs. These data suggested that HBZ needs cellular partners, including bZIP factors, to form HBZ-NBs. Indeed, by cotransfection experiments in COS cells, we have found that the bZIP factor JunB is able to target delocalized form of HBZ (deleted in its nuclear localization subdomains) into the HBZ-NBs. We also show that the viral protein is able to entail a redistribution of JunB into the HBZ-NBs. Moreover, by transfecting HeLa cells (known to express high level of JunB) with a vector expressing HBZ-SP1, the sequestration of JunB to the HBZ-NBs inhibited its transcriptional activity. Lastly, we analyzed the nuclear distribution of HBZ-SP1 in the presence of JunD, a Jun family member known to be activated by HBZ. In this case, no NBs were detected and the HBZ-SP1 protein was diffusely distributed throughout the nucleoplasm. CONCLUSION: Our results suggest that HBZ-mediated sequestration of JunB to the HBZ-NBs may be causing the repression of JunB activity in vivo.

Nuclear localization of HTLV-I bZIP factor (HBZ) is mediated by three distinct motifs.

The genome of the human T-cell leukemia virus type I (HTLV-I) codes for a basic leucine zipper protein, HBZ, capable of repressing JUN activity and viral transcription. Transient expression in mammalian cells showed that HBZ was targeted to the nucleus, where it accumulated in nuclear speckles. By using a complementary set of deletion mutants, we report here that the nuclear targeting of HBZ is mediated by three distinct nuclear localization signals and that at least two are necessary for the translocation of HBZ to the nucleus. Moreover, the resulting mutant proteins distribute throughout the nucleoplasm and/or into the nucleoli, whereas the wild-type HBZ exclusively accumulates in nuclear speckles, suggesting that the integrity of the protein is required for its speckle localization. We also demonstrate that the HBZ-containing speckles do not correspond to Cajal bodies, splicing factor compartments, or promyelocytic leukemia oncoprotein bodies. Unexpectedly, by using immunogold electron microscopy, we found HBZ localized to heterochromatin. Until now, such characteristics had never been described for a transcription factor and could explain the inhibitory activity of HBZ.

HBZ interacts with JunD and stimulates its transcriptional activity.

Human T-cell leukemia virus type I (HTLV-I) bZIP factor (HBZ) is a viral basic leucine zipper protein that was originally described as a partner of cAMP response element binding protein-2 and as a repressor of HTLV-I viral transcription. In addition, HBZ is able to interact with the activator protein-1 (AP-1) transcription factors c-Jun and JunB, the interaction with c-Jun leading to a transcriptional repression of AP-1-regulated genes. Here we show that HBZ also interacts with JunD in vitro and in vivo, and that this association occurs via the bZIP domain of the two proteins. Moreover, we show that HBZ can activate JunD-dependent transcription and that its amino-terminus is required.