Werner helicase interacting protein 1 contributes to G-quadruplex processing in human cells.

Genome replication is frequently impeded by highly stable DNA secondary structures, including G-quadruplex (G4) DNA, that can hinder the progression of the replication fork. Human WRNIP1 (Werner helicase Interacting Protein 1) associates with various components of the replication machinery and plays a crucial role in genome maintenance processes. However, its detailed function is still not fully understood. Here we show that human WRNIP1 interacts with G4 structures and provide evidence for its contribution to G4 processing. The absence of WRNIP1 results in elevated levels of G4 structures, DNA damage and chromosome aberrations following treatment with PhenDC3, a G4-stabilizing ligand. Additionally, we establish a functional and physical relationship between WRNIP1 and the PIF1 helicase in G4 processing. In summary, our results suggest that WRNIP1 aids genome replication and maintenance by regulating G4 processing and this activity relies on Pif1 DNA helicase.

Role of Yme1 in mitochondrial protein homeostasis: from regulation of protein import, OXPHOS function to lipid synthesis and mitochondrial dynamics.

Mitochondria are essential organelles of eukaryotic cells and thus mitochondrial proteome is under constant quality control and remodelling. Yme1 is a multi-functional protein and subunit of the homo-hexametric complex i-AAA proteinase. Yme1 plays vital roles in the regulation of mitochondrial protein homeostasis and mitochondrial plasticity, ranging from substrate degradation to the regulation of protein functions involved in mitochondrial protein biosynthesis, energy production, mitochondrial dynamics, and lipid biosynthesis and signalling. In this mini review, we focus on discussing the current understanding of the roles of Yme1 in mitochondrial protein import via TIM22 and TIM23 pathways, oxidative phosphorylation complex function, as well as mitochondrial lipid biosynthesis and signalling, as well as a brief discussion of the role of Yme1 in modulating mitochondrial dynamics.

The ARK2N-CK2 complex initiates transcription-coupled repair through enhancing the interaction of CSB with lesion-stalled RNAPII.

Transcription is extremely important for cellular processes but can be hindered by RNA polymerase II (RNAPII) pausing and stalling. Cockayne syndrome protein B (CSB) promotes the progression of paused RNAPII or initiates transcription-coupled nucleotide excision repair (TC-NER) to remove stalled RNAPII. However, the specific mechanism by which CSB initiates TC-NER upon damage remains unclear. In this study, we identified the indispensable role of the ARK2N-CK2 complex in the CSB-mediated initiation of TC-NER. The ARK2N-CK2 complex is recruited to damage sites through CSB and then phosphorylates CSB. Phosphorylation of CSB enhances its binding to stalled RNAPII, prolonging the association of CSB with chromatin and promoting CSA-mediated ubiquitination of stalled RNAPII. Consistent with this finding, Ark2n-/- mice exhibit a phenotype resembling Cockayne syndrome. These findings shed light on the pivotal role of the ARK2N-CK2 complex in governing the fate of RNAPII through CSB, bridging a critical gap necessary for initiating TC-NER.

Pathogenic variants in autism gene KATNAL2 cause hydrocephalus and disrupt neuronal connectivity by impairing ciliary microtubule dynamics.

Enlargement of the cerebrospinal fluid (CSF)-filled brain ventricles (cerebral ventriculomegaly), the cardinal feature of congenital hydrocephalus (CH), is increasingly recognized among patients with autism spectrum disorders (ASD). KATNAL2, a member of Katanin family microtubule-severing ATPases, is a known ASD risk gene, but its roles in human brain development remain unclear. Here, we show that nonsense truncation of Katnal2 (Katnal2Δ17) in mice results in classic ciliopathy phenotypes, including impaired spermatogenesis and cerebral ventriculomegaly. In both humans and mice, KATNAL2 is highly expressed in ciliated radial glia of the fetal ventricular-subventricular zone as well as in their postnatal ependymal and neuronal progeny. The ventriculomegaly observed in Katnal2Δ17 mice is associated with disrupted primary cilia and ependymal planar cell polarity that results in impaired cilia-generated CSF flow. Further, prefrontal pyramidal neurons in ventriculomegalic Katnal2Δ17 mice exhibit decreased excitatory drive and reduced high-frequency firing. Consistent with these findings in mice, we identified rare, damaging heterozygous germline variants in KATNAL2 in five unrelated patients with neurosurgically treated CH and comorbid ASD or other neurodevelopmental disorders. Mice engineered with the orthologous ASD-associated KATNAL2 F244L missense variant recapitulated the ventriculomegaly found in human patients. Together, these data suggest KATNAL2 pathogenic variants alter intraventricular CSF homeostasis and parenchymal neuronal connectivity by disrupting microtubule dynamics in fetal radial glia and their postnatal ependymal and neuronal descendants. The results identify a molecular mechanism underlying the development of ventriculomegaly in a genetic subset of patients with ASD and may explain persistence of neurodevelopmental phenotypes in some patients with CH despite neurosurgical CSF shunting.

Human cytomegalovirus deploys molecular mimicry to recruit VPS4A to sites of virus assembly.

The AAA-type ATPase VPS4 is recruited by proteins of the endosomal sorting complex required for transport III (ESCRT-III) to catalyse membrane constriction and membrane fission. VPS4A accumulates at the cytoplasmic viral assembly complex (cVAC) of cells infected with human cytomegalovirus (HCMV), the site where nascent virus particles obtain their membrane envelope. Here we show that VPS4A is recruited to the cVAC via interaction with pUL71. Sequence analysis, deep-learning structure prediction, molecular dynamics and mutagenic analysis identify a short peptide motif in the C-terminal region of pUL71 that is necessary and sufficient for the interaction with VPS4A. This motif is predicted to bind the same groove of the N-terminal VPS4A Microtubule-Interacting and Trafficking (MIT) domain as the Type 2 MIT-Interacting Motif (MIM2) of cellular ESCRT-III components, and this viral MIM2-like motif (vMIM2) is conserved across ß-herpesvirus pUL71 homologues. However, recruitment of VPS4A by pUL71 is dispensable for HCMV morphogenesis or replication and the function of the conserved vMIM2 during infection remains enigmatic. VPS4-recruitment via a vMIM2 represents a previously unknown mechanism of molecular mimicry in viruses, extending previous observations that herpesviruses encode proteins with structural and functional homology to cellular ESCRT-III components.

Impact of Combinatorial Histone Modifications on Acetyllysine Recognition by the ATAD2 and ATAD2B Bromodomains.

The ATPase family AAA+ domain containing 2 (ATAD2) protein and its paralog ATAD2B have a C-terminal bromodomain (BRD) that functions as a reader of acetylated lysine residues on histone proteins. Using a structure-function approach, we investigated the ability of the ATAD2/B BRDs to select acetylated lysine among multiple histone post-translational modifications. The ATAD2B BRD can bind acetylated histone ligands that also contain adjacent methylation or phosphorylation marks, while the presence of these modifications significantly weakened the acetyllysine binding activity of the ATAD2 BRD. Our structural studies provide mechanistic insights into how ATAD2/B BRD-binding pocket residues coordinate the acetyllysine group in the context of adjacent post-translational modifications. Furthermore, we investigated how sequence changes in amino acids of the histone ligands impact the recognition of an adjacent acetyllysine residue. Our study highlights how the interplay between multiple combinations of histone modifications influences the reader activity of the ATAD2/B BRDs, resulting in distinct binding modes.

Conformations of Bcs1L undergoing ATP hydrolysis suggest a concerted translocation mechanism for folded iron-sulfur protein substrate.

The human AAA-ATPase Bcs1L translocates the fully assembled Rieske iron-sulfur protein (ISP) precursor across the mitochondrial inner membrane, enabling respiratory Complex III assembly. Exactly how the folded substrate is bound to and released from Bcs1L has been unclear, and there has been ongoing debate as to whether subunits of Bcs1L act in sequence or in unison hydrolyzing ATP when moving the protein cargo. Here, we captured Bcs1L conformations by cryo-EM during active ATP hydrolysis in the presence or absence of ISP substrate. In contrast to the threading mechanism widely employed by AAA proteins in substrate translocation, subunits of Bcs1L alternate uniformly between ATP and ADP conformations without detectable intermediates that have different, co-existing nucleotide states, indicating that the subunits act in concert. We further show that the ISP can be trapped by Bcs1 when its subunits are all in the ADP-bound state, which we propose to be released in the apo form.

Targeting the mitochondrial protein YME1L to inhibit osteosarcoma cell growth in vitro and in vivo.

Exploring novel diagnostic and therapeutic biomarkers is extremely important for osteosarcoma. YME1 Like 1 ATPase (YME1L), locating in the mitochondrial inner membrane, is key in regulating mitochondrial plasticity and metabolic activity. Its expression and potential functions in osteosarcoma are studied in the present study. We show that YME1L mRNA and protein expression is significantly elevated in osteosarcoma tissues derived from different human patients. Moreover, its expression is upregulated in various primary and immortalized osteosarcoma cells. The Cancer Genome Atlas database results revealed that YME1L overexpression was correlated with poor overall survival and poor disease-specific survival in sarcoma patients. In primary and immortalized osteosarcoma cells, silencing of YME1L through lentiviral shRNA robustly inhibited cell viability, proliferation, and migration. Moreover, cell cycle arrest and apoptosis were detected in YME1L-silenced osteosarcoma cells. YME1L silencing impaired mitochondrial functions in osteosarcoma cells, causing mitochondrial depolarization, oxidative injury, lipid peroxidation and DNA damage as well as mitochondrial respiratory chain complex I activity inhibition and ATP depletion. Contrarily, forced YME1L overexpression exerted pro-cancerous activity and strengthened primary osteosarcoma cell proliferation and migration. YME1L is important for Akt-S6K activation in osteosarcoma cells. Phosphorylation of Akt and S6K was inhibited after YME1L silencing in primary osteosarcoma cells, but was strengthened with YME1L overexpression. Restoring Akt-mTOR activation by S473D constitutively active Akt1 mitigated YME1L shRNA-induced anti-osteosarcoma cell activity. Lastly, intratumoral injection of YME1L shRNA adeno-associated virus inhibited subcutaneous osteosarcoma xenograft growth in nude mice. YME1L depletion, mitochondrial dysfunction, oxidative injury, Akt-S6K inactivation, and apoptosis were detected in YME1L shRNA-treated osteosarcoma xenografts. Together, overexpressed YME1L promotes osteosarcoma cell growth, possibly by maintaining mitochondrial function and Akt-mTOR activation.

The human AAA-ATPase VPS4A isoform and its co-factor VTA1 have a unique function in regulating mammalian cytokinesis abscission.

Mutations in the human AAA-ATPase VPS4 isoform, VPS4A, cause severe neurodevelopmental defects and congenital dyserythropoietic anemia (CDA). VPS4 is a crucial component of the endosomal sorting complex required for transport (ESCRT) system, which drives membrane remodeling in numerous cellular processes, including receptor degradation, cell division, and neural pruning. Notably, while most organisms encode for a single VPS4 gene, human cells have 2 VPS4 paralogs, namely VPS4A and VPS4B, but the functional differences between these paralogs is mostly unknown. Here, we set out to investigate the role of the human VPS4 paralogs in cytokinetic abscission using a series of knockout cell lines. We found that VPS4A and VPS4B hold both overlapping and distinct roles in abscission. VPS4A depletion resulted in a more severe abscission delay than VPS4B and was found to be involved in earlier stages of abscission. Moreover, VPS4A and a monomeric-locked VPS4A mutant bound the abscission checkpoint proteins CHMP4C and ANCHR, while VPS4B did not, indicating a regulatory role for the VPS4A isoform in abscission. Depletion of VTA1, a co-factor of VPS4, disrupted VPS4A-ANCHR interactions and accelerated abscission, suggesting that VTA1 is also involved in the abscission regulation. Our findings reveal a dual role for VPS4A in abscission, one that is canonical and can be compensated by VPS4B, and another that is regulatory and may be delivered by its monomeric form. These observations provide a potential mechanistic explanation for the neurodevelopmental defects and other related disorders reported in VPS4A-mutated patients with a fully functional VPS4B paralog.

Targeting TRIP13 in favorable histology Wilms tumor with nuclear export inhibitors synergizes with doxorubicin.

Wilms tumor (WT) is the most common renal malignancy of childhood. Despite improvements in the overall survival, relapse occurs in ~15% of patients with favorable histology WT (FHWT). Half of these patients will succumb to their disease. Identifying novel targeted therapies remains challenging in part due to the lack of faithful preclinical in vitro models. Here we establish twelve patient-derived WT cell lines and demonstrate that these models faithfully recapitulate WT biology using genomic and transcriptomic techniques. We then perform loss-of-function screens to identify the nuclear export gene, XPO1, as a vulnerability. We find that the FDA approved XPO1 inhibitor, KPT-330, suppresses TRIP13 expression, which is required for survival. We further identify synergy between KPT-330 and doxorubicin, a chemotherapy used in high-risk FHWT. Taken together, we identify XPO1 inhibition with KPT-330 as a potential therapeutic option to treat FHWTs and in combination with doxorubicin, leads to durable remissions in vivo.

PCNA cycling dynamics during DNA replication and repair in mammals.

Proliferating cell nuclear antigen (PCNA) is a eukaryotic replicative DNA clamp. Furthermore, DNA-loaded PCNA functions as a molecular hub during DNA replication and repair. PCNA forms a closed homotrimeric ring that encircles the DNA, and association and dissociation of PCNA from DNA are mediated by clamp-loader complexes. PCNA must be actively released from DNA after completion of its function. If it is not released, abnormal accumulation of PCNA on chromatin will interfere with DNA metabolism. ATAD5 containing replication factor C-like complex (RLC) is a PCNA-unloading clamp-loader complex. ATAD5 deficiency causes various DNA replication and repair problems, leading to genome instability. Here, we review recent progress regarding the understanding of the action mechanisms of PCNA unloading complex in DNA replication/repair pathways.

The SPATA5-SPATA5L1 ATPase complex directs replisome proteostasis to ensure genome integrity.

Ubiquitin-dependent unfolding of the CMG helicase by VCP/p97 is required to terminate DNA replication. Other replisome components are not processed in the same fashion, suggesting that additional mechanisms underlie replication protein turnover. Here, we identify replisome factor interactions with a protein complex composed of AAA+ ATPases SPATA5-SPATA5L1 together with heterodimeric partners C1orf109-CINP (55LCC). An integrative structural biology approach revealed a molecular architecture of SPATA5-SPATA5L1 N-terminal domains interacting with C1orf109-CINP to form a funnel-like structure above a cylindrically shaped ATPase motor. Deficiency in the 55LCC complex elicited ubiquitin-independent proteotoxicity, replication stress, and severe chromosome instability. 55LCC showed ATPase activity that was specifically enhanced by replication fork DNA and was coupled to cysteine protease-dependent cleavage of replisome substrates in response to replication fork damage. These findings define 55LCC-mediated proteostasis as critical for replication fork progression and genome stability and provide a rationale for pathogenic variants seen in associated human neurodevelopmental disorders.

Structural insights into the Clp protein degradation machinery.

The Clp protease system is important for maintaining proteostasis in bacteria. It consists of ClpP serine proteases and an AAA+ Clp-ATPase such as ClpC1. The hexameric ATPase ClpC1 utilizes the energy of ATP binding and hydrolysis to engage, unfold, and translocate substrates into the proteolytic chamber of homo- or hetero-tetradecameric ClpP for degradation. The assembly between the hetero-tetradecameric ClpP1P2 chamber and the Clp-ATPases containing tandem ATPase domains from the same species has not been studied in depth. Here, we present cryo-EM structures of the substrate-bound ClpC1:shClpP1P2 from Streptomyces hawaiiensis, and shClpP1P2 in complex with ADEP1, a natural compound produced by S. hawaiiensis and known to cause over-activation and dysregulation of the ClpP proteolytic core chamber. Our structures provide detailed information on the shClpP1-shClpP2, shClpP2-ClpC1, and ADEP1-shClpP1/P2 interactions, reveal conformational transition of ClpC1 during the substrate translocation, and capture a rotational ATP hydrolysis mechanism likely dominated by the D1 ATPase activity of chaperones.IMPORTANCEThe Clp-dependent proteolysis plays an important role in bacterial homeostasis and pathogenesis. The ClpP protease system is an effective drug target for antibacterial therapy. Streptomyces hawaiiensis can produce a class of potent acyldepsipeptide antibiotics such as ADEP1, which could affect the ClpP protease activity. Although S. hawaiiensis hosts one of the most intricate ClpP systems in nature, very little was known about its Clp protease mechanism and the impact of ADEP molecules on ClpP. The significance of our research is in dissecting the functional mechanism of the assembled Clp degradation machinery, as well as the interaction between ADEP1 and the ClpP proteolytic chamber, by solving high-resolution structures of the substrate-bound Clp system in S. hawaiiensis. The findings shed light on our understanding of the Clp-dependent proteolysis in bacteria, which will enhance the development of antimicrobial drugs targeting the Clp protease system, and help fighting against bacterial multidrug resistance.

Knockdown of hCINAP sensitizes colorectal cancer cells to ionizing radiation.

Colorectal cancer (CRC) poses a significant challenge in terms of treatment due to the prevalence of radiotherapy resistance. However, the underlying mechanisms responsible for radio-resistance in CRC have not been thoroughly explored. This study aimed to shed light on the role of human coilin interacting nuclear ATPase protein (hCINAP) in radiation-resistant HT-29 and SW480 CRC cells (HT-29-IR and SW480-IR) and investigate its potential implications. Firstly, radiation-resistant CRC cell lines were established by subjecting HT-29 and SW480 cells to sequential radiation exposure. Subsequent analysis revealed a notable increase in hCINAP expression in radiation-resistant CRC cells. To elucidate the functional role of hCINAP in radio-resistance, knockdown experiments were conducted. Remarkably, knockdown of hCINAP resulted in an elevation of reactive oxygen species (ROS) generation upon radiation treatment and subsequent activation of apoptosis mediated by mitochondria. These observations indicate that hCINAP depletion enhances the radiosensitivity of CRC cells. Conversely, when hCINAP was overexpressed, it was found to enhance the radio-resistance of CRC cells. This suggests that elevated hCINAP expression contributes to the development of radio-resistance. Further investigation revealed an interaction between hCINAP and ATPase family AAA domain containing 3A (ATAD3A). Importantly, ATAD3A was identified as an essential factor in hCINAP-mediated radio-resistance. These findings establish the involvement of hCINAP and its interaction with ATAD3A in the regulation of radio-resistance in CRC cells. Overall, the results of this study demonstrate that upregulating hCINAP expression may improve the survival of radiation-exposed CRC cells. Understanding the intricate molecular mechanisms underlying hCINAP function holds promise for potential strategies in targeted radiation therapy for CRC. These findings emphasize the importance of further research to gain a comprehensive understanding of hCINAP's precise molecular mechanisms and explore its potential as a therapeutic target in overcoming radio-resistance in CRC. By unraveling the complexities of hCINAP and its interactions, novel therapeutic approaches may be developed to enhance the efficacy of radiation therapy and improve outcomes for CRC patients.

WRNIP1 prevents transcription-associated genomic instability.

R-loops are non-canonical DNA structures that form during transcription and play diverse roles in various physiological processes. Disruption of R-loop homeostasis can lead to genomic instability and replication impairment, contributing to several human diseases, including cancer. Although the molecular mechanisms that protect cells against such events are not fully understood, recent research has identified fork protection factors and DNA damage response proteins as regulators of R-loop dynamics. In this study, we identify the Werner helicase-interacting protein 1 (WRNIP1) as a novel factor that counteracts transcription-associated DNA damage upon replication perturbation. Loss of WRNIP1 leads to R-loop accumulation, resulting in collisions between the replisome and transcription machinery. We observe co-localization of WRNIP1 with transcription/replication complexes and R-loops after replication perturbation, suggesting its involvement in resolving transcription-replication conflicts. Moreover, WRNIP1-deficient cells show impaired replication restart from transcription-induced fork stalling. Notably, transcription inhibition and RNase H1 overexpression rescue all the defects caused by loss of WRNIP1. Importantly, our findings highlight the critical role of WRNIP1 ubiquitin-binding zinc finger (UBZ) domain in preventing pathological persistence of R-loops and limiting DNA damage, thereby safeguarding genome integrity.

Analysis of the Conformational Landscape of the N-Domains of the AAA ATPase p97: Disentangling the Continuous Conformational Variability in Partially Symmetrical Complexes.

Single-particle cryo-electron microscopy (cryo-EM) has been shown to be effective in defining the structure of macromolecules, including protein complexes. Complexes adopt different conformations and compositions to perform their biological functions. In cryo-EM, the protein complexes are observed in solution, enabling the recording of images of the protein in multiple conformations. Various methods exist for capturing the conformational variability through analysis of cryo-EM data. Here, we analyzed the conformational variability in the hexameric AAA + ATPase p97, a complex with a six-fold rotational symmetric core surrounded by six flexible N-domains. We compared the performance of discrete classification methods with our recently developed method, MDSPACE, which uses 3D-to-2D flexible fitting of an atomic structure to images based on molecular dynamics (MD) simulations. Our analysis detected a novel conformation adopted by approximately 2% of the particles in the dataset and determined that the N-domains of p97 sway by up to 60° around a central position. This study demonstrates the application of MDSPACE in analyzing the continuous conformational changes in partially symmetrical protein complexes, systems notoriously difficult to analyze due to the alignment errors caused by their partial symmetry.

PEX1 is essential for glycosome biogenesis and trypanosomatid parasite survival.

Trypanosomatid parasites are kinetoplastid protists that compartmentalize glycolytic enzymes in unique peroxisome-related organelles called glycosomes. The heterohexameric AAA-ATPase complex of PEX1-PEX6 is anchored to the peroxisomal membrane and functions in the export of matrix protein import receptor PEX5 from the peroxisomal membrane. Defects in PEX1, PEX6 or their membrane anchor causes dysfunction of peroxisomal matrix protein import cycle. In this study, we functionally characterized a putative Trypanosoma PEX1 orthologue by bioinformatic and experimental approaches and show that it is a true PEX1 orthologue. Using yeast two-hybrid analysis, we demonstrate that TbPEX1 can bind to TbPEX6. Endogenously tagged TbPEX1 localizes to glycosomes in the T. brucei parasites. Depletion of PEX1 gene expression by RNA interference causes lethality to the bloodstream form trypanosomes, due to a partial mislocalization of glycosomal enzymes to the cytosol and ATP depletion. TbPEX1 RNAi leads to a selective proteasomal degradation of both matrix protein import receptors TbPEX5 and TbPEX7. Unlike in yeast, PEX1 depletion did not result in an accumulation of ubiquitinated TbPEX5 in trypanosomes. As PEX1 turned out to be essential for trypanosomatid parasites, it could provide a suitable drug target for parasitic diseases. The results also suggest that these parasites possess a highly efficient quality control mechanism that exports the import receptors from glycosomes to the cytosol in the absence of a functional TbPEX1-TbPEX6 complex.

Methionine secreted by tumor-associated pericytes supports cancer stem cells in clear cell renal carcinoma.

Here, we identify a subset of vascular pericytes, defined by expression of platelet-derived growth factor receptor beta (PDGFR-ß) and G-protein-coupled receptor 91 (GPR91), that promote tumorigenesis and tyrosine kinase inhibitors (TKIs) resistance by functioning as the primary methionine source for cancer stem cells (CSCs) in clear cell renal cell carcinoma (ccRCC). Tumor-cell-derived succinate binds to GPR91 on pericyte to activate autophagy for methionine production. CSCs use methionine to create stabilizing N6-methyladenosine in ATPase-family-AAA-domain-containing 2 (ATAD2) mRNA, and the resulting ATAD2 protein complexes with SRY-box transcription factor 9 to assemble super enhancers and thereby dictate its target genes that feature prominently in CSCs. Targeting PDGFR-ß+GPR91+ pericytes with specific GRP91 antagonists reduce intratumoral methionine level, eliminate CSCs, and enhance TKIs sensitivity. These results unraveled the mechanisms by which PDGFR-ß+GPR91+ pericytes provide supportive niche for CSCs and could be used to develop targets for treating ccRCC.

AXL/WRNIP1 Mediates Replication Stress Response and Promotes Therapy Resistance and Metachronous Metastasis in HER2+ Breast Cancer.

Therapy resistance and metastatic progression are primary causes of cancer-related mortality. Disseminated tumor cells possess adaptive traits that enable them to reprogram their metabolism, maintain stemness, and resist cell death, facilitating their persistence to drive recurrence. The survival of disseminated tumor cells also depends on their ability to modulate replication stress in response to therapy while colonizing inhospitable microenvironments. In this study, we discovered that the nuclear translocation of AXL, a TAM receptor tyrosine kinase, and its interaction with WRNIP1, a DNA replication stress response factor, promotes the survival of HER2+ breast cancer cells that are resistant to HER2-targeted therapy and metastasize to the brain. In preclinical models, knocking down or pharmacologically inhibiting AXL or WRNIP1 attenuated protection of stalled replication forks. Furthermore, deficiency or inhibition of AXL and WRNIP1 also prolonged metastatic latency and delayed relapse. Together, these findings suggest that targeting the replication stress response, which is a shared adaptive mechanism in therapy-resistant and metastasis-initiating cells, could reduce metachronous metastasis and enhance the response to standard-of-care therapies. SIGNIFICANCE: Nuclear AXL and WRNIP1 interact and mediate replication stress response, promote therapy resistance, and support metastatic progression, indicating that targeting the AXL/WRNIP1 axis is a potentially viable therapeutic strategy for breast cancer.

Autoinhibition of a clamp-loader ATPase revealed by deep mutagenesis and cryo-EM.

Clamp loaders are AAA+ ATPases that facilitate high-speed DNA replication. In eukaryotic and bacteriophage clamp loaders, ATP hydrolysis requires interactions between aspartate residues in one protomer, present in conserved 'DEAD-box' motifs, and arginine residues in adjacent protomers. We show that functional defects resulting from a DEAD-box mutation in the T4 bacteriophage clamp loader can be compensated by widely distributed single mutations in the ATPase domain. Using cryo-EM, we discovered an unsuspected inactive conformation of the clamp loader, in which DNA binding is blocked and the catalytic sites are disassembled. Mutations that restore function map to regions of conformational change upon activation, suggesting that these mutations may increase DNA affinity by altering the energetic balance between inactive and active states. Our results show that there are extensive opportunities for evolution to improve catalytic efficiency when an inactive intermediate is involved.