Biology subjects

Morris, J. R.

Publications and source records attributed to Morris, J. R..

15 recordsLinked to original sources

bioRxiv2025

Inactivation of a protein hydroxylase complex impairs replication fork restart in cancer and neurodevelopmental disorders

Protein hydroxylation is a post-translational modification that is commonly catalysed by enzymes of the oxygen- and 2-oxoglutarate (2OG)-dependent oxygenase family. The Jumonji-C (JmjC)-only sub-family of 2OG-oxygenases catalyse the hydroxylation of protein and tRNA substrates involved in fundamental cellular processes. Jumonji-domain 5 (JMJD5) is a highly conserved and essential 2OG-oxygenase that, thus far, is the only arginyl hydroxylase assigned in eukaryotes. We recently reported that JMJD5 hydroxylase activity is required for DNA replication fidelity, and that pathogenic variants cause replication stress (RS) and genome instability (GIN) in a novel neurodevelopmental disorder. Because of the prevalence of RS and GIN in cancer, and reported roles of JMJD5 in tumorigenesis, we here investigate the impact of JMJD5 cancer mutations on its role in replication fidelity. We describe the structural impact of cancer missense mutations on the hydroxylase activity of JMJD5 and its interaction with RCCD1, an abundant binding partner encoded by a gene associated with susceptibility to a variety of tumour types. We show that the JMJD5:RCCD1 interaction is disrupted by cancer mutations and that the complex is essential for suppressing RS and GIN in tumour cells. Finally, we describe a novel interaction of the complex with RAD51 paralogues and demonstrate the importance of the JMJD5:RCCD1 interaction for normal replication fork restart. Our findings further highlight the importance of protein hydroxylases in fundamental cellular processes and the consequences of JMJD5 and RCCD1 deregulation in human disease.

cancer biology↗

bioRxiv2025

The 7SK small nuclear ribonucleoprotein links the cell responses to transcription and replication stress by promoting replication fork reversal and homologous recombination

The 7SK-small nuclear ribonucleoprotein complex (7SK-snRNP) plays a crucial role in the response to transcription stress, releasing positive transcription elongation factor P-TEFb to sustain RNA polymerase II activity when transcription is blocked. Many conditions that block transcription also block DNA replication, causing replication stress, and 7SK-snRNP components are putative tumour suppressors with roles in transcription-replication conflicts and double-strand break repair. Here, we investigate potential roles of 7SK-snRNP components in the response to replication stress induced by chemotherapy agents with and without additional impact on transcription. We report that HEXIM1 and LARP7 promote replication fork slowing in response to agents that cause both replication- and transcription stress, in a manner consistent with their canonical 7SK-snRNP functions. Our data suggest that this role in fork slowing is mainly through facilitating RAD51-mediated replication fork reversal rather than transcription-replication conflicts. HEXIM1 and LARP7 promote RAD51 recruitment to replication-associated DSBs or post-replicative gaps under conditions of transcription stress such as induced by camptothecin or BET inhibitors and support HR at direct DSBs. In contrast, HEXIM1 and LARP7 are not required for HR in response to hydroxyurea, which does not cause transcription stress. Our data support that LARP7 roles during replication stress are independent of its reported interaction with BRCA1, and that both LARP7 and HEXIM1 promote survival in response to replication stress-inducing agents. 7SK-snRNP components are not recruited to stressed replication forks and RNA polymerase II inhibition phenocopies loss of these proteins. Taken together, our data support a model where 7SK-snRNP modulation of RNA polymerase II activity helps facilitate RAD51 function under transcription stress conditions, thereby connecting the cell responses to transcription- and replication stress.

molecular biology↗

bioRxiv2024

A dual readout embryonic zebrafish xenograft model of rhabdomyosarcoma to assess clinically relevant multi-receptor tyrosine kinase inhibitors

BackgroundRhabdomyosarcoma (RMS) is a highly aggressive soft tissue sarcoma, affecting children and adolescents, with poor prognosis in some patient groups. Better therapeutic regimens and preclinical models to test them in are needed. Multi-receptor tyrosine kinase inhibitors (MRTKIs) are licensed for adult indications and explored in the clinic in sarcoma patients. The MRTKI Regorafenib is currently assessed in the relapse setting in patients with RMS (NCT04625907). Reliable biomarkers of response for MRTKIs are lacking. MRTKIs act not only against the cancer cell, but also the supporting stroma, particularly the vasculature. The embryonic zebrafish is translucent and allows assessment of this interaction with high-throughput in vivo imaging. MethodsA new preclinical embryo zebrafish xenograft model was developed using Tg(flk1:GFP) (blood vessel reporter) transgenic zebrafish embryos inoculated in the yolk with fluorescently labelled cells from 7 different RMS cell lines (fusion-positive (FP): Rh4, Rh30, Rh41, RMS-01, fusion-negative (FN): RD, JR1, SMS-CTR), and patient-derived cells IC-pPDX-104 at 50 hours post-fertilization and incubated at 34{degrees}C for up to 70 hours. Xenografts and vessel beds were imaged and analysed using custom FIJI pipelines. MRTKIs regorafenib and infigratinib were used at a concentration of 0.1uM added to the fish water 4 hours post cell inoculation. Pro-angiogenic growth factors VEFG-A, FGF-2 and PDGF-BB were measured in conditioned media of each cell line. ResultsAll 7 RMS cell lines and the patient-derived cells engrafted with tumour burden assessment by fluorescent imaging and direct cell counting indicating adequate growth and high cell viability during the observation period. RMS tumours induced neo-vascularisation towards the tumour and increased density of proximal vessel beds. MRTKI treatment revealed a greater tumour-intrinsic sensitivity of FP cells, but identified a significant blockade of neo-vascularisation across all RMS lines, with regorafenib response correlated with secretion of VEGF-A. ConclusionWe have developed an embryonic zebrafish xenograft model of RMS, which allows assessment of tumour growth, vascularisation initiation and therapeutic responses to clinically relevant MRTKIs. The identification of VEGF-A secretion as potential biomarker for Regorafenib response and the separation of therapeutic effects on tumour growth and neovascularisation suggests additional value of our model for response prediction to MRTKIs.

cancer biology↗

bioRxiv2024

USP50 suppresses alternative RecQ helicase use and deleterious DNA2 activity during replication.

Mammalian DNA replication employs several RecQ DNA helicases to orchestrate the faithful duplication of genetic information. Helicase function is often coupled to the activity of specific nucleases, but how helicase and nuclease activities are co-directed is unclear. Here we identify the inactive ubiquitin-specific protease, USP50, as a ubiquitin-binding and chromatin-associated protein required for ongoing replication, fork restart, telomere maintenance and cellular survival during replicative stress. USP50 supports WRN:FEN1 at stalled replication forks, suppresses MUS81-dependent fork collapse and restricts double-strand DNA breaks at GC-rich sequences. Surprisingly we find that cells depleted for USP50 and recovering from a replication block exhibit increased DNA2 and RECQL4 foci and that the defects in ongoing replication, poor fork restart and increased fork collapse seen in these cells are mediated by DNA2, RECQL4 and RECQL5. These data define a novel ubiquitin-dependent pathway that promotes the balance of helicase: nuclease use at ongoing and stalled replication forks.

cancer biology↗

bioRxiv2022

Pin1-promoted SUMOylation of RNF168 restrains its chromatin accumulation.

The E3 ubiquitin ligase RNF168 is a rate-limiting component of DNA double-strand break signalling that acts to amplify histone ubiquitylation. The confining of RNF168 chromatin spreading around DNA break sites has been related to constraining its expression levels and to the removal of ubiquitin-conjugates to which it binds. Here we identify a new mechanism that suppresses RNF168 amplification at chromatin. We show that depletion of Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) or mutation of the Pin1-binding site of RNF168 increases DNA-damage site accumulation of RNF168 to supraphysiological levels without impacting RNF168 expression levels. Pin1 promotes SUMOylation of RNF168 by SUMO2/3, and a SUMO conjugation site on RNF168 is required to restrict RNF168 accumulations. We find Pin1-SUMO mediated-regulation of RNF168 is associated with cellular radioresistance. These data demonstrate that the regulation of chromatin ubiquitylation is actively suppressed by modification of RNF168 to counteract excessive RNF168-ubiquitin spreading.

molecular biology↗

bioRxiv2022

SUMO4 regulates DNA double-strand break repair independently of conjugation.

The amplitudes of small-modifier protein signalling through ubiquitin and the Small Ubiquitin-like Modifiers, SUMO1-3, are critical to the correct phasing of DNA repair protein accumulation, activity, and clearance, and for the completion of mammalian DNA double-strand break (DSB) repair. However, how SUMO-conjugate signalling in the response is delineated is poorly understood. At the same time, the role of the non-conjugated SUMO protein, SUMO4, has remained enigmatic. Here we reveal that SUMO4 is required to prevent excessive DNA-damage-induced SUMOylation and deleterious over-accumulation of RAP80. Mechanistically we show SUMO4 acts independently of its conjugation and potentiates SENP1 catalytic activity. These data identify SUMO4 as a SUMO deconjugation component and show SUMO4:SENP1 are critical regulators of DNA-damage-induced SUMO signalling. HighlightsO_LIThe roles of the SUMO family members, SUMO1-4, in DSB repair are not redundant. C_LIO_LISUMO4 conjugation is deleterious. C_LIO_LISUMO4 promotes SENP1 catalytic activity. C_LIO_LISUMO4:SENP1 restrict SUMO-signalling and accumulation of the BRCA1-A complex. C_LI

molecular biology↗

bioRxiv2022

RAD52 underlies the synthetic-lethal relationship between BRCA1/2 and 53BP1 deficiencies and DNA polymerase theta loss.

Introductory paragraphCells lacking several DNA repair proteins, including those promoting homologous recombination (HR), are sensitive to polymerase theta (Pol{theta}) repression1-4. Pol{theta} drives theta-mediated end joining (TMEJ) and suppresses HR but what mediates its synthetic lethal relationships is unclear. Here we examine murine Brca1C61G/C61G 53bp1-/-cells and find they are largely HR proficient by using RNF168 and RAD52. They exhibit no more TMEJ than 53bp1-/- cells yet are more sensitive to targeting of Pol{theta}. We find that RAD52 recruitment to damaged chromatin is increased following Pol{theta} depletion. RAD52 accumulation and cellular sensitivity to Pol{theta} repression can be curbed by the RAD51-binding regions of BARD1 and BRCA2, and sensitivity of BRCA1/2 depleted cells to Pol{theta} repression is suppressed by RAD52 inhibition. 53bp1-/- cells exhibit a smaller increase in RAD52 recruitment following Pol{theta} repression and also become resistant to Pol{theta} repression following RAD52 inhibition. Thus, RAD52 mediates sensitivity to targeting Pol{theta} in these contexts.

molecular biology↗

bioRxiv2022

SUMO monoclonal antibodies vary in sensitivity, specificity, and ability to detect SUMO conjugate types.

Monoclonal antibodies (MAb) to members of the Small Ubiquitin-like modifier (SUMO) family are essential tools in the study of cellular SUMOylation. However, many reagents are poorly validated, and the choice of which antibody to use for which detection format is without an evidence base. Here we test twenty-four anti-SUMO monoclonal antibodies for sensitivity and specificity to SUMO1-4 in monomeric and polymeric states in dot-blots, immunoblots, immunofluorescence and immunoprecipitation. We find substantial variability between SUMO MAbs for different conjugation states, detection of increased SUMOylation in response to thirteen different stress agents and as enrichment reagents for analysis of SUMOylated RanGAP1 or KAP1. All four anti-SUMO4 monoclonal antibodies tested cross-reacted with SUMO2/3, and several SUMO2/3 monoclonal antibodies cross-reacted with SUMO4. ~10% of tested monoclonal antibodies produced specific results across multiple applications.

molecular biology↗

bioRxiv2022

Modification of the SUMO activating enzyme subunit SAE2 directs SUMO isoform bias required for mitotic fidelity.

Mammalian cells possess three conjugatable SUMO variants: SUMO1 and the largely indistinguishable SUMO2 and SUMO3 (designated SUMO2/3). Some SUMOylated substrates are modified by both SUMO1 and SUMO2/3, while others show biased modifications towards SUMO1 or SUMO2/3. How preferential SUMO protein conjugation is coordinated is poorly understood. Here, we examine a modification of the catalytic component of the human SUMO Activation Enzyme, SAE2. We observe that lysine 164 of SAE2 is deacetylated during mitosis in an HDAC6-dependent manner. We find that an acetyl-analogue mutant, SAE2-K164Q, biases the activation and conjugation of SUMO2>SUMO1 and discriminates SUMO1 and SUMO2/3 through their C-terminal tails. Complementation of SAE2-depleted or inhibited cells with SAE2-K164Q restricts mitotic SUMO1-conjugates and increases multipolar spindle formation. We confirm the SUMO E1-dependent modification of the nuclear mitotic apparatus, NuMA, and find that the mitotic defects of both SAE2-K164Q complemented cells and HDAC6-inhibitor-treated cells are corrected by either over-expression of SUMO1 or by expression of a GFP-SUMO1-NuMA-K1766R fusion protein. Our observations suggest a model in which the SAE1:SAE2 enzyme is deacetylated on early mitosis to encourage the conjugation of SUMO1 to support mitotic fidelity. These surprising data reveal that the SUMO-activating enzyme can bias SUMO variant conjugation.

molecular biology↗

bioRxiv2021

Imaging Nanoscale Nuclear Structures with Expansion Microscopy

Commonly applied super-resolution light microscopies have provided insight into subcellular processes at the nanoscale. However, imaging depth, speed, throughput and cost remain significant challenges, reducing the numbers of three-dimensional, nanoscale processes that can be investigated and the number of laboratories able to undertake such analysis. Expansion microscopy solves many of these limitations but its application to imaging nuclear processes has been constrained by concerns of unequal nuclear expansion. Here we demonstrate the conditions for isotropic expansion of the nucleus. Using DNA damage response proteins, BRCA1, 53BP1 and RAD51 as exemplars we quantitatively describe the three-dimensional nanoscale organisation of over 50,000 DNA damage response structures. We demonstrate the ability to assess chromatin regulated events and show the simultaneous assessment of four elements. This study thus provides the means by which expansion microscopy can contribute to the investigation of nanoscale nuclear processes.

molecular biology↗

bioRxiv2020

Phenology and flowering overlap drive specialization in pollinator networks

Variation in diet breadth and specialization stems from fundamental interactions species have with their environment1-3. Consequently, understanding the drivers of this variation is key to understanding ecological and evolutionary processes, and will facilitate the development of predictive tools as ecological networks respond to environmental change4,5. Diet breadth in wild bees has been an area of focus due to both their close mutualistic dependence on plants, and because both groups are under threat from global biodiversity loss6. Though many of the principles governing specialization for pollinators have been identified7,8, they remain largely unvalidated. Using mechanistic models of adaptive foraging in pollinators9,10, we show that while temporal resource overlap has little impact on specialization in pollinators with extended flight periods, reduced overlap increases specialization as pollinator flight periods decrease. These results are corroborated empirically using pollen load data taken from bees with shorter (genus Andrena) and longer (genus Lasioglossum) flight periods across environments with both high and low temporal resource overlap. This approach reveals how interacting phenologies structure plant-pollinator networks and drive pollinator diet breadth via the temporal overlap of floral resources.

ecology↗

bioRxiv2019

PrimPol-dependent single-stranded gap formation mediates homologous recombination at bulky DNA adducts

Obstacles on the DNA template can lead to DNA replication fork stalling and genomic rearrangements. RAD51-mediated homologous recombination (HR) can promote restart and repair of stalled forks, but also post-replicative repair once the obstacle has been bypassed. Bulky DNA adducts are important replication-blocking lesions induced by environmental carcinogens, but it is not known whether they activate HR directly at stalled forks, or at gaps left behind ongoing forks. Here we show that in mammalian cells, bulky adducts predominantly induce HR at post-replicative gaps formed by the DNA/RNA primase PrimPol. Using BPDE and other bulky model lesions, we report that RAD51 is not recruited to stalled or collapsed forks, but instead to long gaps formed by PrimPol re-priming activity and resection by MRE11 and EXO1. In contrast, RAD51 loading at DSBs does not require PrimPol. At bulky adducts, PrimPol is required for the induction of sister chromatid exchanges and genetic recombination. Our data support that HR at bulky adducts in mammalian cells involves post-replicative gap repair and define a role for PrimPol in DNA damage tolerance by homologous recombination.

molecular biology↗

bioRxiv2018

The deSUMOylase SENP2 coordinates homologous recombination and non-homologous end joining by independent mechanisms.

SUMOylation in the DNA double-strand break (DSB) response regulates recruitment, activity and clearance of repair factors. However, our understanding of a role for deSUMOylation in this process is limited. Here we identify different mechanistic roles for deSUMOylation in homologous recombination (HR) and non-homologous enjoining (NHEJ) through the investigation of the deSUMOylase SENP2. We find regulated deSUMOylation of MDC1 prevents excessive SUMOylation and its RNF4-VCP mediated clearance from DSBs, thereby promoting NHEJ. In contrast we show HR is differentially sensitive to SUMO availability and SENP2 activity is needed to provide SUMO. SENP2 is amplified as part of the chromosome 3q amplification in many cancers. Increased SENP2 expression prolongs MDC1 foci retention and increases NHEJ and radioresistance. Collectively our data reveal that deSUMOylation differentially primes cells for responding to DSBs and demonstrates the ability of SENP2 to tune DSB repair responses.

cell biology↗

bioRxiv2018

PIN1 isomerisation of BRCA1 promotes replication fork protection.

BRCA1, BRCA2 and a subset of Fanconis Anaemia proteins act to promote RAD51-mediated protection of newly synthesised DNA at stalled replication forks from degradation by nucleases. How BRCA1 contributes, how it is regulated and whether replication fork protection relates to, or differs from, the roles BRCA1 has in homologous recombination is not clear. Here we show that the canonical BRCA1-PALB2 interaction is not required for fork protection and instead we demonstrate that the ability of BRCA1 to protect nascent DNA is regulated in an unexpected fashion through conformational change mediated by the phosphorylation-directed prolyl isomerase, PIN1. BRCA1 isomerisation enhances BRCA1-BARD1 interaction with RAD51 and consequently RAD51 presence at stalled replication structures. Our data suggest BRCA1-BARD1 promotes fork protection in part by enhancing the RAD51 synapse. Patient missense variants in the regulated BRCA1-BARD1 regions similarly show poor nascent strand protection but proficient homologous recombination, defining novel domains required for fork protection in BRCA1-BARD1 associated with cancer predisposition. Together these findings reveal a previously unrecognised pathway that governs BRCA1-mediated replication fork protection.

cell biology↗

bioRxiv2018

GSK3β-SCF-FBXW7 mediated phosphorylation and ubiquitination of IRF1 are required for its transcription-dependent turnover.

IRF1 (Interferon Regulatory Factor-1) is the prototype of the IRF family of DNA binding transcription factors. IRF1 protein expression is regulated by transient up-regulation in response to external stimuli followed by rapid degradation via the ubiquitin-proteasome system. Here we report that DNA bound IRF1 turnover is promoted by GSK3{beta} (Glycogen Synthase Kinase 3{beta}) via phosphorylation of the T181 residue which generates a phosphodegron for the SCF (Skp-Cul-Fbox) ubiquitin E3-ligase receptor protein Fbxw7 (F-box/WD40 7). This regulated turnover is essential for IRF1 activity, as mutation of T181 results in an improperly stabilised protein that accumulates at target promoters but fails to induce RNA-Pol-II elongation and subsequent transcription of target genes. Consequently, the anti-proliferative activity of IRF1 is lost in cell lines expressing T181A mutant. Further, cell lines with dysfunctional Fbxw7 are less sensitive to IRF1 overexpression, suggesting an important co-activator function for this ligase complex. As T181 phosphorylation requires both DNA binding and RNA-Pol-II elongation, we propose that this event acts to clear \"spent\" molecules of IRF1 from transcriptionally engaged target promoters.

molecular biology↗