Biology subjects

Murray, H.

Publications and source records attributed to Murray, H..

14 recordsLinked to original sources

bioRxiv2026

Decoding human sperm signalling: Phosphoproteomic discovery of kinases governing fertilization competency

Capacitation, the process whereby sperm gain the functional competence to fertilize an egg in the absence of de novo transcription and translation, is orchestrated by a hierarchy of kinases driving the phosphorylation of sperm proteins. While increased phosphorylation, in particular tyrosine phosphorylation, is a revered hallmark of fertilization competency in our species, only a limited repository of phosphorylated substrates and kinases have ever been reported from human sperm. To broaden therapeutic targets for sperm targeted contraceptives and infertility therapies, we adapted a contemporary phosphoproteomic technique termed EasyPhos to generate bespoke methodology for the investigation of human sperm signalling. This approach yielded high depth phosphoproteomes of non-capacitated and capacitated human spermatozoa with in silico investigation of the phosphosites revealing 52 kinases with previously uncharacterized roles in sperm capacitation. Investigating the function of the putative sperm capacitation kinases identified yielded several kinases with novel roles in the regulation of sperm function. Of particular interest, polo like kinase 1 (PLK1) inhibition significantly reduced progressive sperm motility, attenuated capacitation-associated tyrosine phosphorylation and reduced the sperm acrosome reaction, an essential step to achieve fertilization. These findings reveal extensive phosphoproteome remodelling during human sperm capacitation, expanding the landscape of molecular targets for fertility control.

Cell Biology↗

bioRxiv2025

Epitranscriptomic Dysregulation Underpins Artemisinin Mechanism of Action

Artemisinin has long been a first-line antimalarial. Yet, its mode of action is still poorly understood. Emergence of artemisinin-resistant strains highlight the importance of addressing this question so as to develop better drugs and overcome resistance. In this study, we performed RNA-sequencing and proteomics studies on artemisinin treated parasites indicated a striking difference in the codon-usage pattern of differentially translated genes. Using a liquid chromatography-coupled mass spectrometry (LC-MS)-based platform, we have quantified the full spectrum of modified ribonucleosides on tRNA in P. falciparum in response to the drug. We found that N6-threonyl-carbomyladenosine (t6A), a universal tRNA modification found at position 37 is hypomodified in response to artemisinin induced stress. Additionally, we also found that artemisinin treatment resulted in a downregulation of PfSua5, an enzyme involved in the t6A biosynthesis machinery. These findings provide new insights into how artemisinin works. More broadly, the findings exposes the tRNA epitranscriptome as a vulnerability in the parasite that can be exploited for new drugs.

microbiology↗

bioRxiv2025

Substrate stiffness and shear stress collectively regulate the inflammatory phenotype in cultured human brain microvascular endothelial cells

Brain endothelial cells experience mechanical forces in the form of blood flow-mediated shear stress and underlying matrix stiffness, but intersectional contributions of these factors towards blood-brain barrier (BBB) impairment and neurovascular dysfunction have not been extensively studied. Here, we developed in vitro models to examine the sensitivity of primary human brain microvascular endothelial cells (BMECs) to substrate stiffness, with or without exposure to fluid shear stress. Using a combination of molecular profiling techniques, we show that BMECs exhibit an inflammatory signature at both the mRNA and protein level when cultured on gelatin substrates of intermediate stiffness ([~]30 kPa) versus soft substrates ([~]6 kPa). Exposure to modest fluid shear stress (1.7 dyne/cm2) partially attenuated this signature, including reductions in levels of soluble chemoattractants and surface ICAM-1. Overall, our results indicate that increased substrate stiffness promotes an inflammatory phenotype in BMECs that is dampened in the presence of fluid shear stress.

neuroscience↗

bioRxiv2025

Rescuing bacterial genome replication: essential functions to repair a double-strand break and restart DNA synthesis

DNA damage occurs in all cells and must be repaired to maintain genome integrity. Many DNA lesions are targeted for removal by repair systems that excise the damage, thereby generating a temporary single-strand discontinuity in the chromosome. If DNA repair has not been completed prior to a round of genome duplication, the single-strand discontinuity (nick or gap) can be converted to a double-strand break (DSB) by an oncoming replication fork. Because the genomic location of nucleobase damage is stochastic, investigating the fate of replication machinery (replisome) at DNA repair sites with single-strand discontinuities has been limited. Here we have addressed this issue by expressing Cas9 nickases in Bacillus subtilis to create site specific single-strand discontinuities in a bacterial chromosome. We find that a nick in either leading or lagging strand arrests DNA replication, while the fate of the replicative helicase is distinct and depends upon the strand nicked. Genetic, biochemical, and single cell analyses indicate that replisome/nick encounters generate a single-end DSB which requires recombinational repair to enable PriA-dependent replication restart. Together this work defines the physiologically relevant pathway used by B. subtilis to reinitiate DNA synthesis following replication fork inactivation at a single-strand discontinuity. HighlightsO_LISingle-strand discontinuities inactivate the bacterial replisome C_LIO_LINicks on the leading or lagging strand template differentially affect fate of the helicase C_LIO_LIReplication forks are repaired via recombinational repair C_LIO_LIThe single strand binding protein (SSB) acidic tail is essential for replication restart C_LIO_LIDNA replication restart is PriA-dependent C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=182 SRC="FIGDIR/small/640380v2_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@10734e0org.highwire.dtl.DTLVardef@208eddorg.highwire.dtl.DTLVardef@183794aorg.highwire.dtl.DTLVardef@901628_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Conflicts between the DNA replication and repair machineries promote cell death in Gram-positive bacteria

Cellular proliferation relies on successful coordination and completion of genome replication and segregation. To help achieve this, many bacteria utilise regulatory pathways that ensure DNA replication initiation only occurs once per cell cycle. When dysregulated, loss of DNA replication control can have severe consequences. In Escherichia coli it has been established that hyper-initiation of DNA synthesis leads to pleiotropic genome instability and cell death. Therefore, targeting DNA replication initiation proteins to promote hyper-initiation may be an approach to generate novel antimicrobials. However, the pathways and potential consequences of replication hyper-initiation in Gram-positive species remain enigmatic. To address this question, we devised genetic systems to artificially induce hyper-initiation in the model organism Bacillus subtilis and the pathogen Staphylococcus aureus. In both species, hyper-initiation elicited cellular degeneration culminating in growth inhibition by cell death. During this process in B. subtilis, temporal analyses revealed the early onset of the DNA damage response, followed by membrane depolarisation and cell lysis. This phenotype could be supressed by removing pathways that repair damaged DNA, suggesting that cell death is a consequence of conflicts between DNA replication and repair. In S. aureus, cells quickly accumulated striking morphological changes associated with rapid loss of chromosomal DNA and death via a lysis-independent pathway. Moreover, inducing hyper-initiation in S. aureus was observed to decrease bacterial survival during infection of murine macrophages. Taken together, the data suggest that stimulating initiation of bacterial DNA synthesis could be an alternative approach to inhibiting microbial growth, particularly in combination with compounds that inhibit or poison DNA repair, akin to cancer therapies.

microbiology↗

bioRxiv2022

The DNA replication initiation protein DnaD is recruited to a specific strand of the Bacillus subtilis chromosome origin

Genome replication is a fundamental biological activity shared by all organisms. Chromosomal replication proceeds bidirectionally from origins, requiring the loading of two helicases, one for each replisome. The molecular mechanisms for helicase loading at bacterial chromosome origins (oriC) are unclear. Here we investigated the essential DNA replication initiation protein DnaD in the model organism Bacillus subtilis. A set of DnaD residues required for ssDNA binding was identified, and photo-crosslinking revealed that this ssDNA binding region interacts preferentially with one strand of oriC. Biochemical and genetic data support the model that DnaD recognizes a new single-stranded DNA (ssDNA) motif located in oriC (DnaD Recognition Element, "DRE"). Considered with cryo-electron microscopy (cryo-EM) imaging of full length DnaD, we propose that the location of the DRE within the oriC orchestrates strand-specific recruitment of helicase to achieve bidirectional DNA replication. These findings significantly advance our mechanistic understanding of bidirectional replication from a bacterial chromosome origin.

molecular biology↗

bioRxiv2022

SirA inhibits the essential DnaA:DnaD interaction to block helicase recruitment during Bacillus subtilis sporulation

Bidirectional DNA replication from a chromosome origin requires the asymmetric loading of two helicases, one for each replisome. Our understanding of the molecular mechanisms underpinning helicase loading at bacterial chromosome origins is incomplete. Here we report both positive and negative mechanisms for directing helicase recruitment in the model organism Bacillus subtilis. Systematic characterization of the essential initiation protein DnaD revealed distinct protein interfaces required for homo-oligomerization, interaction with the master initiator protein DnaA, and interaction with the helicase co-loader protein DnaB. Informed by these properties of DnaD, we went on to find that the developmentally expressed repressor of DNA replication initiation, SirA, blocks the interaction between DnaD with DnaA, thereby inhibiting helicase recruitment to the origin during sporulation. These results advance our understanding of the mechanisms underpinning DNA replication initiation in B. subtilis, as well as guiding the search for essential cellular activities to target for antimicrobial drug design.

molecular biology↗

bioRxiv2022

Molecular determinants of the Bacillus subtilis chromosome origin basal unwinding system

Genome duplication is essential for cell proliferation and DNA synthesis is generally initiated by dedicated replication proteins at specific loci termed origins. During DNA replication initiation in bacteria, the ubiquitous DnaA protein engages both double-strand DNA (dsDNA) and single-stranded DNA (ssDNA) at the chromosome origin (oriC) to promote DNA duplex unwinding. While the molecular basis for DnaA binding to a specific dsDNA element ("DnaA-box") has been established, the mechanism for DnaA binding to a specific ssDNA motif ("DnaA-trio") is unclear. Here we define specific steps of DnaA-trio engagement by Bacillus subtilis DnaA. Single-molecule total internal reflection fluorescence microscopy indicates that DnaA proteins are loaded onto DnaA-trios using DnaA-boxes located on a shared DNA polymer. Chemical modification of either the phosphodiester backbone or the nucleobases revealed that three DnaA-trio repeats proximal to DnaA-boxes are necessary and sufficient to promote DnaA-dependent strand separation, and that the amino group from the central nucleobase of the DnaA-trio is critical for this reaction. Finally, based on electrophoretic mobility shift assays, we propose that during replication initiation DnaA progresses from DnaA-boxes to nucleobase recognition at DnaA-trios before engaging the phosphodiester backbone and destabilizing the DNA duplex. These results provide new molecular insight into DnaA-dependent Bacterial Unwinding System (BUS) activity at a bacterial chromosome origin.

microbiology↗

bioRxiv2021

Chromosome remodelling by SMC/Condensin in B. subtilis is regulated by Soj/ParA during growth and sporulation

SMC complexes, loaded at ParB-parS sites, are key mediators of chromosome organization in bacteria. ParA/Soj proteins interact with ParB/Spo0J in a pathway involving ATP-dependent dimerization and DNA binding, leading to chromosome segregation and SMC loading. In Bacillus subtilis, ParA/Soj also regulates DNA replication initiation, and along with ParB/Spo0J is involved in cell cycle changes during endospore formation. The first morphological stage in sporulation is the formation of an elongated chromosome structure called an axial filament. We now show that a major redistribution of SMC complexes drives axial filament formation, in a process regulated by ParA/Soj. Unexpectedly, this regulation is dependent on monomeric forms of ParA/Soj that cannot bind DNA or hydrolyse ATP. These results reveal a new role for ParA/Soj proteins in the regulation of SMC dynamics in bacteria, and yet further complexity in the web of interactions involving chromosome replication, segregation, and organization, controlled by ParAB and SMC.

microbiology↗

bioRxiv2021

Enculturating a Community of Action (CoA): A qualitative study of health professions educators' perspectives on teaching with Wikipedia

PurposeThere is a growing desire for health professions educators to engage learners in more meaningful instruction. Many have tapped Wikipedia to offer an applied approach to engage learners, particularly as it relates to evidence-based medicine (EBM). However, little is known about the benefits and challenges of using Wikipedia as a pedagogical tool from the collective experience of educators who have sought to improve their instructional practice with it. This study aims to uncover and synthesize the perspectives of health professions education (HPE) instructors who have incorporated Wikipedia in their HPE courses. MethodsApplying a constructivist approach, the authors conducted semi-structured interviews with 17 participating HPE instructors who had substantively integrated Wikipedia into their curriculum. Participants were interviewed about their experiences of integrating Wikipedia editing into their courses. Thematic analysis was conducted on resulting transcripts. ResultsAuthors observed two broad themes among participants expressed benefits of teaching with Wikipedia: 1) provides a meaningful instructional alternative that also benefits society and develops learners information literacy and EBM skills, and 2) supports learners careers and professional identity formation. Identified challenges included: 1) high effort and time, 2) issues with sourcing references, and 3) challenging interactions with skeptics, editors, and students. DiscussionFindings build on known benefits, such as providing a real-world collaborative project that contextualizes students learning experiences. They also echo known challenges, such as the resource-intensive nature of teaching with Wikipedia. At the same time, findings extend the current literature by revealing a potential opportunity to approach crowd-sourced information tools, like Wikipedia, as a vehicle to engage and enculturate HPE students within a situated learning context. These findings present implications for HPE programs considering implementing Wikipedia and faculty development needed to help instructors harness crowd-sourced information tools pedagogical opportunities as well as anticipate their challenges.

scientific communication and education↗

bioRxiv2021

Positive and negative control of helicase recruitment at a bacterial chromosome origin

The mechanisms responsible for helicase loading during the initiation of chromosome replication in bacteria are unclear. Here we report both a positive and a negative mechanism for directing helicase recruitment in the model organism Bacillus subtilis. Systematic mutagenesis of the essential replication initiation gene dnaD and characterization of DnaD variants revealed protein interfaces required for interacting with the master initiator DnaA and with a specific single-stranded DNA (ssDNA) sequence located in the chromosome origin (DnaD Recognition Element, "DRE"). We propose that the location of the DRE within the replication origin orchestrates recruitment of helicase to achieve bidirectional DNA replication. We also report that the developmentally expressed repressor of DNA replication initiation, SirA, acts by blocking the interaction of DnaD with DnaA, thereby inhibiting helicase recruitment to the origin. These findings significantly advance our mechanistic understanding of helicase recruitment and regulation during bacterial DNA replication initiation. Because DnaD is essential for the viability of clinically relevant Gram-positive pathogens, DnaD is an attractive target for drug development.

microbiology↗

bioRxiv2019

Spatio-temporal control of DNA replication by the pneumococcal cell cycle regulator CcrZ

Most bacteria replicate and segregate their DNA concomitantly while growing, before cell division takes place. How bacteria synchronize these different cell cycle events to ensure faithful chromosome inheritance is poorly understood. Here, we identified a conserved and essential protein in pneumococci and related Firmicutes named CcrZ (for Cell Cycle Regulator protein interacting with FtsZ) that couples cell division with DNA replication by controlling the activity of the master initiator of DNA replication, DnaA. The absence of CcrZ causes mis-timed and reduced initiation of DNA replication, which subsequently results in aberrant cell division. We show that CcrZ from Streptococcus pneumoniae directly interacts with the cytoskeleton protein FtsZ to place it in the middle of the newborn cell where the DnaA-bound origin is positioned. Together, this work uncovers a new mechanism for the control of the bacterial cell cycle in which CcrZ controls DnaA activity to ensure that the chromosome is replicated at the right time during the cell cycle.

microbiology↗

bioRxiv2018

DNA replication initiation in Bacillus subtilis; Structural and functional characterisation of the essential DnaA-DnaD interaction

The homotetrameric DnaD protein is essential in low G+C content gram positive bacteria and is involved in replication initiation at oriC and re-start of collapsed replication forks. It interacts with the ubiquitously conserved bacterial master replication initiation protein DnaA at the oriC but structural and functional details of this interaction are lacking, thus contributing to our incomplete understanding of the molecular details that underpin replication initiation in bacteria. DnaD comprises N-terminal (DDBH1) and C-terminal (DDBH2) domains, with contradicting bacterial two-hybrid and yeast two-hybrid studies suggesting that either the former or the latter interact with DnaA, respectively. Using Nuclear Magnetic Resonance (NMR) we show that both DDBH1 and DDBH2 interact with the N-terminal domain I of DnaA and studied the DDBH2 interaction in structural detail by NMR. We revealed two families of conformations for the DDBH2-DnaA domain I complex and showed that the DnaA-interaction patch of DnaD is distinct from the DNA-interaction patch, suggesting that DnaD can bind simultaneously DNA and DnaA. Using sensitive single-molecule FRET techniques we revealed that DnaD remodels DnaA-DNA filaments consistent with stretching and/or untwisting. Furthermore, the DNA binding activity of DnaD is redundant for this filament remodelling. This in turn suggests that DnaA and DnaD are working collaboratively in the oriC to locally melt the DNA duplex during replication initiation.

biochemistry↗

bioRxiv2017

The C-Terminal Domain Of ParB Is Critical For Dynamic DNA Binding And Bridging Interactions Which Condense The Bacterial Centromere

The ParB protein forms DNA bridging interactions around parS to form networks which condense DNA and earmark the bacterial chromosome for segregation. The mechanism underlying the formation of ParB nucleoprotein complexes is unclear. We show here that the central DNA binding domain is essential for anchoring at parS, and that this interaction is not required for DNA condensation. Structural analysis of the C-terminal domain reveals a dimer with a lysine-rich surface that binds DNA non-specifically and is essential for DNA condensation in vitro. Mutation of either the dimerisation or the DNA binding interface eliminates ParB foci formation in vivo. Moreover, the free C-terminal domain can rapidly decondense ParB networks independently of its ability to bind DNA. Our work reveals a dual role for the C-terminal domain of ParB as both a DNA binding and bridging interface, and highlights the dynamic nature of ParB networks.

biochemistry↗