Biology subjects

Dillingham, M. S.

Publications and source records attributed to Dillingham, M. S..

16 recordsLinked to original sources

bioRxiv2026

Tripartite toxin-antitoxin system regulates the production and release of pneumolysin by Streptococcus pneumoniae.

The production of toxins is critical to the success of bacteria, with their targets and outcomes varying widely. Pneumolysin (Ply), produced by the major human pathogen Streptococcus pneumoniae, is a cytolytic pore forming toxin that plays many diverse roles including inducing tissue damage and inflammation, evasion of many aspects of the human immune system, invasion of the bacteria into deeper tissue, and transmission to new hosts. Despite its importance, many aspects of the biology of this toxin have to date remained unknown, in particular, what role of the three other genes that form part of the ply operon play in Ply production. Here we demonstrate that these genes encode a complex regulatory system, that includes a putative membrane destabilising protein (PlyA) and a MazEF-like toxin-antitoxin system (PlyB and PlyC), the toxin of which (PlyC) destabilises the Ply transcript. We propose a tripartite model whereby when the operon reaches relatively high levels of expression PlyA destabilises the S. pneumoniae membrane creating a stress that triggers a necessary negative feedback loop which results in the degradation of the PlyB antitoxin, release of the PlyC toxin and degradation of the ply transcript, thereby providing a means by which Ply production can be tightly regulated. Given the importance of Ply to the ability of S. pneumoniae to cause infection, identifying the factors that result in the shutdown of Ply production and release by the bacteria would seem promising target for future therapeutic strategies.

microbiology↗

bioRxiv2026

Structural elements required for the efficient loading and activation of HELB on RPA-coated single-stranded DNA

HELB is a human helicase involved in DNA repair and replication that interacts physically with the single-stranded DNA binding protein RPA. ATP-dependent translocation of HELB along ssDNA results in the active displacement of RPA molecules and the formation of ssDNA loops, suggesting that HELB contains at least two DNA binding sites. In this work, we investigated the role of HELB-specific structural elements in facilitating interactions between HELB and RPA-coated DNA. We show that a predicted OB-fold in the N-terminal region of the protein is important both for loop extrusion and RPA displacement. We confirm that a HELB-specific-motif within the RecA-like helicase/translocase domains is critical for binding RPA in solution but that, once HELB is bound to ssDNA, is dispensable for RPA displacement. We propose a model for RPA displacement in which both structural elements play important roles in the recruitment and activation of HELB at RPA-ssDNA filaments. SIGNIFICANCE STATEMENTSingle-stranded DNA generated during replication and repair is rapidly coated by replication protein A (RPA), creating a protected filament that must nevertheless remain accessible to DNA-processing enzymes. We show that the human DNA helicase HELB uses two specialised structural elements to overcome this problem. A HELB-specific RPA-binding motif promotes recruitment, whereas a predicted OB domain enables DNA looping and efficient RPA displacement. Removing these elements modestly enhances activity on naked ssDNA while impairing loading and activation on RPA- coated DNA. This reveals how helicase accessory domains restrain inappropriate motor activity while targeting the intended nucleoprotein substrate. Human HELB variants linked to reproductive ageing map to these regulatory regions, suggesting a connection between impaired RPA dynamics and reproductive health.

biochemistry↗

bioRxiv2025

OT-Curtains: an approach for studying protein interactions with DNA-ends using optical tweezers and confocal fluorescence microscopy

DNA ends generated by double-strand breaks are vulnerable intermediates that must be rapidly recognized, protected and resolved to preserve genome integrity. We present OT-Curtains, a single-molecule method inspired by DNA Curtains that uses a custom branched DNA substrate containing multiple accessible ends for simultaneous observation on dual-trap optical tweezers coupled to confocal fluorescence microscopy. Eliminating DNA surface anchoring, facilitating rapid protein and buffer exchange, and offering force-free experiments, OT-Curtains overcomes common limitations of flow-stretch-based methods. OT-Curtains allows real-time visualization and quantification of end recognition, protection, resection and cleavage at several DNA ends in parallel. We demonstrate compatibility with well-studied DNA binding systems by monitoring Ku-mediated DNA break recognition, AddAB-mediated DNA break resection, ParB-mediated DNA condensation and KpnI-mediated DNA cleavage. We show that kinetic and mechanistic parameters can be extracted from the data under defined forces and solution conditions. OTCurtains offers an accessible and multiplexed route to interrogate DNA-end transactions central to double-stranded DNA break repair pathways and telomere biology, as well as a general framework for benchmarking proteins acting at DNA ends.

biophysics↗

bioRxiv2024

Mechanism of DNA entrapment by the MukBEF SMC complex and its inhibition by a viral DNA mimic

Ring-like structural maintenance of chromosomes (SMC) complexes are crucial for genome organization and operate through mechanisms of DNA entrapment and loop extrusion. Here, we explore the DNA loading process of the bacterial SMC complex MukBEF. Using electron cryomicroscopy (cryo-EM), we demonstrate that ATP binding opens one of MukBEFs three potential DNA entry gates, exposing a DNA capture site that positions DNA at the open neck gate. We discover that the gp5.9 protein of bacteriophage T7 blocks this capture site by DNA mimicry, thereby preventing DNA loading and inactivating MukBEF. We propose a comprehensive and unidirectional loading mechanism in which DNA is first captured at the complexs periphery and then ingested through the DNA entry gate, powered by a single cycle of ATP hydrolysis. These findings illuminate a fundamental aspect of how ubiquitous DNA organizers are primed for genome maintenance and demonstrate how this process can be disrupted by viruses. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=193 HEIGHT=200 SRC="FIGDIR/small/616235v1_ufig1.gif" ALT="Figure 1"> View larger version (68K): org.highwire.dtl.DTLVardef@165a949org.highwire.dtl.DTLVardef@14d1302org.highwire.dtl.DTLVardef@11afb6forg.highwire.dtl.DTLVardef@71694b_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

DNA binding and bridging by human CtIP in the healthy and diseased states

The human DNA repair factor CtIP helps to initiate the resection of double-stranded DNA breaks for repair by homologous recombination, in part through its ability to bind and bridge DNA molecules. However, CtIP is a natively disordered protein that bears no apparent similarity to other DNA-binding proteins and so the structural basis for these activities remains unclear. In this work, we have used bulk DNA binding, single molecule tracking, and DNA bridging assays to study wild-type and variant CtIP proteins to better define the DNA binding domains and the effects of mutations associated with inherited human disease. Our work identifies a monomeric DNA-binding domain in the C-terminal region of CtIP. CtIP binds non-specifically to DNA and can diffuse over thousands of nucleotides. CtIP-mediated bridging of distant DNA segments is observed in single-molecule magnetic tweezers experiments. However, we show that binding alone is insufficient for DNA bridging, which also requires tetramerization via the N-terminal domain. Variant CtIP proteins associated with Seckel and Jawad syndromes display impaired DNA binding and bridging activities. The significance of these findings in the context of facilitating DNA break repair is discussed. Significance StatementCtIP helps to repair broken chromosomes through its ability to bind and bridge DNA molecules. We studied the structural and biochemical basis for these activities and how they are affected by hereditary CtIP mutations associated with developmental disorders. We discovered a minimal domain in the C-terminal region of CtIP which supports DNA binding as a monomer. DNA binding is non-specific and facilitates 1D diffusion, but binding alone is insufficient for intermolecular tethering of DNA molecules which requires tetramerization of CtIP via N-terminal coiled-coil domains. All disease variants tested displayed impaired DNA bridging activity. These results have important implications for understanding the role of CtIP as a hub protein for DNA break repair and its dysfunction in human disease.

biochemistry↗

bioRxiv2022

Structures of RecBCD in complex with phage-encoded inhibitor proteins reveal distinctive strategies for evasion of a bacterial immunity hub

Following infection of bacterial cells, bacteriophage modulate double-stranded DNA break repair pathways to protect themselves from host immunity systems and prioritise their own recombinases. Here we present biochemical and structural analysis of two phage proteins, gp5.9 and Abc2, which target the DNA break resection complex RecBCD. These exemplify two contrasting mechanisms for control of DNA break repair in which the RecBCD complex is either inhibited or co-opted for the benefit of the invading phage. Gp5.9 completely inhibits RecBCD by preventing it from binding to DNA. The RecBCD-gp5.9 structure shows that gp5.9 acts by substrate mimicry, binding predominantly to the RecB arm domain and competing sterically for the DNA binding site. Gp5.9 adopts a parallel coiled-coil architecture that is unprecedented for a natural DNA mimic protein. In contrast, binding of Abc2 does not substantially affect the biochemical activities of isolated RecBCD. The RecBCD-Abc2 structure shows that Abc2 binds to the Chi-recognition domains of the RecC subunit in a position that might enable it to mediate the loading of phage recombinases onto its single-stranded DNA products.

biochemistry↗

bioRxiv2021

Towards a molecular mechanism underlying mitochondrial protein import through the TOM-TIM23 complex

Nearly all mitochondrial proteins need to be targeted for import from the cytosol. For the majority, the first port of call is the translocase of the outer membrane (TOM complex), followed by a procession of alternative molecular machines, conducting transport to their final destination. The pre-sequence translocase of the inner-membrane (TIM23-complex) imports proteins with cleavable pre-sequences, and comes in two flavours: the TIM23SORT complex mediates inner mitochondrial membrane (IMM) protein insertion and the TIM23MOTOR complex delivers proteins to the matrix. Progress in understanding these transport mechanisms has been hampered by the poor sensitivity and time-resolution of import assays. However, with the development of an assay based on split NanoLuc luciferase, we can now explore this process in greater detail. Here, we apply this new methodology to understand how {Delta}{psi} and ATP hydrolysis, the two main driving forces for import through the TIM23MOTOR complex, contribute to the import of pre-sequence-containing precursors (PCPs) with varying properties. Notably, we found that two major rate-limiting steps define PCP import time: passage of PCP across the OMM and initiation of IMM transport by the pre-sequence. The rates of these steps are influenced by PCP properties such as size and net charge, but not total amount of PCP imported - emphasising the importance of collecting rapid kinetic data, achieved here, to elucidate mechanistic detail. The apparent distinction between transport through the two membranes (passage through TOM is substantially complete before PCP-TIM engagement) is in contrast with the current view that import occurs through TOM and TIM in a single continuous step. Our results also indicate that PCPs spend very little time in the TIM23 channel - presumably rapid success or failure of import is critical for maintaining mitochondrial fitness.

biochemistry↗

bioRxiv2021

DNA mismatch and damage detection using a FRET-based assay for monitoring the loading of multiple MutS sliding clamps

We developed a sensitive, homogeneous fluorescence assay for the detection of DNA mismatches and DNA damage based on the mismatch repair (MMR) protein MutS. The assay is based on Forster resonance energy transfer (FRET) between SYBR Green I (SG), non-covalently bound to DNA, and Alexa Fluor 647 (AF647) conjugated to MutS. In contrast to previous assays using only the mismatch binding activity of MutS, we exploited the ATP-dependent loading of multiple MutS sliding clamps provoked by mismatch/damage to the DNA, which increases the overall sensitivity of the assay. The assay was validated using a well-characterized 3 kb circular DNA containing a single G/T mismatch. We also demonstrate that treatment of long (multiple kb) DNA with various chemical or physical agents including non-denaturing bisulfite conversion of cytosine to uracil, cisplatin modification or ultraviolet light (UVC) results in changes in the DNA that can be detected by the FRET-based MutS biosensor.

biochemistry↗

bioRxiv2021

Human HELB is a processive motor protein which catalyses RPA clearance from single-stranded DNA

Human HELB is a poorly-characterised helicase suggested to play both positive and negative regulatory roles in DNA replication and recombination. In this work, we used bulk and single molecule approaches to characterise the biochemical activities of HELB protein with a particular focus on its interactions with RPA and RPA-ssDNA filaments. HELB is a monomeric protein which binds tightly to ssDNA with a site size of [~]20 nucleotides. It couples ATP hydrolysis to translocation along ssDNA in the 5'-to-3' direction accompanied by the formation of DNA loops and with an efficiency of 1 ATP per base. HELB also displays classical helicase activity but this is very weak in the absence of an assisting force. HELB binds specifically to human RPA which enhances its ATPase and ssDNA translocase activities but inhibits DNA unwinding. Direct observation of HELB on RPA nucleoprotein filaments shows that translocating HELB concomitantly clears RPA from single-stranded DNA.

molecular biology↗

bioRxiv2021

Analysis of the PcrA-RNA polymerase complex reveals a helicase interaction motif and a role for PcrA/UvrD helicase in the suppression of R-loops.

The PcrA/UvrD helicase binds directly to RNA polymerase (RNAP) but the structural basis for this interaction and its functional significance have remained unclear. In this work we used biochemical assays and hydrogen-deuterium exchange coupled to mass spectrometry to study the PcrA-RNAP complex. We find that PcrA binds tightly to a transcription elongation complex in a manner dependent on protein:protein interaction with the conserved PcrA C-terminal Tudor domain. The helicase binds predominantly to two positions on the surface of RNAP. The PcrA C-terminal domain engages a conserved region in a lineage-specific insert within the {beta} subunit which we identify as a helicase interaction motif present in many other PcrA partner proteins, including the nucleotide excision repair factor UvrB. The catalytic core of the helicase binds near the RNA and DNA exit channels and blocking PcrA activity in vivo leads to the accumulation of R-loops. We propose a role for PcrA as an R-loop suppression factor that helps to minimise conflicts between transcription and other processes on DNA including replication.

biochemistry↗

bioRxiv2021

Cytidine triphosphate promotes efficient ParB-dependent DNA condensation by facilitating one-dimensional spreading from parS

Faithful segregation of bacterial chromosomes relies on the ParABS partitioning system and the SMC complex. In this work, we used single molecule techniques to investigate the role of cytidine triphosphate (CTP) binding and hydrolysis in the critical interaction between centromere-like parS DNA sequences and the ParB CTPase. Using a combined dual optical tweezers confocal microscope, we observe the specific interaction of ParB with parS directly. Binding around parS is enhanced 4-fold by the presence of CTP or the non-hydrolysable analogue CTP{gamma}S. However, ParB proteins are also detected at a lower density in distal non-specific regions of DNA. This requires the presence of a parS loading site and is prevented by roadblocks on DNA, consistent with one dimensional diffusion by a sliding clamp. Magnetic tweezers experiments show that the spreading activity, which has an absolute requirement for CTP binding but not hydrolysis, results in the condensation of parS-containing DNA molecules at low nanomolar protein concentrations. We propose a model in which ParB-CTP-Mg2+ complexes move along DNA following loading at parS sites and protein:protein interactions result in the localised condensation of DNA within ParB networks.

biophysics↗

bioRxiv2020

Baculovirus-vectored precision delivery of large DNA cargoes in human genomes

Precise gene editing and genome engineering by CRISPR technology requires simultaneous delivery of multiple DNA-encoded components into living cells rapidly exceeding the cargo capacity of currently utilized viral vector systems. Here we exploit the unmatched heterologous DNA cargo capacity of baculovirus to resolve this bottleneck. We implement hybrid DNA techniques (MultiMate) for rapid and error-free assembly of currently up to 25 functional DNA modules in a single baculoviral vector enabling CRISPR-based genome engineering. Utilizing homology-independent targeted integration (HITI), we achieve up to 30% correct genome interventions in human cells, including precision docking of large DNA payloads in the ACTB locus. We demonstrate baculovirus-vectored delivery of prime-editing toolkits for seamless DNA search-and-replace interventions achieving, with a single vector, highly efficient cleavage-free trinucleotide insertion in the HEK3 locus without any detectable indels. Our approach thus unlocks a wide range of editing and engineering applications in human cell genomes.

synthetic biology↗

bioRxiv2020

Refined measurement of SecA-driven protein transport reveals indirect coupling to ATP turnover

The universally conserved Sec system is the primary method cells utilise to transport proteins across membranes. Until recently, measuring the activity - a prerequisite for understanding how biological systems works - has been limited to discontinuous protein transport assays with poor time resolution, or used as reporters large, non-natural tags that interfere with the process. The development of an assay based on a split super-bright luciferase (NanoLuc) changed this. Here, we exploit this technology to unpick the steps that constitute post-translational transport in bacteria. Under the conditions deployed, transport of the model pre-protein substrate proSpy occurs at 200 amino acids per minute with the data best fit by a series of large, [~]30 amino acid, steps each coupled to many (100s) ATP hydrolysis events. Prior to that, there is no evidence for a distinct, rate-limiting initiation event. Kinetic modelling suggests that SecA-driven transport activity is facilitated by the substrate (polypeptide) concentration gradient - in keeping with classical membrane transporters. Furthermore, the features we describe are consistent with a non-deterministic motor mechanism, such as a Brownian ratchet.

biochemistry↗

bioRxiv2019

Bulk and single-molecule analysis of a novel DNA2-like helicase-nuclease reveals a single-stranded DNA looping motor

DNA2 is an essential enzyme involved in DNA replication and repair in eukaryotes. In a search for homologues of this protein, we identified and characterised Geobacillus stearothermophilus Bad, a novel bacterial DNA helicase-nuclease with similarity to human DNA2. We show that Bad contains an Fe-S cluster and identify four cysteine residues that are likely to co-ordinate the cluster by analogy to DNA2. The purified enzyme specifically recognises ss-dsDNA junctions and possesses ssDNA-dependent ATPase, ssDNA binding, ssDNA endonuclease, 5 to 3 ssDNA translocase and 5 to 3 helicase activity. Single molecule analysis reveals that Bad is a highly processive DNA motor capable of moving along DNA for distances of more than 4 kbp at a rate of [~]200 base pairs per second at room temperature. Interestingly, as reported for the homologous human and yeast DNA2 proteins, the DNA unwinding activity of Bad is cryptic and can be unmasked by inactivating the intrinsic nuclease activity. Strikingly, our experiments also show that the enzyme loops DNA while translocating, which is an emerging feature of highly processive DNA unwinding enzymes. The bacterial Bad enzymes will provide an excellent model system for understanding the biochemical properties of DNA2-like helicase-nucleases and DNA looping motor proteins in general.

biochemistry↗

bioRxiv2018

ParB dynamics and the critical role of the CTD in DNA condensation unveiled by combined force-fluorescence measurements

Bacillus subtilis ParB forms multimeric networks involving non-specific DNA binding leading to DNA condensation. In our previous work (Fisher et al., 2017), we found that an excess of the free C-terminal domain (CTD) of ParB impeded DNA condensation or promoted decondensation of pre-assembled networks. However, interpretation of the molecular basis for this phenomenon was complicated by our inability to uncouple protein binding from DNA condensation. Here, we have combined lateral magnetic tweezers with TIRF microscopy to simultaneously control the restrictive force against condensation and to visualize ParB protein binding by fluorescence. At non-permissive forces for condensation, ParB binds non-specifically and highly dynamically to DNA. Our new approach concluded that the free CTD blocks the formation of ParB networks by heterodimerization with full length DNA-bound ParB. This strongly supports a model in which the CTD acts as a key bridging interface between distal DNA binding loci within ParB networks.\n\nSignificance StatementUsing combined Magnetic Tweezers and TIRF microscopy we show that the CTD of ParB blocks ParB network formation by heterodimerization with the full-length protein, which remains bound to the DNA.

molecular biology↗

bioRxiv2018

Human CtIP forms a tetrameric dumbbell-shaped particle which binds and bridges complex DNA end structures for double-strand break repair

CtIP is involved in the resection of double-stranded DNA breaks during the S and G2 phases of the cell cycle for repair by homologous recombination. Acting in concert with the MRN complex, it plays a particularly important role in handling complex DNA end structures by localised nucleolytic processing of DNA termini in preparation for longer range resection. Here we show that human CtIP is a tetrameric protein adopting a dumbbell architecture in which DNA binding domains are connected by long coiled-coils. The protein complex binds two short DNA duplexes with high affinity and bridges DNA molecules in trans. DNA binding is potentiated by dephosphorylation and is not specific for DNA end structures per se. However, the affinity for linear DNA molecules is increased if the DNA terminates with more complex structures including forked ssDNA overhangs and model nucleoprotein conjugates. This work provides a biochemical and structural basis for the function of CtIP at complex DNA breaks.

biochemistry↗