Biology subjects

Moreno-Herrero, F.

Publications and source records attributed to Moreno-Herrero, F..

18 recordsLinked to original sources

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↗

bioRxiv2026

FBH1 Reverses Stalled Replication Forks via Sequential Unwinding of Nascent Strands

Replication fork reversal is a DNA damage tolerance mechanism important for genome stability that entails annealing of parental DNA to push the fork backwards. F-box helicase 1 (FBH1) is a 3'-5' ssDNA translocase and SCF (SKP-CUL1-F box) E3 ubiquitin ligase that catalyzes fork reversal and limits aberrant recombination, yet how its helicase activity drives strand annealing is unknown. Here, using single-molecule and biochemical assays, we show that SCFFBH1 reverses forks through a two-stage reaction in which translocation on the lagging strand template while remaining affixed at the junction destabilizes the leading strand duplex to ultimately displace the nascent leading strand. Reversal is force-sensitive and does not generate a four-way junction, revealing an annealing-independent mechanism distinct from those of SMARCAL1, HLTF, and ZRANB3. These results establish the importance of nascent strand unwinding to fork reversal and suggest the existence of distinct pathways that produce unique DNA structures, which has implications for fork restart and its measurement in cells.

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↗

bioRxiv2025

RPA directly stimulates Mer3/HFM1 helicase processivity to ensure normal crossover formation in meiosis

Meiotic crossover formation is critical for generating viable gametes and enhancing genetic diversity. The helicase Mer3 (HFM1 in humans) is a highly conserved factor essential for promoting crossovers and ensuring their proper distribution. Here, we identify replication protein A (RPA) as a direct interactor of budding yeast Mer3. We demonstrate that this interaction is conserved between human HFM1 and RPA. Cross-linking mass spectrometry and structural modelling with AlphaFold2 reveal a conserved and specific Mer3-RPA interface. Single-molecule magnetic tweezers assays demonstrate that direct RPA interaction is required for Mer3 helicase processivity under conditions of low DNA tension. Consistently, a mer3 mutant deficient in RPA binding exhibit reduced crossover frequencies and accumulate unresolved recombination intermediates during budding yeast meiosis. Via genome-wide localisation experiments, we link this effect to a weakened recruitment to double-strand break sites of the mer3 mutant. Our findings provide mechanistic insights into coordination of meiotic recombination by the Mer3 helicase through interactions with the canonical DNA repair machinery, highlighting a conserved mechanism underlying crossover control during sexual reproduction.

biochemistry↗

bioRxiv2024

Auto-regulation of the real-time kinetics of the human mitochondrial replicative helicase

The human mitochondrial replicative helicase, Twinkle, is essential for the replication and integrity of mitochondrial DNA (mtDNA). Therefore, investigating the real-time kinetics of Twinkles activities and their regulation is crucial for understanding mtDNA maintenance. Here, we combine biochemical and single-molecule manipulation and visualization techniques to investigate the loading of Twinkle onto the DNA fork, its real-time DNA unwinding and rewinding kinetics, and the regulation of these processes by the amino- (N) and carboxyl- (C) terminal ends of the helicase and the mitochondrial SSB protein (mtSSB). We observed that Twinkle rapidly diffuses along dsDNA scanning for the DNA fork, where it establishes specific interactions that halt further diffusion. Our results show that during DNA unwinding, the real-time kinetics of the helicase are downregulated by interactions of its N-terminal Zinc-binding domain (ZBD) with DNA and the control of the ATPase activity by the C-terminal tail. We found that binding of mtSSB to DNA likely outcompetes the ZBD-DNA interactions, alleviating the down regulatory effects of this domain. Furthermore, we show that ZBD-DNA interactions, together with the ATP binding, also control the real-time kinetics of the DNA rewinding events that follow helicase stalling. Our findings reveal that ZBD and C-terminal tail play a major role in regulation of Twinkles real-time kinetics. Their interplay constitutes an auto-regulatory mechanism that may be crucial for coordination of the mtDNA maintenance activities of the helicase.

biophysics↗

bioRxiv2024

Assembly of a segrosome by a CTP-independent ParB-like protein

The ATP- and CTP-dependent ParA-ParB-parS segrosome is a macromolecular complex that segregates chromosomes/plasmids in most bacterial species. CTP binding and hydrolysis enable ParB to slide on DNA and to bridge and condense DNA, thereby dictating the size and dynamics of the tripartite ParABS complex. Several other evolutionarily distinct systems can also segregate DNA, although the full diversity of bacterial DNA partition systems is not yet known. Here, we identify a CTP-independent ParABS system that maintains a conjugative plasmid SCP2 in the filamentous bacterium Streptomyces coelicolor. We demonstrate that an SCP2 ParB-like protein, ParT, loads onto DNA at an 18-bp parS site and diffuses away to the adjacent DNA despite lacking an apparent CTPase domain and detectable NTPase activity. We further show that parS DNA stimulates ParT transition from loading to a diffusing state to accumulate on DNA, and ParT activates the ATPase activity of its cognate partner protein ParA. We also identify numerous structural homologs of ParT, suggesting that CTP-independent diffusion on DNA might be widespread in bacteria despite being previously unappreciated. Overall, our findings uncover a CTP-independent DNA translocation as an alternative and unanticipated mechanism for the assembly of a bacterial DNA segregation complex and suggest that CTP binding and hydrolysis is not a fundamental feature of ParABS-like systems.

microbiology↗

bioRxiv2024

Molecular switching of a DNA-sliding clamp to a repressor mediates long-range gene silencing

Long-range gene regulation is rare in bacteria and is confined to the classical DNA looping model. Here, we use a combination of biophysical approaches, including X-ray crystallography and single-molecule analysis, to show that long-range gene silencing on the plasmid RK2, a source of multidrug resistance across diverse Gram-negative bacteria, is achieved cooperatively by a DNA-sliding clamp, KorB, and a clamp-locking protein, KorA. We find that KorB is a CTPase clamp that can entrap and slide along DNA to reach distal target promoters. We resolved the tripartite crystal structure of a KorB-KorA-DNA co-complex, revealing that KorA latches KorB into a closed-clamp state. KorA thus stimulates repression by stalling KorB sliding at target promoters to occlude RNA polymerase holoenzymes. Altogether, our findings explain the mechanistic basis for KorB role-switching from a DNA-sliding clamp to a co-repressor, and provide a new paradigm for the long-range regulation of gene expression.

microbiology↗

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↗

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

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

Double-stranded RNA bending by AU-tract sequences

Sequence-dependent structural deformations of the DNA double helix (dsDNA) have been extensively studied, where adenine tracts (A-tracts) provide a striking example for global bending in the molecule. In contrast to dsDNA, much less is known about how the nucleotide sequence affects bending deformations of double-stranded RNA (dsRNA). Using all-atom microsecond long molecular dynamics simulations we found a sequence motif consisting of alternating adenines and uracils, or AU-tracts, that bend the dsRNA helix by locally compressing the major groove. We experimentally tested this prediction using atomic force microscopy (AFM) imaging of long dsRNA molecules containing phased AU-tracts. AFM images revealed a clear intrinsic bend in these AU-tracts molecules, as quantified by a significantly lower persistence length compared to dsRNA molecules of arbitrary sequence. The bent structure of AU-tracts here described might play a role in sequence-specific recognition of dsRNAs by dsRNA-interacting proteins or impact the folding of RNA into intricate tertiary and quaternary structures.

biophysics↗

bioRxiv2019

Understanding the paradoxical mechanical response of in-phase A-tracts at different force regimes

A-tracts are A:T rich DNA sequences that exhibit unique structural and mechanical properties associated with several functions in vivo. The crystallographic structure of A-tracts has been well characterized. However, their response to forces remains unknown and the variability of their flexibility reported for different length scales has precluded a comprehensive description of the mechanical properties of these molecules. Here, we rationalize the mechanical properties of A-tracts across multiple length scales using a combination of single-molecule experiments and theoretical polymer models applied to DNA sequences present in the C. elegans genome. Atomic Force Microscopy imaging shows that phased A-tracts induce long-range ([~]200 nm) bending. Moreover, the enhanced bending originates from an intrinsically bent structure rather than as a consequence of larger flexibility. In support of this, our data were well described with a theoretical model based on the worm-like chain model that includes intrinsic bending. Magnetic tweezers experiments confirm that the observed bent is intrinsic to the sequence and does not rely on particular ionic conditions. Using optical tweezers, we assess the local rigidity of A-tracts at high forces and unravel an unusually stiff character of these sequences, as quantified by their large stretch modulus. Our work rationalizes the complex multiscale flexibility of A-tracts, shedding light on the cryptic character of these sequences.

biophysics↗

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↗

bioRxiv2019

Structural and biochemical analyses of Caulobacter crescentus ParB reveal the role of its N-terminal domain in chromosome segregation

Proper chromosome segregation is essential in all living organisms. The ParA-ParB-parS system is widely employed for chromosome segregation in bacteria. Previously, we showed that Caulobacter crescentus ParB requires cytidine triphosphate to escape the nucleation site parS and spread by sliding to the neighboring DNA 1. Here, we provide the structural basis for this transition from nucleation to spreading by solving co-crystal structures of a C-terminal domain truncated C. crescentus ParB with parS and with a CTP analog. Nucleating ParB is an open clamp, in which parS is captured at the DNA-binding domain (the DNA-gate). Upon binding CTP, the N-terminal domain (NTD) self-dimerizes to close the NTD-gate of the clamp. The DNA-gate also closes, thus driving parS into a compartment between the DNA-gate and the C-terminal domain. CTP hydrolysis and/or the release of hydrolytic products are likely associated with re-opening of the gates to release DNA and to recycle ParB. Overall, we suggest a CTP-operated gating mechanism that regulates ParB nucleation, spreading, and recycling.

microbiology↗

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↗

bioRxiv2018

DNA crookedness regulates DNA mechanical properties at short length scales

Sequence-dependent DNA conformation and flexibility play a fundamental role in specificity of DNA-protein interactions. Here we quantify the DNA crookedness: a sequence-dependent deformation of DNA that consists on periodic bends of the base pair centers chain. Using molecular dynamics simulations, we found that DNA crookedness and its associated flexibility are bijective: unveiling a one-to-one relation between DNA structure and dynamics. This allowed us to build a predictive model to compute DNA stretching stiffness from solely its structure. Sequences with very little crookedness show extremely high stiffness and have been previously shown to form unstable nucleosomes and promote gene expression. Interestingly, the crookedness can be tailored by epigenetic modifications, known to affect gene expression. Our results rationalize the idea that the DNA sequence is not only a chemical code, but also a physical one that allows to finely regulate its mechanical properties and, possibly, its 3D arrangement inside the cell.

biophysics↗

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↗