Biology subjects

Dekker, N. H.

Publications and source records attributed to Dekker, N. H..

10 recordsLinked to original sources

bioRxiv2026

Clonal autoantibodies identify microbial antigen as trigger of autoreactive B cells in systemic sclerosis

ObjectivesTransformative observations demonstrate unprecedented success of B cell-depleting interventions in many human autoimmune diseases, calling for a deeper understanding of the triggers leading to B cell-mediated autoimmunity and its perpetuation in human disease. Here, we investigated whether the autoreactive B cell response targeting human topoisomerase 1 (TOP1), a hallmark of systemic sclerosis, could cross-react with TOP1 of microbial origin. MethodsHomologies between human and microbial TOP1 were analyzed using Foldseek. TOP1-reactive monoclonal antibodies from patient-derived, human TOP1-reactive B cell receptors were generated and assessed for reactivity against human TOP1 and TOP1 from a prototypic yeast, Saccharomyces cerevisiae (S. cerevisiae). Reactivity of polyclonal serum IgG from anti-TOP1 autoantibody (ATA)+, anti-centromere autoantibody (ACA)+ SSc patients and healthy donors (HDs) was tested. Finally, B cell lines were generated expressing human ATA to study B cell activation upon antigenic stimulation. ResultsStructural homologues of human TOP1 were found in many microbes, particularly in fungi. Taking TOP1 from S. cerevisiae as a prototype, microbial TOP1 was recognized by polyclonal patient IgG and by several monoclonal ATAs. Importantly, S. cerevisiae TOP1 also activated B cells expressing a patient-derived, human TOP1-reactive B cell receptor. Patients affected by interstitial lung disease most frequently showed recognition of microbial TOP1. ConclusionsThese findings identify fungi as potential drivers of immune dysregulation in human autoimmunity, specifically in SSc, highlighting microbial antigen cross-reactive cells as important therapeutic targets. Moreover, these data provide first functional evidence for a breach of B cell tolerance against human TOP1 triggered by cross-reactivity to fungal TOP1.

immunology↗

bioRxiv2024

Cell Cycle Regulation has Shaped Budding Yeast Replication Origin Structure and Function

Eukaryotic DNA replication initiates from multiple genomic loci known as origins. At budding yeast origins like ARS1, a double hexamer (DH) of the MCM replicative helicase is assembled by Origin Recognition Complex (ORC), Cdc6 and Cdt1 via sequential hexamer loading from two opposed ORC binding sites. Cyclin Dependent Kinase (CDK) inhibits DH assembly, which prevents re-replication by restricting helicase loading to G1 phase. Here we show that an intrinsically disordered region (IDR) in the Orc2 subunit promotes interaction between ORC and the first loaded, closed-ring MCM hexamer (the MO intermediate); CDK phosphorylation of this IDR blocks MO formation and DH assembly. We show that MO functions by stabilising ORC at the lower affinity binding sites required for second hexamer loading. Origins comprising two high affinity ORC sites can assemble DH efficiently without MO by independently loading single hexamers; these origins escape CDK inhibition in vitro and in vivo. Our work reveals mechanistic plasticity in MCM loading with implications for understanding how CDK regulation has shaped yeast origin evolution and how natural origins might escape cell cycle regulation. We also identify key steps common to loading pathways, with implications for understanding how MCM is loaded in other eukaryotes.

biochemistry↗

bioRxiv2023

A chromatinized origin reduces ORC mobility and spatially constrains MCM

Chromatin replication involves the assembly and activity of the replisome within the nucleosomal landscape. At the core of the replisome is the Mcm2-7 complex (MCM), which is loaded onto DNA after binding to the Origin Recognition Complex (ORC). In yeast, ORC is a dynamic protein that diffuses rapidly along DNA, unless halted by origin recognition sequences. However, less is known about the dynamics of ORC proteins in the presence of nucleosomes and attendant consequences for MCM loading. To address this, we harnessed an in vitro single-molecule approach to interrogate a chromatinized origin of replication. We find that ORC binds the origin of replication with similar efficiency independently of whether the origin is chromatinized, despite ORC mobility being reduced by the presence of nucleosomes. Recruitment of MCM also proceeds efficiently on a chromatinized origin, but subsequent movement of MCM away from the origin is severely constrained. These findings suggest that chromatinized origins in yeast are essential for the local retention of MCM, which may facilitate subsequent assembly of the replisome.

biophysics↗

bioRxiv2023

Principles and best practices of optimizing a micromirror-based multicolor TIRF microscopy system

TIRF (Total Internal Reflection Fluorescence) microscopy is a powerful tool for measuring the intra- and intermolecular dynamics of fluorescently-labeled single molecules. As TIRF measurements move to more complex biological systems with more fluorescent probes, the multi-band-pass dichroic that separates excitation from emission becomes limiting for the microscopes detection efficiency. To avoid this, multicolor colocalization-based experiments can employ "micromirror" (mm)TIRF, which replaces the dichroic with two 45{degrees}-angled rod mirrors that control the TIR excitation beam(s). Whereas a dichroic spectrally separates excitation and emission wavelengths, the micromirrors act to spatially separate the excitation beams from the collected emission photons within the objective lens itself. Comprehensive control of the TIR beam in mmTIRF can yield excellent signal to noise, and hence data quality, but at the price of increased optical complexity. Here, we introduce the theory behind these additional optical components and provide practical advice from our experience on the best way to set up, align, optimize, and maintain a mmTIRF instrument. We also demonstrate the practical effects of small misalignments to illustrate both the optimized signal quality and the degree of accuracy required to achieve it. We hope that this guide increases the accessibility of this type of instrumentation and helps researchers use it to produce data of the highest quality possible.

biophysics↗

bioRxiv2022

Nucleotide binding halts diffusion of the eukaryotic replicative helicase during activation

The eukaryotic replicative helicase CMG centrally orchestrates the replisome and leads the way at the front of replication forks1. Understanding the motion of CMG on the DNA is therefore key to our understanding of DNA replication. In vivo, CMG is assembled and activated through a cell-cycle-regulated mechanism involving 36 polypeptides that has been reconstituted from purified proteins in ensemble biochemical studies2,3. Conversely, single-molecule studies of CMG motion have thus far4-6 relied on pre-formed CMG assembled through an unknown mechanism upon overexpression of individual constituents7,8. Here, we report the first activation at the single-molecule level of CMG fully reconstituted from purified yeast proteins and the quantification of its motion. We observe that CMG can move on DNA in two ways: by unidirectional translocation and by diffusion. We demonstrate that CMG preferentially exhibits unidirectional translocation in the presence of ATP, whereas it preferentially exhibits diffusive motion in the absence of ATP. We also demonstrate that nucleotide binding halts diffusive CMG. Taken together, our findings support a mechanism by which nucleotide binding allows newly assembled CMG to engage with the DNA within its central channel without melting it, halting its diffusion and facilitating the initial DNA melting required to initiate DNA replication.

biophysics↗

bioRxiv2022

CAF-1 deposits newly synthesized histones during DNA replication using distinct mechanisms on the leading and lagging strands

During every cell cycle, both the genome and the associated chromatin must be accurately replicated. Chromatin Assembly Factor-1 (CAF-1) is a key regulator of chromatin replication, but how CAF-1 cooperates with the DNA replication machinery is unknown. Here, we reveal that this crosstalk differs between the leading and lagging strand at replication forks. Using biochemical reconstitution, we show that DNA and histones promote CAF-1 recruitment to its binding partner PCNA and reveal that two CAF-1 complexes are required for efficient nucleosome assembly under these conditions. Remarkably, in the context of the replisome, CAF-1 competes with the leading strand DNA polymerase epsilon (Pol{varepsilon}) for PCNA binding, but not with the lagging strand DNA polymerase Delta (Pol{delta}). Yet, in cells, CAF-1 deposits newly synthesized histones equally on both daughter strands. Thus, on the leading strand, chromatin assembly by CAF-1 cannot occur simultaneously to DNA synthesis, while on the lagging strand both processes are coupled. We propose that these differences may facilitate distinct parental histone recycling mechanisms and accommodate the inherent asymmetry of DNA replication.

biochemistry↗

bioRxiv2020

Induced copy-back RNA synthesis as a novel therapeutic mechanism against RNA viruses

The viral RNA-dependent RNA polymerase (RdRp) is a well-established target for development of broad-spectrum antiviral therapeutics. Incorporation of ribonucleotide analogues by the RdRp will either cause termination of RNA synthesis or mutagenesis of the RNA product. We demonstrated recently that incorporation of a pyrazine-carboxamide ribonucleotide into nascent RNA leads to pausing and backtracking of the elongating RdRp. Here, we provide evidence for the single-stranded RNA product of backtracking serving as an intermediate in RdRp-catalyzed, template-switching reactions. This intermediate is used for both intramolecular template-switching (copy-back RNA synthesis) and intermolecular template-switching (homologous RNA recombination). The use of a magnetic-tweezers platform to monitor RdRp elongation dynamics permitted direct observation of copy-back synthesis and illuminated properties of the RdRp that promote copy-back synthesis, including stability of the RdRp-nascent-RNA complex and the dimensions of the RdRp nucleic-acid-binding channel. In cells, recombination was stimulated by the presence of a pyrazine-carboxamide ribonucleotide. The effect of the drug on recombination was diminished for a recombination-defective virus, but this virus was not resistant to the drug. The discovery that a ribonucleotide analogue can induce copy-back RNA synthesis suggests that this third mechanistic class of compounds may function by promoting formation of defective viral genomes. This study identifies RdRp-catalyzed intra- and intermolecular template switching as a viable new mechanistic target with potentially broad-spectrum appeal.

microbiology↗

bioRxiv2019

A unifying mechanistic model of bacterial transcription with three interconnected pause states and non-diffusive backtrack recovery

Pausing by bacterial RNA polymerase (RNAp) is vital in the recruitment of regulatory factors, RNA folding, and coupled translation. While backtracking and intra-structural isomerization have been proposed to trigger pausing, our understanding of backtrack-associated pauses and catalytic recovery remains incomplete. Using high-throughput magnetic tweezers, we examined the E. coli RNAp transcription dynamics over a wide range of forces and NTP concentrations. Dwell-time analysis and stochastic modeling identified, in addition to a short-lived elemental pause, two distinct long-lived backtrack pause states differing in recovery rates. We further identified two stochastic sources of transcription heterogeneity: alterations in short-pause frequency that underlie elongation-rate switching, and RNA cleavage deficiency that underpins different long-lived backtrack states. Together with effects of force and Gre factors, we demonstrate that recovery from deep backtracks is governed by intrinsic RNA cleavage rather than diffusional Brownian dynamics. We introduce a consensus mechanistic model that unifies our findings with prior models.

biophysics↗

bioRxiv2019

DNA sequence is a major determinant of tetrasome dynamics

Eukaryotic genomes are hierarchically organized into protein-DNA assemblies for compaction into the nucleus. Nucleosomes, with the (H3-H4)2 tetrasome as a likely intermediate, are highly dynamic in nature by way of several different mechanisms. We have recently shown that tetrasomes spontaneously change the direction of their DNA wrapping between left- and right-handed conformations, which may prevent torque build-up in chromatin during active transcription or replication. DNA sequence has been shown to strongly affect nucleosome positioning throughout chromatin. It is not known, however, whether DNA sequence also impacts the dynamic properties of tetrasomes. To address this question, we examined tetrasomes assembled on a high-affinity DNA sequence using freely orbiting magnetic tweezers. In this context, we also studied the effects of mono- and divalent salts on the flipping dynamics. We found that neither DNA sequence nor altered buffer conditions affect overall tetrasome structure. In contrast, tetrasomes bound to high-affinity DNA sequences showed significantly altered flipping kinetics, predominantly via a reduction in the lifetime of the canonical state of left-handed wrapping. Increased mono- and divalent salt concentrations counteracted this behaviour. Thus, our study indicates that high-affinity DNA sequences impact not only the positioning of the nucleosome, but that they also endow the subnucleosomal tetrasome with enhanced conformational plasticity. This may provide a means to prevent histone loss upon exposure to torsional stress, thereby contributing to the integrity of chromatin at high-affinity sites.\n\nSTATEMENT OF SIGNIFICANCECanonical (H3-H4)2 tetrasomes possess high conformational flexibility, as evidenced by their spontaneous flipping between states of left- and right-handed DNA wrapping. Here, we show that these conformational dynamics of tetrasomes cannot be described by a fixed set of rates over all conditions. Instead, an accurate description of their behavior must take into account details of their loading, in particular the underlying DNA sequence. In vivo, differences in tetrasome flexibility could be regulated by modifications of the histone core or the tetrasomal DNA, and as such constitute an intriguing, potentially adjustable mechanism for chromatin to accommodate the torsional stress generated by processes such as transcription and replication.

biophysics↗

bioRxiv2018

Predicting Intra- and Intertypic Recombination in Enterovirus 71

Enteroviruses are well known for their ability to cause neurological damage and paralysis. The model enterovirus is poliovirus (PV), the causative agent of poliomyelitis, a condition characterized by acute flaccid paralysis. A related virus, enterovirus 71 (EV-A71), causes similar clinical outcomes in recurrent outbreaks throughout Asia. Retrospective phylogenetic analysis has shown that recombination between circulating strains of EV-A71 produces the outbreak-associated strains which exhibit increased virulence and/or transmissibility. While studies on the mechanism(s) of recombination in PV are ongoing in several laboratories, little is known about factors that influence recombination in EV-A71. We have developed a cell-based assay to study recombination of EV-A71 based upon previously reported assays for poliovirus recombination. Our results show that: (1) EV-A71 strain-type and RNA sequence diversity impacts recombination frequency in a predictable manner that mimics the observations found in nature; (2) recombination is primarily a replicative process mediated by the RNA-dependent RNA polymerase (RdRp); (3) a mutation shown to reduce recombination in PV (L420A) similarly reduces EV-A71 recombination suggesting conservation in mechanism(s); and (4) sequencing of intertypic recombinant genomes indicates that template-switching is by a mechanism that requires some sequence homology at the recombination junction and that the triggers for template-switching may be sequence independent. The development of this recombination assay will permit further investigation on the interplay between replication, recombination and disease.

microbiology↗