Biology subjects

Tisma, M.

Publications and source records attributed to Tisma, M..

7 recordsLinked to original sources

bioRxiv2026

Chromosome topology gates productive RecA homology search

In homologous recombination, DNA repair depends on recombinase filaments finding homologous templates on chromosomes whose topology is continually remodeled by replication and transcription. How dynamic chromosome topology affects homology search and DNA repair remains unknown. By combining live-cell imaging of RecA-mediated homology search with single-molecule assays on topologically-defined DNA substrates, we show that DNA supercoiling acts as a selectivity filter for productive homology search. In Caulobacter crescentus cells, supercoiling is dispensable for the filament movement required for homology search, but required for homology target capture and repair. In vitro, filaments transiently sample both relaxed and supercoiled DNA, yet selectively commit to capture only on negatively supercoiled targets. Mechanistically, we find an extreme kinetic preference (~100-fold) of filaments towards negatively supercoiled DNA targets; this topological preference is independent of DNA compaction, pins plectonemes at the capture site, and is conserved across diverse bacterial RecA homologs. Our findings establish that chromosome topology physically regulates homology search and DNA repair.

biophysics↗

bioRxiv2024

DNA supercoiling enhances DNA condensation by ParB proteins

The ParABS system plays a critical role in bacterial chromosome segregation. The key component of this system, ParB, loads and spreads along DNA to form a local protein-DNA condensate known as a partition complex. As bacterial chromosomes are heavily supercoiled due to the continuous action of RNA polymerases, gyrases, and nucleoid-associated proteins, it is important to study the impact of DNA supercoiling on the ParB-DNA partition complex formation. Here, we use an in vitro single- molecule assay to visualize ParB on supercoiled DNA. Unlike most DNA-binding proteins, individual ParB proteins are found to not pin plectonemes on supercoiled DNA, but freely diffuse along supercoiled DNA. We find that DNA supercoiling enhances ParB-DNA condensation which initiates at lower ParB concentrations than on DNA that is torsionally relaxed. ParB proteins induce a DNA- protein condensate that strikingly absorbs all supercoiling writhe. Our findings provide mechanistic insights that have important implications for our understanding of bacterial chromosome organization and segregation.

biophysics↗

bioRxiv2023

Topology-dependent DNA binding

DNA stores our genetic information and is ubiquitous in biological and biotechnological applications, where it interacts with binding partners ranging from small molecules to large macromolecular complexes. Binding is modulated by mechanical strains in the molecule and, in turn, can change the local DNA structure. Frequently, DNA occurs in closed topological forms where topology and supercoiling add a global constraint to the interplay of binding-induced deformations and strain-modulated binding. Here, we present a quantitative model of how the global constraints introduced by DNA topology modulate binding and create a complex interplay between topology and affinity. We focus on fluorescent intercalators, which unwind DNA and enable direct quantification via fluorescence detection. Using bulk measurements, we show that DNA supercoiling can increase or decrease intercalation relative to an open topology depending on ligand concentration and the initial topology. Our model quantitatively accounts for observations obtained using psoralen for UV-induced DNA crosslinking, which is frequently used to quantify supercoiling in vivo. Finally, we observe topology-dependent binding in a single-molecule assay, which provides direct access to binding kinetics and DNA supercoil dynamics. Our results have broad implications for the detection and quantification of DNA and for the modulation of DNA binding in cellular contexts.

biophysics↗

bioRxiv2023

Dynamic ParB-DNA interactions initiate and maintain a partition condensate for bacterial chromosome segregation

In most bacteria, chromosome segregation is driven by the ParABS system where the CTPase protein ParB loads at the parS site to trigger the formation of a large partition complex. Here, we present in vitro studies of the partition complex for Bacillus subtilis ParB, using single-molecule fluorescence microscopy and AFM imaging to show that transient ParB-ParB bridges are essential for forming DNA condensates. Molecular Dynamics simulations confirm that condensation occurs abruptly at a critical concentration of ParB and show that multimerization is a prerequisite for forming the partition complex. Magnetic tweezer force spectroscopy on mutant ParB proteins demonstrates that CTP hydrolysis at the N-terminal domain is essential for DNA condensation. Finally, we show that transcribing RNA polymerases can steadily traverse the ParB-DNA partition complex. These findings uncover how ParB forms a stable yet dynamic partition complex for chromosome segregation that induces DNA condensation and segregation while enabling replication and transcription.

biophysics↗

bioRxiv2023

Direct observation of a crescent-shape chromosome in Bacillus subtilis

Bacterial chromosomes are folded into tightly regulated three-dimensional structures to ensure proper transcription, replication, and segregation of the genomic information. Direct visualization of the chromosomal shape within bacterial cells is hampered by cell-wall confinement and the optical diffraction limit. Here, we combine cell-shape manipulation strategies, high-resolution fluorescence microscopy techniques, and genetic engineering to visualize the intrinsic shape of the bacterial chromosome in real-time in live Bacillus subtilis cells. We show that the chromosome exhibits a crescent shape with a non-uniform DNA density that is increased near the origin of replication (oriC). Additionally, we localized ParB and BsSMC proteins - the key drivers of chromosomal organization - along the contour of the crescent chromosome, showing the highest density near oriC. Opening of the BsSMC ring complex disrupted the crescent chromosome shape and instead yielded a torus shape. These findings help to understand the threedimensional organization of the chromosome and the main protein complexes that underlie its structure.

microbiology↗

bioRxiv2021

ParS-independent recruitment of the bacterial chromosome-partitioning protein ParB

The ParABS system plays an essential role in prokaryotic chromosome segregation. After loading at the parS site on the genome, ParB proteins rapidly redistribute to distances of ~15 kb away from the loading site. It has remained puzzling how this large-distance spreading can occur along DNA that is loaded with hundreds of proteins. Using single-molecule in vitro visualization, we here show that, unexpectedly, ParB can load onto DNA independently and distantly of parS, whereby loaded ParB molecules are themselves able to recruit additional ParB proteins from bulk. Strikingly, this recruitment can occur in-cis but also in-trans whereby, at low tensions within the DNA, newly recruited ParB can bypass roadblocks as it gets loaded to spatially proximal but genomically distant DNA regions. The data are supported by Molecular Dynamics simulations which also show that cooperative ParB-ParB recruitment enhances spreading. ParS-independent recruitment explains how ParB can cover substantial genomic distance during chromosome segregation which is vital for the bacterial cell cycle.

biophysics↗

bioRxiv2020

Direct visualization of four diffusive LexA states controlling SOS response strength during antibiotic treatment

In bacteria, the key mechanism governing mutation, adaptation and survival upon DNA damage is the SOS response. Through autoproteolytic digestion triggered by single-stranded DNA caused by most antibiotics, the transcriptional repressor LexA controls over 50 SOS genes including DNA repair pathways and drivers of mutagenesis. Efforts to inhibit this response and thereby combat antibiotic resistance rely on a broad understanding of its behavior in vivo, which is still limited. Here, we develop a single-molecule localization microscopy assay to directly visualize LexA mobility in Escherichia coli and monitor the SOS response on the level of transcription factor activity. We identify four diffusive populations and monitor their temporal evolution upon ciprofloxacin-induced continuous DNA damage. With LexA mutants, we assign target bound, non-specifically DNA bound, freely diffusing and cleaved repressors. We develop a strategy to count LexA in fixed cells at different time points after antibiotic stress and combine the time-evolution of LexA sub-populations and the repressors overall abundance. Through fitting a detailed kinetic model we obtain in vivo synthesis, cleavage and binding rates and determined that the regulatory feedback system reaches a new equilibrium in [~]100 min. LexA concentrations showed non-constant heterogeneity during SOS response and designate LexA expression, and thereby regulation of downstream SOS proteins, as drivers of evolutionary adaptation. Even under low antibiotic stress, we observed a strong SOS response on the LexA level, suggestion that small amounts of antibiotics can trigger adaptation in E. coli.

biophysics↗