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In response to double strand breaks (DSB), repair proteins accumulate at damaged sites, forming membrane-less sub-compartments or foci. Here we explored the physical nature of these foci, using single molecule microscopy in living cells. Rad52, the functional homolog of BRCA2 in yeast, accumulates at DSB sites and diffuses ~6 times faster within repair foci than the focus itself, exhibiting confined motion. The Rad52 confinement radius coincides with the focus size: foci resulting from 2 DSBs are twice larger in volume that the ones induced by a unique DSB and the Rad52 confinement radius scales accordingly. In contrast, molecules of the single strand binding protein Rfa1 follow anomalous diffusion similar to the focus itself or damaged chromatin. We conclude that while most Rfa1 molecules are bound to the ssDNA, Rad52 molecules are free to explore the entire focus reflecting the existence of a liquid droplet around damaged DNA.

DNA replication is a challenge for the faithful transmission of parental information to daughter cells, as both DNA and chromatin organization must be duplicated. Replication stress further complicates the safeguard of epigenome integrity. Here, we investigate the transmission of the histone variants H3.3 and H3.1 during replication. We follow their distribution relative to replication timing, first in the genome and, second, in 3D using super-resolution microscopy. We find that H3.3 and H3.1 mark early- and late-replicating chromatin, respectively. In the nucleus, H3.3 forms domains, which decrease in density throughout replication, while H3.1 domains increase in density. Hydroxyurea impairs local recycling of parental histones at replication sites. Similarly, depleting the histone chaperone ASF1 affects recycling, leading to an impaired histone variant landscape. We discuss how faithful transmission of histone variants involves ASF1 and can be impacted by replication stress, with ensuing consequences for cell fate and tumorigenesis. Epigenetic modifications are a key contributor to cell identity, and their propagation is crucial for proper development. Here the authors use a super-resolution microscopy approach to reveal how histone variants are faithfully transmitted during genome duplication, and reveal an important role for the histone chaperone ASF1 in the redistribution of parental histones.

The analysis of live-cell single-molecule imaging experiments can reveal valuable information about the heterogeneity of transport processes and interactions between cell components. These characteristics are seen as motion changes in the particle trajectories. Despite the existence of multiple approaches to carry out this type of analysis, no objective assessment of these methods has been performed so far. Here, we report the results of a competition to characterize and rank the performance of these methods when analyzing the dynamic behavior of single molecules. To run this competition, we implemented a software library that simulates realistic data corresponding to widespread diffusion and interaction models, both in the form of trajectories and videos obtained in typical experimental conditions. The competition constitutes the first assessment of these methods, providing insights into the current limitations of the field, fostering the development of new approaches, and guiding researchers to identify optimal tools for analyzing their experiments. Muñoz-Gil and colleagues report the results of an open challenge where they benchmarked algorithms for the characterization of motion changes in single-particle tracking. By ranking methods on simulations, the competition revealed strengths and limitations of AI and classic approaches, guiding researchers toward optimal tools.

Timely access to DNA lesions is crucial for genome integrity. This process requires profound remodeling of densely packed chromatin to establish a repair-competent architecture. However, limited resolution has made it impossible to fully understand these remodeling events. Here, combining microirradiation with live-cell multiscale imaging, we report that DNA damage-induced changes in genome packing rely on the conformational behaviour of the chromatin fiber. Immediately after damage, a transient increase in nucleosome mobility switches chromatin from a densely-packed state to a looser conformation, making it accessible to repair. While histone poly-ADP-ribosylation is required to trigger this switch, mono-ADP-ribosylation is sufficient to maintain the open-chromatin state. The removal of these histone marks by the ARH3 hydrolase then leads to chromatin recondensation. Together, our multiscale study of chromatin dynamics establishes a global model: distinct waves of histone ADP-ribosylation control nucleosome mobility, triggering a transient breathing of chromatin, crucial for initiating the DNA damage response. Chromatin “breathing” at DNA lesions is controlled by different waves of histone ADP-ribosylation.

The spatial organization of complex biochemical reactions is essential for the regulation of cellular processes. Membrane-less structures called foci containing high concentrations of specific proteins have been reported in a variety of contexts, but the mechanism of their formation is not fully understood. Several competing mechanisms exist that are difficult to distinguish empirically, including liquid-liquid phase separation, and the trapping of molecules by multiple binding sites. Here, we propose a theoretical framework and outline observables to differentiate between these scenarios from single molecule tracking experiments. In the binding site model, we derive relations between the distribution of proteins, their diffusion properties, and their radial displacement. We predict that protein search times can be reduced for targets inside a liquid droplet, but not in an aggregate of slowly moving binding sites. We use our results to reject the multiple binding site model for Rad52 foci, and find a picture consistent with a liquid-liquid phase separation. These results are applicable to future experiments and suggest different biological roles for liquid droplet and binding site foci.

Homologous recombination, an essential process for preserving genomic integrity, uses intact homologous sequences to repair broken chromosomes. To explore the mechanism of homologous pairing in vivo , we tagged two homologous loci in diploid yeast Saccharomyces cerevisiae cells and investigated their dynamic organization in the absence and presence of DNA damage. When neither locus is damaged, homologous loci occupy largely separate regions, exploring only 2.7% of the nuclear volume. Following the induction of a double-strand break, homologous loci co-localize ten times more often. The mobility of the cut chromosome markedly increases, allowing it to explore a nuclear volume that is more than ten times larger. Interestingly, the mobility of uncut chromosomes also increases, allowing them to explore a four times larger volume. We propose a model for homology search in which increased chromosome mobility facilitates homologous pairing. Finally, we find that the increase in DNA dynamics is dependent on early steps of homologous recombination. Repair of double-strand breaks (DSBs) by homologous recombination is thought to involve the movement of damaged chromosomes to facilitate pairing of homologues. Rothstein and colleagues have now followed the movement of damaged loci in diploid yeast by time-lapse microscopy, revealing the dynamics of damage-induced movement and the requirement for repair proteins in this process.

The human recombinase hRad51 is a key protein for the maintenance of genome integrity and for cancer development. Polymerization and depolymerization of hRad51 on duplex DNA were studied here using a new generation of magnetic tweezers, measuring DNA twist in real time with a resolution of 5°. Our results combined with earlier structural information suggest that DNA is somewhat less extended by hRad51 than by RecA (4.5 vs. 5.1 Å per base pair) and untwisted by 18.2° per base pair. They also confirm a stoichiometry of 3-4 bp per protein in the hRad51-dsDNA nucleoprotein filament. At odds with earlier claims, we show that after initial deposition of a multimeric nucleus, nucleoprotein filament growth occurs by addition/release of single proteins, involving DNA twisting steps of 65° ± 5°. Simple numeric simulations show that this mechanism is an efficient way to minimize nucleoprotein filament defects. Nucleoprotein filament growth from a preformed nucleus was observed at hRad51 concentrations down to 10 nM, whereas nucleation was never observed below 100 nM in the same buffer. This behavior can be associated with the different stoichiometries of nucleation and growth. It may be instrumental in vivo to permit efficient continuation of strand exchange by hRad51 alone while requiring additional proteins such as Rad52 for its initiation, thus keeping the latter under the strict control of regulatory pathways.

A chemical regularly used to image cells can dramatically alter the way cellular compartments called condensates look under the microscope.A chemical regularly used to image cells can dramatically alter the way cellular compartments called condensates look under the microscope.

Chromatin is now regarded as a heterogeneous and dynamic structure occupying a non-random position within the cell nucleus, where it plays a key role in regulating various functions of the genome. This current view of chromatin has emerged thanks to high spatiotemporal resolution imaging, among other new technologies developed in the last decade. In addition to challenging early assumptions of chromatin being regular and static, high spatiotemporal resolution imaging made it possible to visualize and characterize different chromatin structures such as clutches, domains and compartments. More specifically, super-resolution microscopy facilitates the study of different cellular processes at a nucleosome scale, providing a multi-scale view of chromatin behavior within the nucleus in different environments. In this review, we describe recent imaging techniques to study the dynamic organization of chromatin at high spatiotemporal resolution. We also discuss recent findings, elucidated by these techniques, on the chromatin landscape during different cellular processes, with an emphasis on the DNA damage response.

Gene targeting depends on the ability of cells to use homologous recombination to integrate exogenous DNA into their own genome. A robust mechanistic model of homologous recombination is necessary to fully exploit gene targeting for therapeutic benefit. In this work, our recently developed numerical simulation model for homology search is employed to develop rules for the design of oligonucleotides used in gene targeting. A Metropolis Monte-Carlo algorithm is used to predict the pairing dynamics of an oligonucleotide with the target double-stranded DNA. The model calculates the base-alignment between a long, target double-stranded DNA and a probe nucleoprotein filament comprised of homologous recombination proteins (Rad51 or RecA) polymerized on a single strand DNA. In this study, we considered different sizes of oligonucleotides containing 1 or 3 base heterologies with the target; different positions on the probe were tested to investigate the effect of the mismatch position on the pairing dynamics and stability. We show that the optimal design is a compromise between the mean time to reach a perfect alignment between the two molecules and the stability of the complex. A single heterology can be placed anywhere without significantly affecting the stability of the triplex. In the case of three consecutive heterologies, our modeling recommends using long oligonucleotides (at least 35 bases) in which the heterologous sequences are positioned at an intermediate position. Oligonucleotides should not contain more than 10% consecutive heterologies to guarantee a stable pairing with the target dsDNA. Theoretical modeling cannot replace experiments, but we believe that our model can considerably accelerate optimization of oligonucleotides for gene therapy by predicting their pairing dynamics with the target dsDNA.