Multi-Scale Imaging of the Dynamic Organization of Chromatin.

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.

In vivo tracking of functionally tagged Rad51 unveils a robust strategy of homology search.

Homologous recombination (HR) is a major pathway to repair DNA double-strand breaks (DSB). HR uses an undamaged homologous DNA sequence as a template for copying the missing information, which requires identifying a homologous sequence among megabases of DNA within the crowded nucleus. In eukaryotes, the conserved Rad51-single-stranded DNA nucleoprotein filament (NPF) performs this homology search.

When fixation creates fiction.

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

Repair Foci as Liquid Phase Separation: Evidence and Limitations.

In response to DNA double strand breaks (DSB), repair proteins accumulate at damaged sites, forming membrane-less condensates or "foci". The formation of these foci and their disassembly within the proper time window are essential for genome integrity. However, how these membrane-less sub-compartments are formed, maintained and disassembled remains unclear.

Physical observables to determine the nature of membrane-less cellular sub-compartments.

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.

Single molecule microscopy reveals key physical features of repair foci in living cells.

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.

Complex Chromatin Motions for DNA Repair.

A number of studies across different model systems revealed that chromatin undergoes significant changes in dynamics in response to DNA damage. These include local motion changes at damage sites, increased nuclear exploration of both damaged and undamaged loci, and directed motions to new nuclear locations associated with certain repair pathways. These studies also revealed the need for new analytical methods to identify directed motions in a context of mixed trajectories, and the importance of investigating nuclear dynamics over different time scales to identify diffusion regimes.

Physical principles and functional consequences of nuclear compartmentalization in budding yeast.

One striking feature of eukaryotic nuclei is the existence of discrete regions, in which specific factors concentrate while others are excluded, thus forming microenvironments with different molecular compositions and biological functions. These domains are often referred to as subcompartments even though they are not membrane enclosed. Despite their functional importance the physical nature of these structures remains largely unknown.

High-resolution visualization of H3 variants during replication reveals their controlled recycling.

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.

Multi-scale tracking reveals scale-dependent chromatin dynamics after DNA damage.

The dynamic organization of genes inside the nucleus is an important determinant for their function. Using fast DNA tracking microscopy in cells and improved analysis of mean square displacements, we quantified DNA motion at time scales ranging from 10 milliseconds to minute and found that following DNA damage, DNA exhibits distinct sub-diffusive regimes. In response to double-strand breaks, chromatin is more mobile at large time scales but, surprisingly, its mobility is reduced at short time scales.

DNA in motion during double-strand break repair.

DNA organization and dynamics profoundly affect many biological processes such as gene regulation and DNA repair. In this review, we present the latest studies on DNA mobility in the context of DNA damage. Recent studies demonstrate that DNA mobility is dramatically increased in the presence of double-strand breaks (DSBs) in the yeast Saccharomyces cerevisiae.

Increased chromosome mobility facilitates homology search during recombination.

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.

Optimizing the design of oligonucleotides for homology directed gene targeting.

Background: 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.

Methodology/principal Findings: 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.

Separation of time scales in one-dimensional directed nucleation-growth processes.

Proteins involved in homologous recombination such as RecA and hRad51 polymerize on single- and double-stranded DNA according to a nucleation-growth kinetics, which can be monitored by single-molecule in vitro assays. The basic models currently used to extract biochemical rates rely on ensemble averages and are typically based on an underlying process of bidirectional polymerization, in contrast with the often observed anisotropic polymerization of similar proteins. For these reasons, if one considers single-molecule experiments, the available models are useful to understand observations only in some regimes.

Direct observation of twisting steps during Rad51 polymerization on DNA.

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 degrees . Our results combined with earlier structural information suggest that DNA is somewhat less extended by hRad51 than by RecA (4.