Jason C. Bell

Direct imaging of RecA nucleation and growth on single molecules of SSB-coated ssDNA

Escherichia coli RecA is the defining member of a ubiquitous class of DNA strand-exchange proteins that are essential for homologous recombination, a pathway that maintains genomic integrity by repairing broken DNA. To function, filaments of RecA must nucleate and grow on single-stranded DNA (ssDNA) in direct competition with ssDNA-binding protein (SSB), which rapidly binds and continuously sequesters ssDNA, kinetically blocking RecA assembly. This dynamic self-assembly on a DNA lattice, in competition with another protein, is unique for the RecA family compared to other filament-forming proteins such as actin and tubulin. The complexity of this process has hindered our understanding of RecA filament assembly because ensemble measurements cannot reliably distinguish between the nucleation and growth phases, despite extensive and diverse attempts. Previous single-molecule assays have measured the nucleation and growth of RecA—and its eukaryotic homologue RAD51—on naked double-stranded DNA and ssDNA; however, the template for RecA self-assembly in vivo is SSB-coated ssDNA. Using single-molecule microscopy, here we directly visualize RecA filament assembly on single molecules of SSB-coated ssDNA, simultaneously measuring nucleation and growth. We establish that a dimer of RecA is required for nucleation, followed by growth of the filament through monomer addition, consistent with the finding that nucleation, but not growth, is modulated by nucleotide and magnesium ion cofactors. Filament growth is bidirectional, albeit faster in the 5′→3′ direction. Both nucleation and growth are repressed at physiological conditions, highlighting the essential role of recombination mediators in potentiating assembly in vivo. We define a two-step kinetic mechanism in which RecA nucleates on transiently exposed ssDNA during SSB sliding and/or partial dissociation (DNA unwrapping) and then the RecA filament grows. We further demonstrate that the recombination mediator protein pair, RecOR (RecO and RecR), accelerates both RecA nucleation and filament growth, and that the introduction of RecF further stimulates RecA nucleation.

Fluorescent Single-Stranded DNA Binding Protein as a Probe for Sensitive, Real-Time Assays of Helicase Activity

The formation and maintenance of single-stranded DNA (ssDNA) are essential parts of many processes involving DNA. For example, strand separation of double-stranded DNA (dsDNA) is catalyzed by helicases, and this exposure of the bases on the DNA allows further processing, such as replication, recombination, or repair. Assays of helicase activity and probes for their mechanism are essential for understanding related biological processes. Here we describe the development and use of a fluorescent probe to measure ssDNA formation specifically and in real time, with high sensitivity and time resolution. The reagentless biosensor is based on the ssDNA binding protein (SSB) from Escherichia coli, labeled at a specific site with a coumarin fluorophore. Its use in the study of DNA manipulations involving ssDNA intermediates is demonstrated in assays for DNA unwinding, catalyzed by DNA helicases.

Mechanics and Single-Molecule Interrogation of DNA Recombination

The repair of DNA by homologous recombination is an essential, efficient, and high-fidelity process that mends DNA lesions formed during cellular metabolism; these lesions include double-stranded DNA breaks, daughter-strand gaps, and DNA cross-links. Genetic defects in the homologous recombination pathway undermine genomic integrity and cause the accumulation of gross chromosomal abnormalities-including rearrangements, deletions, and aneuploidy-that contribute to cancer formation. Recombination proceeds through the formation of joint DNA molecules-homologously paired but metastable DNA intermediates that are processed by several alternative subpathways-making recombination a versatile and robust mechanism to repair damaged chromosomes. Modern biophysical methods make it possible to visualize, probe, and manipulate the individual molecules participating in the intermediate steps of recombination, revealing new details about the mechanics of genetic recombination. We review and discuss the individual stages of homologous recombination, focusing on common pathways in bacteria, yeast, and humans, and place particular emphasis on the molecular mechanisms illuminated by single-molecule methods.

Imaging and energetics of single SSB-ssDNA molecules reveal intramolecular condensation and insight into RecOR function

Escherichia coli single-stranded DNA (ssDNA) binding protein (SSB) is the defining bacterial member of ssDNA binding proteins essential for DNA maintenance. SSB binds ssDNA with a variable footprint of ∼30–70 nucleotides, reflecting partial or full wrapping of ssDNA around a tetramer of SSB. We directly imaged single molecules of SSB-coated ssDNA using total internal reflection fluorescence (TIRF) microscopy and observed intramolecular condensation of nucleoprotein complexes exceeding expectations based on simple wrapping transitions. We further examined this unexpected property by single-molecule force spectroscopy using magnetic tweezers. In conditions favoring complete wrapping, SSB engages in long-range reversible intramolecular interactions resulting in condensation of the SSB-ssDNA complex. RecO and RecOR, which interact with SSB, further condensed the complex. Our data support the idea that RecOR--and possibly other SSB-interacting proteins—function(s) in part to alter long-range, macroscopic interactions between or throughout nucleoprotein complexes by microscopically altering wrapping and bridging distant sites. DOI: http://dx.doi.org/10.7554/eLife.08646.001

RNA-dependent stabilization of SUV39H1 at constitutive heterochromatin

Heterochromatin formed by the SUV39 histone methyltransferases represses transcription from repetitive DNA sequences and ensures genomic stability. How SUV39 enzymes localize to their target genomic loci remains unclear. Here, we demonstrate that chromatin-associated RNA contributes to the stable association of SUV39H1 with constitutive heterochromatin in human cells. We find that RNA associated with mitotic chromosomes is concentrated at pericentric heterochromatin, and is encoded, in part, by repetitive α-satellite sequences, which are retained in cis at their transcription sites. Purified SUV39H1 directly binds nucleic acids through its chromodomain; and in cells, SUV39H1 associates with α-satellite RNA transcripts. Furthermore, nucleic acid binding mutants destabilize the association of SUV39H1 with chromatin in mitotic and interphase cells – effects that can be recapitulated by RNase treatment or RNA polymerase inhibition – and cause defects in heterochromatin function. Collectively, our findings uncover a previously unrealized function for chromatin-associated RNA in regulating constitutive heterochromatin in human cells. DOI: http://dx.doi.org/10.7554/eLife.25299.001

Chromatin-associated RNA sequencing (ChAR-seq) maps genome-wide RNA-to-DNA contacts

RNA is a critical component of chromatin in eukaryotes, both as a product of transcription, and as an essential constituent of ribonucleoprotein complexes that regulate both local and global chromatin states. Here, we present a proximity ligation and sequencing method called Chromatin-Associated RNA sequencing (ChAR-seq) that maps all RNA-to-DNA contacts across the genome. Using Drosophila cells, we show that ChAR-seq provides unbiased, de novo identification of targets of chromatin-bound RNAs including nascent transcripts, chromosome-specific dosage compensation ncRNAs, and genome-wide trans-associated RNAs involved in co-transcriptional RNA processing.

Condensing chromosome condensation

Mitotic chromosome condensation has fascinated biologists since Flemmings early illustrations of mitosis in the late nineteenth century. Now — 130 years later — chromatid condensation is reconstituted in vitro with the minimum components. The results are remarkably and beautifully simple, requiring only core histones, three histone chaperones, topoisomerase II and condensin I.

Intramolecular dynamics of single molecules in free diffusion

Biomolecular systems such as multiprotein complexes or biopolymers can span several tens to several hundreds of nanometers, but the dynamics of such “mesocale” molecules remain challenging to probe. We have developed a single-molecule technique that uses Tracking Fluorescence Correlation Spectroscopy (tFCS) to measure the conformation and dynamics of molecular assemblies specifically at the mesoscale level (~100-1000 nm). tFCS is non-perturbative, as molecules, which are tracked in real-time, are untethered and freely diffusing. To achieve sub-diffraction spatial resolution, we use a feedback scheme which allows us to maintain the molecule at an optimal position within the laser intensity gradient. We find that tFCS is sufficiently sensitive to measure the distance fluctuations between two sites within a DNA molecule separated by distances as short as 1000 bp. We demonstrate that tFCS detects changes in the compaction of reconstituted chromatin, and can assay transient protein mediated interactions between distant sites in an individual DNA molecule. Our measurements highlight the impact that tFCS can have in the study of a wide variety of biochemical processes involving mesoscale conformational dynamics.

Direct Visualization of Fluorescent SSB on Single Molecules of ssDNA as a Mechanistic Probe in the Early Stages of Homologous Recombination

In all organisms homologous recombination (HR) is essential for the efficient and error-free repair of DNA lesions. Defects in HR result in genomic instability, which often manifests in humans as a genetic disposition to cancer. Central to the process of homologous recombination is the strand exchange activity of the RecA/Rad51 class of proteins. Through the formation of a pre-synaptic filament on single stranded DNA (ssDNA), RecA/Rad51 aligns a broken chromosome with an intact one (a process called synapsis), allowing for a subsequent array of potential repair pathways. During the presynaptic stage, filament formation is inhibited by the diffusion-limited association of ssDNA with the high-affinity single stranded DNA binding protein, SSB/RPA. A class of positive regulators called mediators facilitate filament formation by alleviating this biochemical inhibition. These mediators include RecF/O/R (E. coli), Rad52 (S. cerevisiae and H. sapiens), and BRCA2 (H. sapiens). We have fluorescently modified several of the key proteins involved in pre-synaptic filament formation in E. coli, specifically SSB and RecA. Here we present their characterization and utility as fluorescent biochemical sensors for single-stranded DNA in single molecule assays designed to mechanistically probe the early, pre-synaptic stage of homologous recombination.