BRCA2 chaperones RAD51 to single molecules of RPA-coated ssDNA.

Mutations in the breast cancer susceptibility gene, BRCA2, greatly increase an individual's lifetime risk of developing breast and ovarian cancers. BRCA2 suppresses tumor formation by potentiating DNA repair via homologous recombination. Central to recombination is the assembly of a RAD51 nucleoprotein filament, which forms on single-stranded DNA (ssDNA) generated at or near the site of chromosomal damage. However, replication protein-A (RPA) rapidly binds to and continuously sequesters this ssDNA, imposing a kinetic barrier to RAD51 filament assembly that suppresses unregulated recombination. Recombination mediator proteins-of which BRCA2 is the defining member in humans-alleviate this kinetic barrier to catalyze RAD51 filament formation. We combined microfluidics, microscopy, and micromanipulation to directly measure both the binding of full-length BRCA2 to-and the assembly of RAD51 filaments on-a region of RPA-coated ssDNA within individual DNA molecules designed to mimic a resected DNA lesion common in replication-coupled recombinational repair. We demonstrate that a dimer of RAD51 is minimally required for spontaneous nucleation; however, growth self-terminates below the diffraction limit. BRCA2 accelerates nucleation of RAD51 to a rate that approaches the rapid association of RAD51 to naked ssDNA, thereby overcoming the kinetic block imposed by RPA. Furthermore, BRCA2 eliminates the need for the rate-limiting nucleation of RAD51 by chaperoning a short preassembled RAD51 filament onto the ssDNA complexed with RPA. Therefore, BRCA2 regulates recombination by initiating RAD51 filament formation.

Assembly mechanism and cryoEM structure of RecA recombination nucleofilaments from Streptococcus pneumoniae.

RecA-mediated homologous recombination (HR) is a key mechanism for genome maintenance and plasticity in bacteria. It proceeds through RecA assembly into a dynamic filament on ssDNA, the presynaptic filament, which mediates DNA homology search and ordered DNA strand exchange. Here, we combined structural, single molecule and biochemical approaches to characterize the ATP-dependent assembly mechanism of the presynaptic filament of RecA from Streptococcus pneumoniae (SpRecA), in comparison to the Escherichia coli RecA (EcRecA) paradigm. EcRecA polymerization on ssDNA is assisted by the Single-Stranded DNA Binding (SSB) protein, which unwinds ssDNA secondary structures that block EcRecA nucleofilament growth. We report by direct microscopic analysis of SpRecA filamentation on ssDNA that neither of the two paralogous pneumococcal SSBs could assist the extension of SpRecA nucleopolymers. Instead, we found that the conserved RadA helicase promotes SpRecA nucleofilamentation in an ATP-dependent manner. This allowed us to solve the atomic structure of such a long native SpRecA nucleopolymer by cryoEM stabilized with ATPγS. It was found to be equivalent to the crystal structure of the EcRecA filament with a marked difference in how RecA mediates nucleotide orientation in the stretched ssDNA. Then, our results show that SpRecA and EcRecA HR activities are different, in correlation with their distinct ATP-dependent ssDNA binding modes.

Massively parallel single-cell chromatin landscapes of human immune cell development and intratumoral T cell exhaustion.

Understanding complex tissues requires single-cell deconstruction of gene regulation with precision and scale. Here, we assess the performance of a massively parallel droplet-based method for mapping transposase-accessible chromatin in single cells using sequencing (scATAC-seq). We apply scATAC-seq to obtain chromatin profiles of more than 200,000 single cells in human blood and basal cell carcinoma. In blood, application of scATAC-seq enables marker-free identification of cell type-specific cis- and trans-regulatory elements, mapping of disease-associated enhancer activity and reconstruction of trajectories of cellular differentiation. In basal cell carcinoma, application of scATAC-seq reveals regulatory networks in malignant, stromal and immune cells in the tumor microenvironment. Analysis of scATAC-seq profiles from serial tumor biopsies before and after programmed cell death protein 1 blockade identifies chromatin regulators of therapy-responsive T cell subsets and reveals a shared regulatory program that governs intratumoral CD8+ T cell exhaustion and CD4+ T follicular helper cell development. We anticipate that scATAC-seq will enable the unbiased discovery of gene regulatory factors across diverse biological systems.

Chromatin-Associated RNA Sequencing (ChAR-seq).

RNA is a fundamental component of chromatin. Noncoding RNAs (ncRNAs) can associate with chromatin to influence gene expression and chromatin state; many also act at long distances from their transcriptional origin. Yet we know almost nothing about the functions or sites of action for most ncRNAs. Current methods to identify sites of RNA interaction with the genome are limited to the study of a single RNA at a time. Here we describe a protocol for ChAR-seq, a strategy to identify all chromatin-associated RNAs and map their DNA contacts genome-wide. In ChAR-seq, proximity ligation of RNA and DNA to a linker molecule is used to construct a chimeric RNA-DNA molecule that is converted to DNA for sequencing. In a single assay, ChAR-seq can discover de novo chromatin interactions of distinct RNAs, including nascent transcripts, splicing RNAs, and long noncoding RNAs (lncRNAs). Resulting "maps" of genome-bound RNAs should provide new insights into RNA biology. © 2019 by John Wiley & Sons, Inc.

Sequential assembly of the septal cell envelope prior to V snapping in Corynebacterium glutamicum.

Members of the Corynebacterineae, including Corynebacterium and Mycobacterium, have an atypical cell envelope characterized by an additional mycomembrane outside of the peptidoglycan layer. How this multilayered cell envelope is assembled remains unclear. Here, we tracked the assembly dynamics of different envelope layers in Corynebacterium glutamicum and Mycobacterium smegmatis by using metabolic labeling and found that the septal cell envelope is assembled sequentially in both species. Additionally, we demonstrate that in C. glutamicum, the peripheral peptidoglycan layer at the septal junction remains contiguous throughout septation, forming a diffusion barrier for the fluid mycomembrane. This diffusion barrier is resolved through perforations in the peripheral peptidoglycan, thus leading to the confluency of the mycomembrane before daughter cell separation (V snapping). Furthermore, the same junctional peptidoglycan also serves as a mechanical link holding the daughter cells together and undergoes mechanical fracture during V snapping. Finally, we show that normal V snapping in C. glutamicum depends on complete assembly of the septal cell envelope.

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.

Measurement of Mesoscale Conformational Dynamics of Freely Diffusing Molecules with Tracking FCS.

Few techniques are suited to probe the structure and dynamics of molecular complexes at the mesoscale level (∼100-1000 nm). We have developed a single-molecule technique that uses tracking fluorescence correlation spectroscopy (tFCS) to probe the conformation and dynamics of mesoscale molecular assemblies. tFCS measures the distance fluctuations between two fluorescently labeled sites within an untethered, freely diffusing biomolecule. To achieve subdiffraction spatial resolution, we developed a feedback scheme that allows us to maintain the molecule at an optimal position within the laser intensity gradient for fluorescence correlation spectroscopy. We characterized tFCS spatial sensitivity by measuring the Brownian end-to-end dynamics of DNA molecules as short as 1000 bp. We demonstrate that tFCS detects changes in the compaction of reconstituted nucleosome arrays and can assay transient protein-mediated interactions between distant sites in an individual DNA molecule. Our measurements highlight the applicability of tFCS to a wide variety of biochemical processes involving mesoscale conformational dynamics.

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.

RecA: Regulation and Mechanism of a Molecular Search Engine.

Homologous recombination maintains genomic integrity by repairing broken chromosomes. The broken chromosome is partially resected to produce single-stranded DNA (ssDNA) that is used to search for homologous double-stranded DNA (dsDNA). This homology driven 'search and rescue' is catalyzed by a class of DNA strand exchange proteins that are defined in relation to Escherichia coli RecA, which forms a filament on ssDNA. Here, we review the regulation of RecA filament assembly and the mechanism by which RecA quickly and efficiently searches for and identifies a unique homologous sequence among a vast excess of heterologous DNA. Given that RecA is the prototypic DNA strand exchange protein, its behavior affords insight into the actions of eukaryotic RAD51 orthologs and their regulators, BRCA2 and other tumor suppressors.

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.

Condensing chromosome condensation.

Mitotic chromosome condensation has fascinated biologists since Flemming's 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.

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.