Mechanism of single-stranded DNA annealing by RAD52-RPA complex.

RAD52 is important for the repair of DNA double-stranded breaks1,2, mitotic DNA synthesis3-5 and alternative telomere length maintenance6,7. Central to these functions, RAD52 promotes the annealing of complementary single-stranded DNA (ssDNA)8,9 and provides an alternative to BRCA2/RAD51-dependent homologous recombination repair10. Inactivation of RAD52 in homologous-recombination-deficient BRCA1- or BRCA2-defective cells is synthetically lethal11,12, and aberrant expression of RAD52 is associated with poor cancer prognosis13,14. As a consequence, RAD52 is an attractive therapeutic target against homologous-recombination-deficient breast, ovarian and prostate cancers15-17. Here we describe the structure of RAD52 and define the mechanism of annealing. As reported previously18-20, RAD52 forms undecameric (11-subunit) ring structures, but these rings do not represent the active form of the enzyme. Instead, cryo-electron microscopy and biochemical analyses revealed that ssDNA annealing is driven by RAD52 open rings in association with replication protein-A (RPA). Atomic models of the RAD52-ssDNA complex show that ssDNA sits in a positively charged channel around the ring. Annealing is driven by the RAD52 N-terminal domains, whereas the C-terminal regions modulate the open-ring conformation and RPA interaction. RPA associates with RAD52 at the site of ring opening with critical interactions occurring between the RPA-interacting domain of RAD52 and the winged helix domain of RPA2. Our studies provide structural snapshots throughout the annealing process and define the molecular mechanism of ssDNA annealing by the RAD52-RPA complex.

Structural insights into BCDX2 complex function in homologous recombination.

Homologous recombination (HR) fulfils a pivotal role in the repair of DNA double-strand breaks and collapsed replication forks1. HR depends on the products of several paralogues of RAD51, including the tetrameric complex of RAD51B, RAD51C, RAD51D and XRCC2 (BCDX2)2. BCDX2 functions as a mediator of nucleoprotein filament assembly by RAD51 and single-stranded DNA (ssDNA) during HR, but its mechanism remains undefined. Here we report cryogenic electron microscopy reconstructions of human BCDX2 in apo and ssDNA-bound states. The structures reveal how the amino-terminal domains of RAD51B, RAD51C and RAD51D participate in inter-subunit interactions that underpin complex formation and ssDNA-binding specificity. Single-molecule DNA curtain analysis yields insights into how BCDX2 enhances RAD51-ssDNA nucleoprotein filament assembly. Moreover, our cryogenic electron microscopy and functional analyses explain how RAD51C alterations found in patients with cancer3-6 inactivate DNA binding and the HR mediator activity of BCDX2. Our findings shed light on the role of BCDX2 in HR and provide a foundation for understanding how pathogenic alterations in BCDX2 impact genome repair.

Mechanism of AAA+ ATPase-mediated RuvAB-Holliday junction branch migration.

The Holliday junction is a key intermediate formed during DNA recombination across all kingdoms of life1. In bacteria, the Holliday junction is processed by two homo-hexameric AAA+ ATPase RuvB motors, which assemble together with the RuvA-Holliday junction complex to energize the strand-exchange reaction2. Despite its importance for chromosome maintenance, the structure and mechanism by which this complex facilitates branch migration are unknown. Here, using time-resolved cryo-electron microscopy, we obtained structures of the ATP-hydrolysing RuvAB complex in seven distinct conformational states, captured during assembly and processing of a Holliday junction. Five structures together resolve the complete nucleotide cycle and reveal the spatiotemporal relationship between ATP hydrolysis, nucleotide exchange and context-specific conformational changes in RuvB. Coordinated motions in a converter formed by DNA-disengaged RuvB subunits stimulate hydrolysis and nucleotide exchange. Immobilization of the converter enables RuvB to convert the ATP-contained energy into a lever motion, which generates the pulling force driving the branch migration. We show that RuvB motors rotate together with the DNA substrate, which, together with a progressing nucleotide cycle, forms the mechanistic basis for DNA recombination by continuous branch migration. Together, our data decipher the molecular principles of homologous recombination by the RuvAB complex, elucidate discrete and sequential transition-state intermediates for chemo-mechanical coupling of hexameric AAA+ motors and provide a blueprint for the design of state-specific compounds targeting AAA+ motors.

A rapid method to visualize human mitochondrial DNA replication through rotary shadowing and transmission electron microscopy.

We report a rapid experimental procedure based on high-density in vivo psoralen inter-strand DNA cross-linking coupled to spreading of naked purified DNA, positive staining, low-angle rotary shadowing, and transmission electron microscopy (TEM) that allows quick visualization of the dynamic of heavy strand (HS) and light strand (LS) human mitochondrial DNA replication. Replication maps built on linearized mitochondrial genomes and optimized rotary shadowing conditions enable clear visualization of the progression of the mitochondrial DNA synthesis and visualization of replication intermediates carrying long single-strand DNA stretches. One variant of this technique, called denaturing spreading, allowed the inspection of the fine chromatin structure of the mitochondrial genome and was applied to visualize the in vivo three-strand DNA structure of the human mitochondrial D-loop intermediate with unprecedented clarity.

The large bat Helitron DNA transposase forms a compact monomeric assembly that buries and protects its covalently bound 5'-transposon end.

Helitrons are widespread eukaryotic DNA transposons that have significantly contributed to genome variability and evolution, in part because of their distinctive, replicative rolling-circle mechanism, which often mobilizes adjacent genes. Although most eukaryotic transposases form oligomers and use RNase H-like domains to break and rejoin double-stranded DNA (dsDNA), Helitron transposases contain a single-stranded DNA (ssDNA)-specific HUH endonuclease domain. Here, we report the cryo-electron microscopy structure of a Helitron transposase bound to the 5'-transposon end, providing insight into its multidomain architecture and function. The monomeric transposase forms a tightly packed assembly that buries the covalently attached cleaved end, protecting it until the second end becomes available. The structure reveals unexpected architectural similarity to TraI, a bacterial relaxase that also catalyzes ssDNA movement. The HUH active site suggests how two juxtaposed tyrosines, a feature of many replication initiators that use HUH nucleases, couple the conformational shift of an α-helix to control strand cleavage and ligation reactions.

Optimizing protein V untranslated region sequence in M13 phage for increased production of single-stranded DNA for origami.

DNA origami requires long scaffold DNA to be aligned with the guidance of short staple DNA strands. Scaffold DNA is produced in Escherichia coli as a form of the M13 bacteriophage by rolling circle amplification (RCA). This study shows that RCA can be reconfigured by reducing phage protein V (pV) expression, improving the production throughput of scaffold DNA by at least 5.66-fold. The change in pV expression was executed by modifying the untranslated region sequence and monitored using a reporter green fluorescence protein fused to pV. In a separate experiment, pV expression was controlled by an inducer. In both experiments, reduced pV expression was correlated with improved M13 bacteriophage production. High-cell-density cultivation was attempted for mass scaffold DNA production, and the produced scaffold DNA was successfully folded into a barrel shape without compromising structural quality. This result suggested that scaffold DNA production throughput can be significantly improved by reprogramming the RCA in E. coli.

Genetically Encoding Ultrastable Virus-like Particles Encapsulating Functional DNA Nanostructures in Living Bacteria.

DNA nanotechnology is leading the field of in vitro molecular-scale device engineering, accumulating to a dazzling array of applications. However, while DNA nanostructures' function is robust under in vitro settings, their implementation in real-world conditions requires overcoming their rapid degradation and subsequent loss of function. Viruses are sophisticated supramolecular assemblies, able to protect their nucleic acid content in inhospitable biological environments. Inspired by this natural ability, we engineered in vitro and in vivo technologies, enabling the encapsulation and protection of functional DNA nanostructures inside MS2 bacteriophage virus-like particles (VLPs). We demonstrate the ssDNA-VLPs nanocomposites' (NCs) abilities to encapsulate single-stranded-DNA (ssDNA) in a variety of sizes (200-1500 nucleotides (nt)), sequences, and structures while retaining their functionality. Moreover, by exposing these NCs to hostile biological conditions, such as human blood serum, we exhibit that the VLPs serve as an excellent protective shell. These engineered NCs pose critical properties that are yet unattainable by current fabrication methods.

Sugar-Pucker Force-Induced Transition in Single-Stranded DNA.

The accurate knowledge of the elastic properties of single-stranded DNA (ssDNA) is key to characterize the thermodynamics of molecular reactions that are studied by force spectroscopy methods where DNA is mechanically unfolded. Examples range from DNA hybridization, DNA ligand binding, DNA unwinding by helicases, etc. To date, ssDNA elasticity has been studied with different methods in molecules of varying sequence and contour length. A dispersion of results has been reported and the value of the persistence length has been found to be larger for shorter ssDNA molecules. We carried out pulling experiments with optical tweezers to characterize the elastic response of ssDNA over three orders of magnitude in length (60-14 k bases). By fitting the force-extension curves (FECs) to the Worm-Like Chain model we confirmed the above trend:the persistence length nearly doubles for the shortest molecule (60 b) with respect to the longest one (14 kb). We demonstrate that the observed trend is due to the different force regimes fitted for long and short molecules, which translates into two distinct elastic regimes at low and high forces. We interpret this behavior in terms of a force-induced sugar pucker conformational transition (C3'-endo to C2'-endo) upon pulling ssDNA.

Single-molecule visualization of human RECQ5 interactions with single-stranded DNA recombination intermediates.

RECQ5 is one of five RecQ helicases found in humans and is thought to participate in homologous DNA recombination by acting as a negative regulator of the recombinase protein RAD51. Here, we use kinetic and single molecule imaging methods to monitor RECQ5 behavior on various nucleoprotein complexes. Our data demonstrate that RECQ5 can act as an ATP-dependent single-stranded DNA (ssDNA) motor protein and can translocate on ssDNA that is bound by replication protein A (RPA). RECQ5 can also translocate on RAD51-coated ssDNA and readily dismantles RAD51-ssDNA filaments. RECQ5 interacts with RAD51 through protein-protein contacts, and disruption of this interface through a RECQ5-F666A mutation reduces translocation velocity by ∼50%. However, RECQ5 readily removes the ATP hydrolysis-deficient mutant RAD51-K133R from ssDNA, suggesting that filament disruption is not coupled to the RAD51 ATP hydrolysis cycle. RECQ5 also readily removes RAD51-I287T, a RAD51 mutant with enhanced ssDNA-binding activity, from ssDNA. Surprisingly, RECQ5 can bind to double-stranded DNA (dsDNA), but it is unable to translocate. Similarly, RECQ5 cannot dismantle RAD51-bound heteroduplex joint molecules. Our results suggest that the roles of RECQ5 in genome maintenance may be regulated in part at the level of substrate specificity.

The Cryo-EM structure of AAV2 Rep68 in complex with ssDNA reveals a malleable AAA+ machine that can switch between oligomeric states.

The adeno-associated virus (AAV) non-structural Rep proteins catalyze all the DNA transactions required for virus viability including, DNA replication, transcription regulation, genome packaging, and during the latent phase, site-specific integration. Rep proteins contain two multifunctional domains: an Origin Binding Domain (OBD) and a SF3 helicase domain (HD). Studies have shown that Rep proteins have a dynamic oligomeric behavior where the nature of the DNA substrate molecule modulates its oligomeric state. In the presence of ssDNA, Rep68 forms a large double-octameric ring complex. To understand the mechanisms underlying AAV Rep function, we investigated the cryo-EM and X-ray structures of Rep68-ssDNA complexes. Surprisingly, Rep68 generates hybrid ring structures where the OBD forms octameric rings while the HD forms heptamers. Moreover, the binding to ATPγS promotes a large conformational change in the entire AAA+ domain that leads the HD to form both heptamer and hexamers. The HD oligomerization is driven by an interdomain linker region that acts as a latch to 'catch' the neighboring HD subunit and is flexible enough to permit the formation of different stoichiometric ring structures. Overall, our studies show the structural basis of AAV Rep's structural flexibility required to fulfill its multifunctional role during the AAV life cycle.

The TFIIH subunits p44/p62 act as a damage sensor during nucleotide excision repair.

Nucleotide excision repair (NER) in eukaryotes is orchestrated by the core form of the general transcription factor TFIIH, containing the helicases XPB, XPD and five 'structural' subunits, p62, p44, p34, p52 and p8. Recent cryo-EM structures show that p62 makes extensive contacts with p44 and in part occupies XPD's DNA binding site. While p44 is known to regulate the helicase activity of XPD during NER, p62 is thought to be purely structural. Here, using helicase and adenosine triphosphatase assays we show that a complex containing p44 and p62 enhances XPD's affinity for dsDNA 3-fold over p44 alone. Remarkably, the relative affinity is further increased to 60-fold by dsDNA damage. Direct binding studies show this preference derives from p44/p62's high affinity (20 nM) for damaged ssDNA. Single molecule imaging of p44/p62 complexes without XPD reveals they bind to and randomly diffuse on DNA, however, in the presence of UV-induced DNA lesions these complexes stall. Combined with the analysis of a recent cryo-EM structure, we suggest that p44/p62 acts as a novel DNA-binding entity that enhances damage recognition in TFIIH. This revises our understanding of TFIIH and prompts investigation into the core subunits for an active role during DNA repair and/or transcription.

Mechanism of Type IA Topoisomerases.

Topoisomerases in the type IA subfamily can catalyze change in topology for both DNA and RNA substrates. A type IA topoisomerase may have been present in a last universal common ancestor (LUCA) with an RNA genome. Type IA topoisomerases have since evolved to catalyze the resolution of topological barriers encountered by genomes that require the passing of nucleic acid strand(s) through a break on a single DNA or RNA strand. Here, based on available structural and biochemical data, we discuss how a type IA topoisomerase may recognize and bind single-stranded DNA or RNA to initiate its required catalytic function. Active site residues assist in the nucleophilic attack of a phosphodiester bond between two nucleotides to form a covalent intermediate with a 5'-phosphotyrosine linkage to the cleaved nucleic acid. A divalent ion interaction helps to position the 3'-hydroxyl group at the precise location required for the cleaved phosphodiester bond to be rejoined following the passage of another nucleic acid strand through the break. In addition to type IA topoisomerase structures observed by X-ray crystallography, we now have evidence from biophysical studies for the dynamic conformations that are required for type IA topoisomerases to catalyze the change in the topology of the nucleic acid substrates.

DNA origami-based single-molecule force spectroscopy elucidates RNA Polymerase III pre-initiation complex stability.

The TATA-binding protein (TBP) and a transcription factor (TF) IIB-like factor are important constituents of all eukaryotic initiation complexes. The reason for the emergence and strict requirement of the additional initiation factor Bdp1 in the RNA polymerase (RNAP) III system, however, remained elusive. A poorly studied aspect in this context is the effect of DNA strain arising from DNA compaction and transcriptional activity on initiation complex formation. We made use of a DNA origami-based force clamp to follow the assembly of human initiation complexes in the RNAP II and RNAP III systems at the single-molecule level under piconewton forces. We demonstrate that TBP-DNA complexes are force-sensitive and TFIIB is sufficient to stabilise TBP on a strained promoter. In contrast, Bdp1 is the pivotal component that ensures stable anchoring of initiation factors, and thus the polymerase itself, in the RNAP III system. Thereby, we offer an explanation for the crucial role of Bdp1 for the high transcriptional output of RNAP III.

Capturing transient antibody conformations with DNA origami epitopes.

Revealing antibody-antigen interactions at the single-molecule level will deepen our understanding of immunology. However, structural determination under crystal or cryogenic conditions does not provide temporal resolution for resolving transient, physiologically or pathologically relevant functional antibody-antigen complexes. Here, we develop a triangular DNA origami framework with site-specifically anchored and spatially organized artificial epitopes to capture transient conformations of immunoglobulin Gs (IgGs) at room temperature. The DNA origami epitopes (DOEs) allows programmed spatial distribution of epitope spikes, which enables direct imaging of functional complexes with atomic force microscopy (AFM). We establish the critical dependence of the IgG avidity on the lateral distance of epitopes within 3-20 nm at the single-molecule level. High-speed AFM imaging of transient conformations further provides structural and dynamic evidence for the IgG avidity from monovalent to bivalent in a single event, which sheds light on various applications including virus neutralization, diagnostic detection and cancer immunotherapy.

A single-molecule study reveals novel rod-like structures formed by a thrombin aptamer repeat sequence.

Thrombin aptamers (TBAs) have attracted much attention due to their various applications. The structures and properties of long ssDNA chains with multiple TBA repeat sequences are interesting and distinct from those of their monomers. Due to the complexity of the sample system, it is quite difficult to reveal the structure of such a long-chain ssDNA using traditional methods. In this work, we investigated the repeated ssDNA by using single-molecule magnetic tweezers and AFM imaging. To do that we developed the polymerase change-rolling circle amplification (PC-RCA) synthetic method and prepared two-end modified repeated ssDNA. The rod-like G4 structures formed by intramolecular stacking of the repeat sequence were for the first time identified. This novel structure is different from those higher-order quadruplex structures formed by G-tetrads or loop-mediated interactions. It is also quite interesting to find that the increase of the TBA copy number can unitize the diversity of TBA conformation to the best-fit binding structure for thrombin. The methodology developed in this work can be used for studying other repeat sequences in the genome, such as telomeric DNA as well as interactions of ssDNA with the binding molecule.

Differential Charging in Photoemission from Mercurated DNA Monolayers on Ferromagnetic Films.

Spin-dependent and enantioselective electron-molecule scattering occurs in photoelectron transmission through chiral molecular films. This spin selectivity leads to electron spin filtering by molecular helices, with increasing magnitude concomitant with increasing numbers of helical turns. Using ultraviolet photoelectron spectroscopy, we measured spin-selective surface charging accompanying photoemission from ferromagnetic substrates functionalized with monolayers of mercurated DNA hairpins that constitute only one helical turn. Mercury ions bind specifically at thymine-thymine mismatches within self-hybridized single-stranded DNA, enabling precise control over the number and position of Hg2+ along the helical axis. Differential charging of the organic layers, manifested as substrate-magnetization-dependent photoionization energies, was observed for DNA hairpins containing Hg2+; no differences were measured for hairpin monolayers in the absence of Hg2+. Inversion of the DNA helical secondary structure at increased metal loading led to complementary inversion in spin selectivity. We attribute these results to increased scattering probabilities from relativistic enhancement of spin-orbit interactions in mercurated DNA.

On the role of flexibility in linker-mediated DNA hydrogels.

Three-dimensional DNA networks, composed of tri- or higher valent nanostars with sticky, single-stranded DNA overhangs, have been previously studied in the context of designing thermally responsive, viscoelastic hydrogels. In this work, we use linker-mediated gels, where the sticky ends of two trivalent nanostars are connected through the complementary sticky ends of a linear DNA duplex. We can design this connection to be either rigid or flexible by introducing flexible, non-binding bases. The additional flexibility provided by these non-binding bases influences the effective elasticity of the percolating gel formed at low temperatures. Here we show that by choosing the right length of the linear duplex and non-binding flexible joints, we obtain a completely different phase behaviour to that observed for rigid linkers. In particular, we use dynamic light scattering as a microrheological tool to monitor the self-assembly of DNA nanostars with linear linkers as a function of temperature. While we observe classical gelation when using rigid linkers, the presence of flexible joints leads to a cluster fluid with a much-reduced viscosity. Using both the oxDNA model and a coarse-grained simulation to investigate the nanostar-linker topology, we hypothesise on the possible structure formed by the DNA clusters. Moreover, we present a systematic study of the strong viscosity increase of aqueous solutions in the presence of these DNA building blocks.

In vitro role of Rad54 in Rad51-ssDNA filament-dependent homology search and synaptic complexes formation.

Homologous recombination (HR) uses a homologous template to accurately repair DNA double-strand breaks and stalled replication forks to maintain genome stability. During homology search, Rad51 nucleoprotein filaments probe and interact with dsDNA, forming the synaptic complex that is stabilized on a homologous sequence. Strand intertwining leads to the formation of a displacement-loop (D-loop). In yeast, Rad54 is essential for HR in vivo and required for D-loop formation in vitro, but its exact role remains to be fully elucidated. Using electron microscopy to visualize the DNA-protein complexes, here we find that Rad54 is crucial for Rad51-mediated synaptic complex formation and homology search. The Rad54-K341R ATPase-deficient mutant protein promotes formation of synaptic complexes but not D-loops and leads to the accumulation of stable heterologous associations, suggesting that the Rad54 ATPase is involved in preventing non-productive intermediates. We propose that Rad51/Rad54 form a functional unit operating in homology search, synaptic complex and D-loop formation.

Resolution-Free Accurate DNA Contour Length Estimation from Atomic Force Microscopy Images.

This research presented an accurate and efficient contour length estimation method developed for DNA digital curves acquired from Atomic Force Microscopy (AFM) images. This automation method is calibrated against different AFM resolutions and ideal to be extended to all different kinds of biopolymer samples, encompassing all different sample stiffnesses. The methodology considers the digital curve local geometric relationship, as these digital shape segments and pixel connections represent the actual morphology of the biopolymer sample as it is being imaged from the AFM scanning. In order to incorporate the true local geometry relationship that is embedded in the continuous form of the original sample, one needs to find this geometry counterpart in the digitized image. This counterpart is realized by taking the skeleton backbone of the sample contour and by using these digitized pixels' connection relationship to find its local shape representation. In this research, one uses the 8-connect Freeman Chain Code (CC) to describe the directional connection between DNA image pixels, in order to account for the local shapes of four connected pixels. The result is a novel shape number (SN) system derived from CC, which is a fully automated algorithm that can be applied to DNA samples of any length for accurate estimation, with efficient computational cost. This shape-wise consideration is weighted to modify the local length with great precision, accounting for all the different morphologies of the biopolymer sample, and resulted with accurate length estimation, as the error falls below 0.07%, an order of magnitude improvement compared to previous findings.

First principle approach towards logic design using hydrogen-doped single-strand DNA.

Molecular logic gate has been proposed using single-strand DNA (ssDNA) consisting of basic four nucleobases. In this study, density functional theory and non-equilibrium Green's function based first principle approach is applied to investigate the electronic transmission characteristics of ssDNA chain. The heavily hydrogen-doped-ssDNA (H-ssDNA) chain is connected with gold electrode to achieve enhanced quantum-ballistic transmission along 〈1 1 1〉 direction. Logic gates OR, Ex-OR, NXOR have been implemented using this analytical model of H-ssDNA device. Enhanced logic properties have been observed for ssDNA after H adsorption due to improved electronic transmission. Dense electron cloud is considered as logic 'high' (1) output in presence of hydrogen molecule and on the contrary sparse cloud indicate logic 'low' (0) in the absence of hydrogen molecule. Device current is significantly increased from 0.2 nA to 2.4 µA (approx.) when ssDNA chain is heavily doped with hydrogen molecule. The current-voltage characteristics confirm the formation of various Boolean logic gate operations.