Graphene quantum point contact transistor for DNA sensing

By using the nonequilibrium Green’s function technique, we show that the shape of the edge, the carrier concentration, and the position and size of a nanopore in graphene nanoribbons can strongly affect its electronic conductance as well as its sensitivity to external charges. This technique, combined with a self-consistent Poisson–Boltzmann formalism to account for ion charge screening in solution, is able to detect the rotational and positional conformation of a DNA strand inside the nanopore. In particular, we show that a graphene membrane with quantum point contact geometry exhibits greater electrical sensitivity than a uniform armchair geometry provided that the carrier concentration is tuned to enhance charge detection. We propose a membrane design that contains an electrical gate in a configuration similar to a field-effect transistor for a graphene-based DNA sensing device.Over the past few years the need has grown for low-cost, high-speed, and accurate biomolecule sensing, propelling the so-called third generation of genome sequencing devices (1–4). Many associated technologies have been developed, but recent advances in the fabrication of solid-state nanopores have shown that the translocation of biomolecules such as DNA through such pores is a promising alternative to traditional sensing methods (5–9). Some of these methods include measuring (i) ionic blockade current fluctuations through nanopores in the presence of nucleotides (10), (ii) tunneling currents across nanopores containing biomolecules (11), and (iii) direct transverse-current measurements (12). Graphene is a prime candidate for such measurements. Theoretical studies suggest that functionalized graphene nanopores can be used to differentiate passing ions, demonstrating the potential use of graphene membranes in nanofluidics and molecular sensing (13). In addition, its atomic-scale thickness allows a molecule passing through it to be scanned at the highest possible resolution, and the feasibility of using graphene nanopores for DNA detection has been demonstrated experimentally (14–17). Lastly, electrically active graphene can, in principle, both control and probe translocating molecules, acting as a gate as well as a charge sensor, which passive, oxide-based nanopore devices are incapable of doing.Molecular dynamics studies describing the electrophoresis of DNA translocation through graphene nanopores demonstrated that DNA sequencing by measuring ionic current blockades is possible in principle (18, 19). Additionally, several groups have reported first-principles–based studies to identify base pairs using tunneling currents or transverse conductance-based approaches (12, 20, 21). Saha et al. reported transverse edge current variations of the order of 1 µA through graphene nanoribbons (GNRs) caused by the presence of isolated nucleotides in a nanopore, and reported base pair specific edge currents (12). These studies, however, do not account for solvent or screening effects; the latter effects are due to the presence of ions in the solution and can reduce the ability of the nanoribbon to discern individual nucleotides. Very recently, Avdoshenko et al. investigated the influence of single-stranded DNA on sheet currents in GNRs with nanopores (22). However, their study does not consider the carrier concentration modulation of the current, the influence of the GNR-edge boundary condition on the nanopore sensitivity, or a self-consistent treatment of screening due to charged ions in solution.GNRs are strips of graphene with a finite width that quantizes the energy states of the conduction electrons (23–26). Unlike traditional quantum wells, the boundary conditions of GNRs are complicated functions of position and momentum resulting from the dual sublattice symmetry of graphene, giving rise to a unique band structure. Because of this, the shape of the boundary as well as the presence of nanopores profoundly affects the electronic states of GNRs (27–30), for example, leading to a difference in band structure for zigzag and armchair-edged GNRs (31).The edge of a GNR can be patterned with near-atomic precision, opening up the possibility to investigate many different geometries (32). In the case of complicated edge shapes, the current displays an extremely nonlinear and not strictly increasing dependence on carrier concentration. The graphene quantum point contact (g-QPC) is a perfect example in this regard, as its irregular edge yields a complex band structure and rich conductance spectrum with many regions of high sensitivity and negative differential transconductance (NDTC) as shown below. In addition, the g-QPC electronic properties are not limited by stringent GNR uniformity (armchair or zigzag) in the boundary conditions. Moreover, the carrier concentration itself, which can be controlled by the presence of a back gate embedded within a g-QPC device as in a field-effect transistor (FET), can profoundly affect the sensitivity and nonlinearity of the current. As a result, changes in external electric fields, including changes due to rotation and translation of external molecular charges, alter the local carrier concentration and can dramatically influence the g-QPC conductance. In the following we demonstrate the complex and nonlinear effects of altering boundary shapes, graphene carrier concentrations, and electric potentials due to DNA translocation on the conductance of such a device. We propose to sense DNA by performing transport measurements in a g-QPC device and demonstrate that the sensitivity of the conductance can be geometrically and electronically tuned to detect small differences in the charge geometry of biomolecules such as DNA.     

Detection of genomic variations and DNA polymorphisms and impact on analysis of meiotic recombination and genetic mapping

DNA polymorphisms are important markers in genetic analyses and are increasingly detected by using genome resequencing. However, the presence of repetitive sequences and structural variants can lead to false positives in the identification of polymorphic alleles. Here, we describe an analysis strategy that minimizes false positives in allelic detection and present analyses of recently published resequencing data from Arabidopsis meiotic products and individual humans. Our analysis enables the accurate detection of sequencing errors, small insertions and deletions (indels), and structural variants, including large reciprocal indels and copy number variants, from comparisons between the resequenced and reference genomes. We offer an alternative interpretation of the sequencing data of meiotic products, including the number and type of recombination events, to illustrate the potential for mistakes in single-nucleotide polymorphism calling. Using these examples, we propose that the detection of DNA polymorphisms using resequencing data needs to account for nonallelic homologous sequences.DNA polymorphisms are ubiquitous genetic variations among individuals and include single nucleotide polymorphisms (SNPs), insertions and deletions (indels), and other larger rearrangements (1–3) (Fig. 1 A and B). They can have phenotypic consequences and also serve as molecular markers for genetic analyses, facilitating linkage and association studies of genetic diseases, and other traits in humans (4–6), animals, plants, (7–10) and other organisms. Using DNA polymorphisms for modern genetic applications requires low-error, high-throughput analytical strategies. Here, we illustrate the use of short-read next-generation sequencing (NGS) data to detect DNA polymorphisms in the context of whole-genome analysis of meiotic products.Open in a separate windowFig. 1.(A) SNPs and small indels between two ecotype genomes. (B) Possible types of SVs. Col genotypes are marked in blue and Ler in red. Arrows indicate DNA segments involved in SVs between the two ecotypes. (C) Meiotic recombination events including a CO and a GC (NCO). Centromeres are denoted by yellow dots.There are many methods for detecting SNPs (11–14) and structural variants (SVs) (15–25), including NGS, which can capture nearly all DNA polymorphisms (26–28). This approach has been widely used to analyze markers in crop species such as rice (29), genes associated with diseases (6, 26), and meiotic recombination in yeast and plants (30, 31). However, accurate identification of DNA polymorphisms can be challenging, in part because short-read sequencing data have limited information for inferring chromosomal context.Genomes usually contain repetitive sequences that can differ in copy number between individuals (26–28, 31); therefore, resequencing analyses must account for chromosomal context to avoid mistaking highly similar paralogous sequences for polymorphisms. Here, we use recently published datasets to describe several DNA sequence features that can be mistaken as allelic (32, 33) and describe a strategy for differentiating between repetitive sequences and polymorphic alleles. We illustrate the effectiveness of these analyses by examining the reported polymorphisms from the published datasets.Meiotic recombination is initiated by DNA double-strand breaks (DSBs) catalyzed by the topoisomerase-like SPORULATION 11 (SPO11). DSBs are repaired as either crossovers (COs) between chromosomes (Fig. 1C), or noncrossovers (NCOs). Both COs and NCOs can be accompanied by gene conversion (GC) events, which are the nonreciprocal transfer of sequence information due to the repair of heteroduplex DNA during meiotic recombination. Understanding the control of frequency and distribution of CO and NCO (including GC) events has important implications for human health (including cancer and aneuploidy), crop breeding, and the potential for use in genome engineering. COs can be detected relatively easily by using polymorphic markers in the flanking sequences, but NCO products can only be detected if they are accompanied by a GC event. Because GCs associated with NCO result in allelic changes at polymorphic sites without exchange of flanking sequences, they are more difficult to detect. Recent advances in DNA sequencing have made the analysis of meiotic NCOs more feasible (30–32, 34); however, SVs present a challenge in these analyses. We recommend a set of guidelines for detection of DNA polymorphisms by using genomic resequencing short-read datasets. These measures improve the accuracy of a wide range of analyses by using genomic resequencing, including estimation of COs, NCOs, and GCs.     

Self-assembly of amphiphilic Janus dendrimers into uniform onion-like dendrimersomes with predictable size and number of bilayers

A constitutional isomeric library synthesized by a modular approach has been used to discover six amphiphilic Janus dendrimer primary structures, which self-assemble into uniform onion-like vesicles with predictable dimensions and number of internal bilayers. These vesicles, denoted onion-like dendrimersomes, are assembled by simple injection of a solution of Janus dendrimer in a water-miscible solvent into water or buffer. These dendrimersomes provide mimics of double-bilayer and multibilayer biological membranes with dimensions and number of bilayers predicted by the Janus compound concentration in water. The simple injection method of preparation is accessible without any special equipment, generating uniform vesicles, and thus provides a promising tool for fundamental studies as well as technological applications in nanomedicine and other fields.Most living organisms contain single-bilayer membranes composed of lipids, glycolipids, cholesterol, transmembrane proteins, and glycoproteins (1). Gram-negative bacteria (2, 3) and the cell nucleus (4), however, exhibit a strikingly special envelope that consists of a concentric double-bilayer membrane. More complex membranes are also encountered in cells and their various organelles, such as multivesicular structures of eukaryotic cells (5) and endosomes (6), and multibilayer structures of endoplasmic reticulum (7, 8), myelin (9, 10), and multilamellar bodies (11, 12). This diversity of biological membranes inspired corresponding biological mimics. Liposomes (Fig. 1) self-assembled from phospholipids are the first mimics of single-bilayer biological membranes (13–16), but they are polydisperse, unstable, and permeable (14). Stealth liposomes coassembled from phospholipids, cholesterol, and phospholipids conjugated with poly(ethylene glycol) exhibit improved stability, permeability, and mechanical properties (17–20). Polymersomes (21–24) assembled from amphiphilic block copolymers exhibit better mechanical properties and permeability, but are not always biocompatible and are polydisperse. Dendrimersomes (25–28) self-assembled from amphiphilic Janus dendrimers and minidendrimers (26–28) have also been elaborated to mimic single-bilayer biological membranes. Amphiphilic Janus dendrimers take advantage of multivalency both in their hydrophobic and hydrophilic parts (23, 29–32). Dendrimersomes are assembled by simple injection (33) of a solution of an amphiphilic Janus dendrimer (26) in a water-soluble solvent into water or buffer and produce uniform (34), impermeable, and stable vesicles with excellent mechanical properties. In addition, their size and properties can be predicted by their primary structure (27). Amphiphilic Janus glycodendrimers self-assemble into glycodendrimersomes that mimic the glycan ligands of biological membranes (35). They have been demonstrated to be bioactive toward biomedically relevant bacterial, plant, and human lectins, and could have numerous applications in nanomedicine (20).Open in a separate windowFig. 1.Strategies for the preparation of single-bilayer vesicles and multibilayer onion-like vesicles.More complex and functional cell mimics such as multivesicular vesicles (36, 37) and multibilayer onion-like vesicles (38–40) have also been discovered. Multivesicular vesicles compartmentalize a larger vesicle (37) whereas multibilayer onion-like vesicles consist of concentric alternating bilayers (40). Currently multibilayer vesicles are obtained by very complex and time-consuming methods that do not control their size (39) and size distribution (40) in a precise way. Here we report the discovery of “single–single” (28) amphiphilic Janus dendrimer primary structures that self-assemble into uniform multibilayer onion-like dendrimersomes (Fig. 1) with predictable size and number of bilayers by simple injection of their solution into water or buffer.     

Kaposi's sarcoma-associated herpesvirus LANA recruits the DNA polymerase clamp loader to mediate efficient replication and virus persistence

Kaposi''s sarcoma-associated herpesvirus (KSHV) latently infects tumor cells and persists as a multiple-copy, extrachromosomal, circular episome. To persist, the viral genome must replicate with each cell cycle. The KSHV latency-associated nuclear antigen (LANA) mediates viral DNA replication and persistence, but little is known regarding the underlying mechanisms. We find that LANA recruits replication factor C (RFC), the DNA polymerase clamp [proliferating cell nuclear antigen (PCNA)] loader, to drive DNA replication efficiently. Mutated LANA lacking RFC interaction was deficient for LANA-mediated DNA replication and episome persistence. RFC depletion had a negative impact on LANA’s ability to replicate and maintain viral DNA in cells containing artificial KSHV episomes or in infected cells, leading to loss of virus. LANA substantially increased PCNA loading onto DNA in vitro and recruited RFC and PCNA to KSHV DNA in cells. These findings suggest that PCNA loading is a rate-limiting step in DNA replication that is incompatible with viral survival. LANA enhancement of PCNA loading permits efficient virus replication and persistence, revealing a previously unidentified mechanism for KSHV latency.Kaposi''s sarcoma-associated herpesvirus (KSHV or human herpesvirus 8) has a causative role in Kaposi''s sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman disease (1–4). KSHV infection of tumor cells is predominantly latent. During latent infection, the viral genome persists as a multicopy, circular, extrachromosomal episome (plasmid) (5, 6). To persist, the genome must replicate and segregate to progeny nuclei with each cell division. Latency-associated nuclear antigen (LANA), a 1,162-residue protein, is one of a few KSHV genes expressed in latency. LANA is necessary and sufficient for episome persistence in the absence of other viral genes (7, 8). Both N-terminal LANA (N-LANA) and C-terminal LANA (C-LANA) are essential for function. N-LANA associates with mitotic chromosomes via binding histones H2A/H2B, and C-LANA simultaneously binds KSHV terminal repeat (TR) DNA (7, 9–20). Thus, LANA tethers the viral genome to host chromosomes and distributes viral DNA to daughter nuclei during mitosis. Importantly, LANA, which lacks enzymatic function, also mediates KSHV TR DNA replication (10, 21–23) through recruitment of host cell machinery, but little is known regarding the details of this process.We recently identified an internal 59-aa LANA region critical for efficient DNA replication and persistence (24, 25). Here, we find that LANA recruits replication factor C (RFC), the DNA polymerase clamp [proliferating cell nuclear antigen (PCNA)] loader (26), through this sequence to mediate viral DNA replication and episome persistence.     

Stabilization of graphene nanopore

Graphene is an ultrathin, impervious membrane. The controlled introduction of nanoscale pores in graphene would lead to applications that involve water purification, chemical separation, and DNA sequencing. However, graphene nanopores are unstable against filling by carbon adatoms. Here, using aberration-corrected scanning transmission electron microscopy and density-functional calculations, we report that Si atoms stabilize graphene nanopores by bridging the dangling bonds around the perimeter of the hole. Si‐passivated pores remain intact even under intense electron beam irradiation, and they were observed several months after the sample fabrication, demonstrating that these structures are intrinsically robust and stable against carbon filling. Theoretical calculations reveal the underlying mechanism for this stabilization effect: Si atoms bond strongly to the graphene edge, and their preference for tetrahedral coordination forces C adatoms to form dendrites sticking out of the graphene plane, instead of filling the nanopore. Our results provide a novel way to develop stable nanopores, which is a major step toward reliable graphene-based molecular translocation devices.Extensive experimental and theoretical work has been done on vacancy clusters and nanopores in graphene (1–6). In particular, nanopore technology has emerged as a powerful tool for single-ion channels, single-molecule detection, and liquid purification. A monolayer graphene nanopore (7–9) compared with a typical solid-state nanopore of a 30-nm thickness (10–14) provides an optimal approach for very high-resolution, high-throughput, rapid DNA sequencing.Rapid progress has been made in experimental studies exploring a wide variety of methods for introducing nanopores in graphene. The most recent methods have used diblock copolymer templating, helium-ion beam drilling, and chemical etching to achieve both higher porosity and a more precise pore size control (15–18). However, it is observed that small holes in graphene are subject to reconstruction and partial or total filling by diffusing carbon or other adatoms (known as the self-healing process) (19–21). Stabilization of pores for extended periods of time has not been achieved so far.Here, we report that Si atoms stabilize graphene nanopores by bridging the dangling bonds around the perimeter of the hole. Without passivation, we find that small pores in graphene fill within hours even under ultrahigh-vacuum conditions. This pore stabilization is understood in terms of the fact that Si atoms prefer tetrahedral coordination so that C adatoms that might bond to them would not lie in the graphene plane (22, 23). Our molecular-dynamics (MD) simulations show that the C adatoms would form dendrites sticking out of the graphene plane, instead of filling the nanopore, suggesting that Si passivation is indeed effective in preventing the self-healing process. Furthermore, experimental and theoretical evidence suggests that Si-stabilized nanopores are stable in ambient atmosphere and liquids.     

Programmable motion of DNA origami mechanisms

DNA origami enables the precise fabrication of nanoscale geometries. We demonstrate an approach to engineer complex and reversible motion of nanoscale DNA origami machine elements. We first design, fabricate, and characterize the mechanical behavior of flexible DNA origami rotational and linear joints that integrate stiff double-stranded DNA components and flexible single-stranded DNA components to constrain motion along a single degree of freedom and demonstrate the ability to tune the flexibility and range of motion. Multiple joints with simple 1D motion were then integrated into higher order mechanisms. One mechanism is a crank–slider that couples rotational and linear motion, and the other is a Bennett linkage that moves between a compacted bundle and an expanded frame configuration with a constrained 3D motion path. Finally, we demonstrate distributed actuation of the linkage using DNA input strands to achieve reversible conformational changes of the entire structure on ∼minute timescales. Our results demonstrate programmable motion of 2D and 3D DNA origami mechanisms constructed following a macroscopic machine design approach.The ability to control, manipulate, and organize matter at the nanoscale has demonstrated immense potential for advancements in industrial technology, medicine, and materials (1–3). Bottom-up self-assembly has become a particularly promising area for nanofabrication (4, 5); however, to date designing complex motion at the nanoscale remains a challenge (6–9). Amino acid polymers exhibit well-defined and complex dynamics in natural systems and have been assembled into designed structures including nanotubes, sheets, and networks (10–12), although the complexity of interactions that govern amino acid folding make designing complex geometries extremely challenging. DNA nanotechnology, on the other hand, has exploited well-understood assembly properties of DNA to create a variety of increasingly complex designed nanostructures (13–15).Scaffolded DNA origami, the process of folding a long single-stranded DNA (ssDNA) strand into a custom structure (16–18), has enabled the fabrication of nanoscale objects with unprecedented geometric complexity that have recently been implemented in applications such as containers for drug delivery (19, 20), nanopores for single-molecule sensing (21–23), and templates for nanoparticles (24, 25) or proteins (26–28). The majority of these and other applications of DNA origami have largely focused on static structures. Natural biomolecular machines, in contrast, have a rich diversity of functionalities that rely on complex but well-defined and reversible conformational changes. Currently, the scope of biomolecular nanotechnology is limited by an inability to achieve similar motion in designed nanosystems.DNA nanotechnology has enabled critical steps toward that goal starting with the work of Mao et al. (29), who developed a DNA nanostructure that took advantage of the B–Z transition of DNA to switch states. Since then, efforts to fabricate dynamic DNA systems have primarily focused on strand displacement approaches (30) mainly on systems comprising a few strands or arrays of strands undergoing ∼nm-scale motions (31–37) in some cases guided by DNA origami templates (38–40). More recently, strand displacement has been used to reconfigure DNA origami nanostructures, for example opening DNA containers (19, 41, 42), controlling molecular binding (43, 44), or reconfiguring structures (45). The largest triggerable structural change was achieved by Han et al. in a DNA origami Möbius strip (one-sided ribbon structure) that could be opened to approximately double in size (45). Constrained motion has been achieved in systems with rotational motion (19, 20, 32, 41, 44, 46, 47) in some cases to open lid-like components (19, 20, 41) or detect molecular binding (44, 48, 49). A few of these systems achieved reversible conformational changes (32, 41, 44, 46), although the motion path and flexibility were not studied. Constrained linear motion has remained largely unexplored. Linear displacements on the scale of a few nanometers have been demonstrated via conformational changes of DNA structure motifs (50–55), strand invasion to open DNA hairpins (36, 55, 56), or the reversible sliding motion of a DNA tile actuator (56); these cases also did not investigate the motion path or flexibility of motion.Building on these prior studies, this work implements concepts from macroscopic machine design to build modular parts with constrained motion. We demonstrate an ability to tune the flexibility and range of motion and then integrate these parts into prototype mechanisms with designed 2D and 3D motion. We further demonstrate reversible actuation of a mechanism with complex conformational changes on minute timescales.     

From the Cover: PNAS Plus: Single-molecule visualization of RecQ helicase reveals DNA melting,nucleation, and assembly are required for processive DNA unwinding

DNA helicases are motor proteins that unwind double-stranded DNA (dsDNA) to reveal single-stranded DNA (ssDNA) needed for many biological processes. The RecQ helicase is involved in repairing damage caused by DNA breaks and stalled replication forks via homologous recombination. Here, the helicase activity of RecQ was visualized on single molecules of DNA using a fluorescent sensor that directly detects ssDNA. By monitoring the formation and progression of individual unwinding forks, we observed that both the frequency of initiation and the rate of unwinding are highly dependent on RecQ concentration. We establish that unwinding forks can initiate internally by melting dsDNA and can proceed in both directions at up to 40–60 bp/s. The findings suggest that initiation requires a RecQ dimer, and that continued processive unwinding of several kilobases involves multiple monomers at the DNA unwinding fork. We propose a distinctive model wherein RecQ melts dsDNA internally to initiate unwinding and subsequently assembles at the fork into a distribution of multimeric species, each encompassing a broad distribution of rates, to unwind DNA. These studies define the species that promote resection of DNA, proofreading of homologous pairing, and migration of Holliday junctions, and they suggest that various functional forms of RecQ can be assembled that unwind at rates tailored to the diverse biological functions of RecQ helicase.DNA helicases are ubiquitous enzymes involved in many aspects of DNA metabolism, including DNA replication, repair, and recombination. These enzymes work by coupling the hydrolysis of nucleoside triphosphates (NTPs) to unwinding of double-stranded DNA (dsDNA) to produce single-stranded DNA (ssDNA) (1). This activity allows the cell’s machinery to access the information stored within the bases of the double helix. RecQ helicase from Escherichia coli is the founding member of the RecQ family of helicases (2). These enzymes belong to the superfamily 2 (SF2) group of helicases, yet share greater sequence homology with their own family members, and they play important roles in the maintenance of genomic integrity by DNA recombination and repair (1, 3). Mutations in the human RecQ-like helicases, Bloom (BLM), Werner (WRN), and RecQ4 proteins, lead to Bloom’s, Werner’s, and Rothmund–Thomson syndromes, respectively. These genetic disorders are characterized by genomic instability and an increased incidence of cancers (4).E. coli RecQ is a 3′ → 5′ helicase that functions in DNA-break repair by homologous recombination (2, 5, 6). RecQ and RecJ, a 5′ → 3′ exonuclease, process ssDNA gaps or dsDNA breaks into ssDNA for recombinational repair by RecA (7–10). In addition, RecQ ensures recombination fidelity in vivo by removing inappropriately paired joint molecules to prevent illegitimate recombination and also by disrupting joint molecule intermediates to facilitate repair by synthesis-dependent strand annealing, preventing chromosomal crossing over (7, 9, 11, 12). RecQ also functions with topoisomerase III (Topo III), a type I topoisomerase, to catenate and decatenate DNA molecules and to separate converged replication forks (13, 14). RecQ and Topo III provide an alternative to the RuvABC pathway for disengaging double Holliday junctions and do so without producing chromosomal crossovers (15).In vitro, RecQ can unwind a multitude of DNA substrates and does not require a ssDNA tail, or even a dsDNA end, to initiate unwinding; consequently, it is distinctive in being able to unwind covalently closed circular plasmid DNA (16, 17). The winged-helix domain of RecQ is important for the recognition of this broad array of DNA substrates (18). This domain binds to dsDNA yet it adopts a flexible conformation that allows it to adapt to many DNA structures. A curious feature of RecQ is that maximal unwinding requires nearly stoichiometric amounts of protein relative to the DNA (one protein per ∼10 bp), which can be partially mitigated (one protein per ∼30 bp) by including the ssDNA binding protein, SSB (5, 6, 16, 19). This behavior is compatible with the low processivity of ssDNA translocation (∼30–100 nucleotides) by RecQ (20–22) and other RecQ members (23). Paradoxically, at limiting concentrations, most RecQ helicases nonetheless efficiently unwind several kilobases of dsDNA in the course of their normal functions (9, 16, 24–26), suggesting a dynamic unwinding process. Although RecQ can unwind DNA as a monomer, it also shows a functional cooperativity when unwinding DNA with an ssDNA tail (27–29). Thus, multiple monomers can bind to ssDNA to unwind long dsDNA regions.Fluorescence techniques coupled with single-molecule microscopy have emerged as a powerful method for studying the unwinding mechanism of helicases (30–35). These assays measure individual enzymes directly or indirectly through their actions on individual DNA molecules, thus alleviating many of the drawbacks of ensemble experiments. In particular, total internal reflection fluorescence (TIRF) microscopy permits the detection of individual fluorophores with high sensitivity in the presence of a high background (30). To visualize the activity of DNA binding proteins and helicases, TIRF microscopy can be coupled with microfluidic techniques to facilitate visualization of molecules and exchange of solution components (36–39).Ensemble assays have elucidated many features of DNA unwinding by the RecQ helicase family, but the need to average over a heterogeneous and unsynchronized population of enzymes has precluded a thorough understanding of this diverse and universally important helicase family. To permit a more insightful analysis, we directly visualized unwinding of individual molecules of DNA by RecQ helicase. Unwinding was monitored using fluorescent SSB to visualize generation of ssDNA. On single DNA molecules, we could see tracks of the fluorescent SSB binding to ssDNA produced by the helicase activity of RecQ. We show that RecQ initiates DNA unwinding via melting of duplex DNA at internal sites. Once initiated, DNA unwinding propagates either uni- or bidirectionally via the cooperative action of multiple RecQ molecules at the junction of ssDNA with dsDNA. Collectively, these observations define a stable oligomeric complex of subunits involved in processive helicase action, which is concordant with both biochemical and biological function of RecQ helicases and other helicases.     

Higher 5-hydroxymethylcytosine identifies immortal DNA strand chromosomes in asymmetrically self-renewing distributed stem cells

Immortal strands are the targeted chromosomal DNA strands of nonrandom sister chromatid segregation, a mitotic chromosome segregation pattern unique to asymmetrically self-renewing distributed stem cells (DSCs). By nonrandom segregation, immortal DNA strands become the oldest DNA strands in asymmetrically self-renewing DSCs. Nonrandom segregation of immortal DNA strands may limit DSC mutagenesis, preserve DSC fate, and contribute to DSC aging. The mechanisms responsible for specification and maintenance of immortal DNA strands are unknown. To discover clues to these mechanisms, we investigated the 5-methylcytosine and 5-hydroxymethylcytosine (5hmC) content on chromosomes in mouse hair follicle DSCs during nonrandom segregation. Although 5-methylcytosine content did not differ significantly, the relative content of 5hmC was significantly higher in chromosomes containing immortal DNA strands than in opposed mitotic chromosomes containing younger mortal DNA strands. The difference in relative 5hmC content was caused by the loss of 5hmC from mortal chromosomes. These findings implicate higher 5hmC as a specific molecular determinant of immortal DNA strand chromosomes. Because 5hmC is an intermediate during DNA demethylation, we propose a ten-eleven translocase enzyme mechanism for both the specification and maintenance of nonrandomly segregated immortal DNA strands. The proposed mechanism reveals a means by which DSCs “know” the generational age of immortal DNA strands. The mechanism is supported by molecular expression data and accounts for the selection of newly replicated DNA strands when nonrandom segregation is initiated. These mechanistic insights also provide a possible basis for another characteristic property of immortal DNA strands, their guanine ribonucleotide dependency.Although distributed stem cells (DSCs) are universally essential for normal tissue function, health, and longevity (1–5), understanding of the cellular mechanisms responsible for their unique tissue functions is quite limited. In particular, the mechanisms responsible for the defining properties of DSCs—asymmetric self-renewal (ASR) and nonrandom sister chromatid segregation—are unknown.ASR is a special subclass of asymmetric cell division by which DSCs continuously renew mature differentiated tissue cells (6–8). During ASR, DSCs divide asymmetrically with retention of their own stem cell phenotype while simultaneously producing nonstem sisters that are precursors for short-lived tissue-specific differentiating cell lineages. This long-lived role of DSCs in the cell kinetics architecture of renewing tissues is the basis for the hypothesis that they are the predominant cells of origin for tumors induced by gene mutations (9, 10).Nonrandom sister chromatid segregation is tightly associated with ASR (11, 12). During mitosis, asymmetrically self-renewing DSCs (aDSCs) continuously cosegregate to themselves the set of mitotic chromosomes that contain the older of the two parental template DNA strands. By retaining the same set of template DNA strands over many successive ASR divisions, long-lived DSCs are proposed to reduce their rate of accrual of carcinogenic mutations by 100–1,000-fold compared with their shorter-lived differentiating progeny cells, which retain unrepaired and misrepaired replication errors as a consequence of random segregation (9, 13). Nonrandom segregation also may be an important factor contributing to tissue aging. Accrued chemical damage in the cosegregated template DNA strands, called “immortal DNA strands” (9), may compromise the function and viability of tissue DSCs (1). The immortal DNA strands themselves also might organize epigenomic regulators that are responsible for maintaining the DSC fate (3).Nonrandom segregation of mitotic sister chromatids was discovered in experiments with cultured mouse fetal fibroblasts (14) and the root tips of legumes and wheats (15, 16). More recently, nonrandom segregation has been described in a diverse range of mammalian species and normal (3, 4, 11, 12, 17–27) and cancerous (28–30) tissue types. Thus, far, these studies have shed only limited light on the nature of the responsible molecular mechanisms. In particular, the molecular basis for the specification and maintenance of immortal DNA strands during nonrandom segregation remains unknown nearly a half century after their discovery (16).As proposed initially (9), we have confirmed that nonrandom segregation occurs specifically when DSCs adopt an ASR program (4, 11, 12). Five specific cellular proteins—p53, inosine 5′ monophosphate dehydrogenase type II (EC 1.2.1.14) (12), left-right dynein (25), histone H2A.Z (3), and PIM-1 kinase (26)—have been identified as playing a role in nonrandom chromosome segregation mechanisms. In addition, we have shown that guanine ribonucleotide precursors and high cell density are physiological regulators of ASR and nonrandom segregation (4, 12, 31, 32). However, so far, none of these factors has led to an understanding of how DSCs achieve nonrandom segregation.Two recently discovered properties of the histone H2A variant H2A.Z motivated us to investigate the 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) content of immortal and mortal chromosomes in mouse hair follicle DSCs undergoing nonrandom segregation. H2A.Z chromosomal detection shows a reciprocal relationship with 5mC sites (33), and H2A.Z is molecularly masked on the mortal set of chromosomes in nonrandomly segregating DSCs (3). These analyses reveal that immortal chromosomes, which contain immortal DNA strands, have a significantly higher level of 5hmC modification. The identification of this chromosomal 5hmC asymmetry suggests a molecular mechanism that can account for immortal DNA strand specification and maintenance as well as their long-enigmatic guanine ribonucleotide dependency (12, 32).     

Plasma DNA tissue mapping by genome-wide methylation sequencing for noninvasive prenatal,cancer, and transplantation assessments

Plasma consists of DNA released from multiple tissues within the body. Using genome-wide bisulfite sequencing of plasma DNA and deconvolution of the sequencing data with reference to methylation profiles of different tissues, we developed a general approach for studying the major tissue contributors to the circulating DNA pool. We tested this method in pregnant women, patients with hepatocellular carcinoma, and subjects following bone marrow and liver transplantation. In most subjects, white blood cells were the predominant contributors to the circulating DNA pool. The placental contributions in the plasma of pregnant women correlated with the proportional contributions as revealed by fetal-specific genetic markers. The graft-derived contributions to the plasma in the transplant recipients correlated with those determined using donor-specific genetic markers. Patients with hepatocellular carcinoma showed elevated plasma DNA contributions from the liver, which correlated with measurements made using tumor-associated copy number aberrations. In hepatocellular carcinoma patients and in pregnant women exhibiting copy number aberrations in plasma, comparison of methylation deconvolution results using genomic regions with different copy number status pinpointed the tissue type responsible for the aberrations. In a pregnant woman diagnosed as having follicular lymphoma during pregnancy, methylation deconvolution indicated a grossly elevated contribution from B cells into the plasma DNA pool and localized B cells as the origin of the copy number aberrations observed in plasma. This method may serve as a powerful tool for assessing a wide range of physiological and pathological conditions based on the identification of perturbed proportional contributions of different tissues into plasma.There is much recent interest in the diagnostic applications of cell-free DNA in plasma. Cell-free fetal DNA has been found in the plasma of pregnant women (1). Its detection has made noninvasive prenatal testing, most notably for chromosomal aneuploidies, a clinical reality (2–7). Tumor-derived DNA has been found in the plasma of cancer patients (8–12), offering the possibility of performing “liquid biopsy” for cancer assessment and monitoring. Following organ transplantation, donor-derived DNA from the transplanted organs has been detected in the plasma of the recipients (13) and has been used for monitoring graft rejection (14).Plasma DNA is generally regarded as consisting of a mixture of DNA released from cells from different tissues of the body. Through the analysis of genetic differences between the minor and major background circulating DNA species, researchers have shown that a number of bodily organs made contributions to the plasma DNA pool. For example, studies on pregnant cases in which the fetus and placenta exhibit different karyotypes have demonstrated that the placenta is the origin of the cell-free fetal DNA detectable in the maternal circulation (15, 16). The detection of tumor-associated genetic alterations has allowed the detection of tumor DNA originating from cancer at different body organs in plasma (17). The detection of donor-derived genetic signatures in the plasma of patients following bone marrow (18) and solid organ transplantation (e.g., liver transplantation) (13, 19) has provided a glimpse of the contribution by these various organs into the circulating DNA pool.On the other hand, different DNA methylation signatures can be found in different tissues (20, 21) and even between different cell types within a particular tissue (22). Therefore, the use of such signatures is a potential method for tracing the tissue of origin of plasma DNA. Indeed, researchers have detected organ-specific DNA methylation signatures in plasma, e.g., placental methylation signatures in maternal plasma (23–26) and tumor-associated methylation changes in the plasma of cancer patients (27, 28). These studies have generally focused on signatures of one tissue or organ at a time.We reason that it would be of great biological and potential diagnostic interest if an approach can be developed that simultaneously determines the relative contributions of DNA from multiple tissue types to the plasma DNA pool. Such an approach would provide a “bird’s eye view” of the plasma DNA contributions by different tissues. We based this approach on the performance of genome-wide bisulfite sequencing of plasma DNA (24, 28) (Fig. 1). Then, we used the recent availability of high-resolution methylation profiles of multiple tissue types (21, 24, 29) to deconvolute the plasma bisulfite sequencing data into the percentage contributions by different tissues into plasma. Through this approach, we obtained a “tissue map” of plasma DNA. We applied this approach to study plasma samples obtained from pregnant women, cancer patients, patients following transplantation, and healthy controls. Finally, we demonstrated that this method could be used to trace the tissue of origin of copy number aberrations observed in plasma and demonstrated its potential clinical utility.Open in a separate windowFig. 1.Schematic illustration of the principle of plasma DNA tissue mapping by genome-wide methylation sequencing and its applications.     

The evolution of self-control

Cognition presents evolutionary research with one of its greatest challenges. Cognitive evolution has been explained at the proximate level by shifts in absolute and relative brain volume and at the ultimate level by differences in social and dietary complexity. However, no study has integrated the experimental and phylogenetic approach at the scale required to rigorously test these explanations. Instead, previous research has largely relied on various measures of brain size as proxies for cognitive abilities. We experimentally evaluated these major evolutionary explanations by quantitatively comparing the cognitive performance of 567 individuals representing 36 species on two problem-solving tasks measuring self-control. Phylogenetic analysis revealed that absolute brain volume best predicted performance across species and accounted for considerably more variance than brain volume controlling for body mass. This result corroborates recent advances in evolutionary neurobiology and illustrates the cognitive consequences of cortical reorganization through increases in brain volume. Within primates, dietary breadth but not social group size was a strong predictor of species differences in self-control. Our results implicate robust evolutionary relationships between dietary breadth, absolute brain volume, and self-control. These findings provide a significant first step toward quantifying the primate cognitive phenome and explaining the process of cognitive evolution.Since Darwin, understanding the evolution of cognition has been widely regarded as one of the greatest challenges for evolutionary research (1). Although researchers have identified surprising cognitive flexibility in a range of species (2–40) and potentially derived features of human psychology (41–61), we know much less about the major forces shaping cognitive evolution (62–71). With the notable exception of Bitterman’s landmark studies conducted several decades ago (63, 72–74), most research comparing cognition across species has been limited to small taxonomic samples (70, 75). With limited comparable experimental data on how cognition varies across species, previous research has largely relied on proxies for cognition (e.g., brain size) or metaanalyses when testing hypotheses about cognitive evolution (76–92). The lack of cognitive data collected with similar methods across large samples of species precludes meaningful species comparisons that can reveal the major forces shaping cognitive evolution across species, including humans (48, 70, 89, 93–98).To address these challenges we measured cognitive skills for self-control in 36 species of mammals and birds (Fig. 1 and Tables S1–S4) tested using the same experimental procedures, and evaluated the leading hypotheses for the neuroanatomical underpinnings and ecological drivers of variance in animal cognition. At the proximate level, both absolute (77, 99–107) and relative brain size (108–112) have been proposed as mechanisms supporting cognitive evolution. Evolutionary increases in brain size (both absolute and relative) and cortical reorganization are hallmarks of the human lineage and are believed to index commensurate changes in cognitive abilities (52, 105, 113–115). Further, given the high metabolic costs of brain tissue (116–121) and remarkable variance in brain size across species (108, 122), it is expected that the energetic costs of large brains are offset by the advantages of improved cognition. The cortical reorganization hypothesis suggests that selection for absolutely larger brains—and concomitant cortical reorganization—was the predominant mechanism supporting cognitive evolution (77, 91, 100–106, 120). In contrast, the encephalization hypothesis argues that an increase in brain volume relative to body size was of primary importance (108, 110, 111, 123). Both of these hypotheses have received support through analyses aggregating data from published studies of primate cognition and reports of “intelligent” behavior in nature—both of which correlate with measures of brain size (76, 77, 84, 92, 110, 124).Open in a separate windowFig. 1.A phylogeny of the species included in this study. Branch lengths are proportional to time except where long branches have been truncated by parallel diagonal lines (split between mammals and birds ∼292 Mya).With respect to selective pressures, both social and dietary complexities have been proposed as ultimate causes of cognitive evolution. The social intelligence hypothesis proposes that increased social complexity (frequently indexed by social group size) was the major selective pressure in primate cognitive evolution (6, 44, 48, 50, 87, 115, 120, 125–141). This hypothesis is supported by studies showing a positive correlation between a species’ typical group size and the neocortex ratio (80, 81, 85–87, 129, 142–145), cognitive differences between closely related species with different group sizes (130, 137, 146, 147), and evidence for cognitive convergence between highly social species (26, 31, 148–150). The foraging hypothesis posits that dietary complexity, indexed by field reports of dietary breadth and reliance on fruit (a spatiotemporally distributed resource), was the primary driver of primate cognitive evolution (151–154). This hypothesis is supported by studies linking diet quality and brain size in primates (79, 81, 86, 142, 155), and experimental studies documenting species differences in cognition that relate to feeding ecology (94, 156–166).Although each of these hypotheses has received empirical support, a comparison of the relative contributions of the different proximate and ultimate explanations requires (i) a cognitive dataset covering a large number of species tested using comparable experimental procedures; (ii) cognitive tasks that allow valid measurement across a range of species with differing morphology, perception, and temperament; (iii) a representative sample within each species to obtain accurate estimates of species-typical cognition; (iv) phylogenetic comparative methods appropriate for testing evolutionary hypotheses; and (v) unprecedented collaboration to collect these data from populations of animals around the world (70).Here, we present, to our knowledge, the first large-scale collaborative dataset and comparative analysis of this kind, focusing on the evolution of self-control. We chose to measure self-control—the ability to inhibit a prepotent but ultimately counterproductive behavior—because it is a crucial and well-studied component of executive function and is involved in diverse decision-making processes (167–169). For example, animals require self-control when avoiding feeding or mating in view of a higher-ranking individual, sharing food with kin, or searching for food in a new area rather than a previously rewarding foraging site. In humans, self-control has been linked to health, economic, social, and academic achievement, and is known to be heritable (170–172). In song sparrows, a study using one of the tasks reported here found a correlation between self-control and song repertoire size, a predictor of fitness in this species (173). In primates, performance on a series of nonsocial self-control control tasks was related to variability in social systems (174), illustrating the potential link between these skills and socioecology. Thus, tasks that quantify self-control are ideal for comparison across taxa given its robust behavioral correlates, heritable basis, and potential impact on reproductive success.In this study we tested subjects on two previously implemented self-control tasks. In the A-not-B task (27 species, n = 344), subjects were first familiarized with finding food in one location (container A) for three consecutive trials. In the test trial, subjects initially saw the food hidden in the same location (container A), but then moved to a new location (container B) before they were allowed to search (Movie S1). In the cylinder task (32 species, n = 439), subjects were first familiarized with finding a piece of food hidden inside an opaque cylinder. In the following 10 test trials, a transparent cylinder was substituted for the opaque cylinder. To successfully retrieve the food, subjects needed to inhibit the impulse to reach for the food directly (bumping into the cylinder) in favor of the detour response they had used during the familiarization phase (Movie S2).Thus, the test trials in both tasks required subjects to inhibit a prepotent motor response (searching in the previously rewarded location or reaching directly for the visible food), but the nature of the correct response varied between tasks. Specifically, in the A-not-B task subjects were required to inhibit the response that was previously successful (searching in location A) whereas in the cylinder task subjects were required to perform the same response as in familiarization trials (detour response), but in the context of novel task demands (visible food directly in front of the subject).     

Nonequilibrium dynamics and ultraslow relaxation of confined DNA during viral packaging

Many viruses use molecular motors that generate large forces to package DNA to near-crystalline densities inside preformed viral proheads. Besides being a key step in viral assembly, this process is of interest as a model for understanding the physics of charged polymers under tight 3D confinement. A large number of theoretical studies have modeled DNA packaging, and the nature of the molecular dynamics and the forces resisting the tight confinement is a subject of wide debate. Here, we directly measure the packaging of single DNA molecules in bacteriophage phi29 with optical tweezers. Using a new technique in which we stall the motor and restart it after increasing waiting periods, we show that the DNA undergoes nonequilibrium conformational dynamics during packaging. We show that the relaxation time of the confined DNA is >10 min, which is longer than the time to package the viral genome and 60,000 times longer than that of the unconfined DNA in solution. Thus, the confined DNA molecule becomes kinetically constrained on the timescale of packaging, exhibiting glassy dynamics, which slows the motor, causes significant heterogeneity in packaging rates of individual viruses, and explains the frequent pausing observed in DNA translocation. These results support several recent hypotheses proposed based on polymer dynamics simulations and show that packaging cannot be fully understood by quasistatic thermodynamic models.DNA packaging is both a critical step in viral assembly and a unique model for understanding the physics of polymers under strong confinement. Before packaging, the DNA (∼6–60 µm long) forms a loose random coil of diameter ∼1–3 µm. After translocation into the viral prohead (∼50–100 nm in diameter), a ∼10,000-fold volume compaction is achieved. Packaging is driven by a powerful molecular motor that must work against the large forces resisting confinement arising from DNA bending, repulsion between DNA segments, and entropy loss (1–8).DNA packaging in bacteriophages phi29, lambda, and T4 has been directly measured via single-molecule manipulation with optical tweezers and the packaging motors have been shown to generate forces of >60 pN, among the highest known for biomotors, while translocating DNA at rates ranging from ∼100 bp (for phage phi29, which packages a 19.3-kbp genome into a 42 × 54-nm prohead shell) up to as high as ∼2,000 bp/s (for phage T4, which packages a 171-kbp genome into a 120 × 86-nm prohead) (9–15). The force resisting packaging rises steeply with prohead filling and has been proposed to play an important role in driving viral DNA ejection (16).Recently, a variety of theoretical models for viral DNA packaging have been proposed (3–5, 17–21). The simplest treat DNA as an elastic rod with repulsive self-interactions and assume that packaging is a quasistatic thermodynamic process, i.e., that the DNA is able to continuously relax to a free-energy minimum state (3–5, 19–21). The DNA arrangement is generally assumed to be an inverse spool with local hexagonal close packing between DNA segments, as suggested by electron microscopy and X-ray scattering studies (22, 23). Such models yield exact analytical predictions that reproduce many of the experimental trends, including the sharp rise in resistance during the latter stages of packaging (3–5, 20).Dynamic simulations, however, predict differing results. Depending on model and simulation protocol, some predict rapid equilibration into ordered spool or folded toroid conformations, whereas others predict nonequilibrium dynamics and disordered conformations (3, 6, 24–31). The packaged DNA conformation also depends on ionic conditions, capsid size and shape, and shape of the internal core structure found in some phages (6, 30). Notably, some electron microscopy studies have also been interpreted as suggesting ordered spooled conformations (22), whereas others have been interpreted as suggesting partly disordered conformations (29). Although some simulations predict nonequilibrium dynamics, several potential caveats are that (i) the DNA has been represented by coarse-grained polymer models with various approximations for physical interactions (6), (ii) the packaging rate used in the simulations is >10⁵ times higher than the measured packaging rate due to computational constraints (3, 26–28), and (iii) it has been pointed out by some authors that simulation timescale cannot be directly related to experimental timescale because of the use of coarse-grained models for DNA (25, 28). As noted in early modeling studies, the calculations based on quasistatic models may represent a lower bound on the required packaging forces due to dissipative dynamic losses (4). Whether nonequilibrium dynamics play a significant role in real systems has thus remained an important open question.     

Mismatch repair protein hMSH2–hMSH6 recognizes mismatches and forms sliding clamps within a D-loop recombination intermediate

High fidelity homologous DNA recombination depends on mismatch repair (MMR), which antagonizes recombination between divergent sequences by rejecting heteroduplex DNA containing excessive nucleotide mismatches. The hMSH2–hMSH6 heterodimer is the first responder in postreplicative MMR and also plays a prominent role in heteroduplex rejection. Whether a similar molecular mechanism underlies its function in these two processes remains enigmatic. We have determined that hMSH2–hMSH6 efficiently recognizes mismatches within a D-loop recombination initiation intermediate. Mismatch recognition by hMSH2–hMSH6 is not abrogated by human replication protein A (HsRPA) bound to the displaced single-stranded DNA (ssDNA) or by HsRAD51. In addition, ATP-bound hMSH2–hMSH6 sliding clamps that are essential for downstream MMR processes are formed and constrained within the heteroduplex region of the D-loop. Moreover, the hMSH2–hMSH6 sliding clamps are stabilized on the D-loop by HsRPA bound to the displaced ssDNA. Our findings reveal similarities and differences in hMSH2–hMSH6 mismatch recognition and sliding-clamp formation between a D-loop recombination intermediate and linear duplex DNA.DNA mismatch repair (MMR) ensures the fidelity of both DNA replication and homologous recombination (HR) (1, 2) and is responsible for DNA damage checkpoint signaling (3). Cells deficient in MMR have a mutator phenotype characterized by an elevated spontaneous mutation rate and chromosome rearrangements. Alterations in human MMR genes predispose to Lynch syndrome or hereditary nonpolyposis colorectal cancer (LS/HNPCC) (4).MMR catalyzes a replication-coupled excision reaction that removes mispaired nucleotides from newly synthesized DNA (5, 6). The mispaired nucleotides are recognized by the MutS homodimer in bacteria (7, 8) and by two partially redundant MutS-homolog (MSH) heterodimers, MSH2-MSH6 and MSH2-MSH3, in eukaryotes (1, 9). Sequence analysis indicates that MSH proteins are members of the ATP binding cassette (ABC) superfamily of ATPases (10). Multiple crystal structures of the prokaryotic MutS and human hMSH2–hMSH6 revealed nearly identical, sometimes ADP-bound, clamp-like architectures that encircle a mismatch-containing DNA (11–13). Single-molecule studies have suggested that this initial clamp-like structure persists for a few seconds during the mismatch search and recognition process (14). The structure of an ATP-bound MSH dimer has yet to be solved. However, numerous biochemical and single-molecule studies have demonstrated that mismatch recognition triggers ADP→ATP nucleotide exchange, which results in a conformational transition that converts the MSH dimer into an ATP-bound stable sliding clamp that disengages from the mismatch and freely diffuses on the adjacent dsDNA for 8–10 min (14–21). The formation of an ATP-bound MSH sliding clamp is followed by recruitment of MutL homologs (MLH/PMS) (22, 23). With the exception of Gram-negative enteric bacteria such as Escherichia coli, the MLH/PMS of most species including the human hMLH1-hPMS2 contain an endogenous endonuclease activity required for processing the error-containing DNA strand (17, 24).RAD51 is the prototypical eukaryotic DNA strand exchange protein. It mediates strand invasion into homologous dsDNA during HR (25, 26). If the parental DNAs contain sequence heterology, the strand-exchange process results in the formation of a mismatch-containing heteroduplex DNA (27). A role for MMR in controlling recombination between divergent DNA sequences was initially suggested in λ heteroduplex transfection studies and then clearly demonstrated during intergeneric recombination between E. coli and Salmonella typhimurium (28, 29). In these latter experiments, the recombination frequency between an Hfr donor increased nearly 10,000-fold when the F⁻ recipient bacteria contained a mutation in one of the core bacterial MMR genes MutS, MutL, MutH, or UvrD (MutU) (29). Subsequent studies in bacteria, yeast, and human cells have confirmed the central role of MMR in suppressing recombination between divergent sequences (termed: homeologous recombination) (3, 30, 31). This replication-independent function of MMR is critical for preventing potentially lethal or tumorigenic genome rearrangements such as chromosomal translocations, deletions, or inversions mediated by repetitive genomic elements (32).Despite the critical role of MMR in ensuring recombination fidelity, how heteroduplex rejection is initiated remains poorly defined. MutS and MutL inhibit RecA-catalyzed strand transfer between heterologous DNAs in vitro presumably by blocking the RecA-mediated branch migration (33) and act in concert with UvrD helicase to antagonize illegitimate recombination (34). These studies brought forward the idea that the MMR proteins may operate in the context of the recombination intermediate, where the structure of the D-loop and the presence of the postsynaptic filament may interfere with the formation or stability of the MutS–mismatch complex. This idea, however, has not been confirmed directly. Moreover, the biochemical differences between bacterial RecA and human RAD51 recombinases may lead to differences in the initiation of the hetroduplex rejection reactions. Here, we have reconstituted the initial recognition step of human heteroduplex rejection by constructing mismatch-containing D-loop structures and examining their interaction with combinations of hMSH2–hMSH6, HsRPA, and HsRAD51 proteins. The data suggest a simple model that highlights the similarities and differences in downstream processing during postreplication MMR and heteroduplex rejection.     

Exonuclease TREX1 degrades double-stranded DNA to prevent spontaneous lupus-like inflammatory disease

The TREX1 gene encodes a potent DNA exonuclease, and mutations in TREX1 cause a spectrum of lupus-like autoimmune diseases. Most lupus patients develop autoantibodies to double-stranded DNA (dsDNA), but the source of DNA antigen is unknown. The TREX1 D18N mutation causes a monogenic, cutaneous form of lupus called familial chilblain lupus, and the TREX1 D18N enzyme exhibits dysfunctional dsDNA-degrading activity, providing a link between dsDNA degradation and nucleic acid-mediated autoimmune disease. We determined the structure of the TREX1 D18N protein in complex with dsDNA, revealing how this exonuclease uses a novel DNA-unwinding mechanism to separate the polynucleotide strands for single-stranded DNA (ssDNA) loading into the active site. The TREX1 D18N dsDNA interactions coupled with catalytic deficiency explain how this mutant nuclease prevents dsDNA degradation. We tested the effects of TREX1 D18N in vivo by replacing the TREX1 WT gene in mice with the TREX1 D18N allele. The TREX1 D18N mice exhibit systemic inflammation, lymphoid hyperplasia, vasculitis, and kidney disease. The observed lupus-like inflammatory disease is associated with immune activation, production of autoantibodies to dsDNA, and deposition of immune complexes in the kidney. Thus, dysfunctional dsDNA degradation by TREX1 D18N induces disease in mice that recapitulates many characteristics of human lupus. Failure to clear DNA has long been linked to lupus in humans, and these data point to dsDNA as a key substrate for TREX1 and a major antigen source in mice with dysfunctional TREX1 enzyme.The TREX1 gene encodes a powerful DNA exonuclease (1–7). The amino terminal domain of the TREX1 enzyme contains all of the structural elements for full exonuclease activity, and the carboxy terminal region controls cellular trafficking to the perinuclear space (8–10). Mutations in TREX1 cause a spectrum of autoimmune disorders, including Aicardi–Goutieres syndrome, familial chilblain lupus, and retinal vasculopathy with cerebral leukodystrophy and are associated with systemic lupus erythematosus (9, 11–19). The TREX1 disease-causing alleles locate to positions throughout the gene, exhibit dominant and recessive genetics, include inherited and de novo mutations, and cause varied effects on catalytic function and cellular localization. These genetic discoveries have established a causal relationship between TREX1 mutation and nucleic acid-mediated immune activation disease. The spectrum of TREX1-associated disease parallels the diverse effects on enzyme function and localization, indicating multiple mechanisms of TREX1 dysfunction might explain the overlapping clinical symptoms related to failed DNA degradation and immune activation.The TREX1 enzyme structure reveals the uniquely stable dimer that is relevant to its function and to disease mechanisms. The backbone contacts between the protomer β3-strands generate a stable, central antiparallel β-sheet that stretches the length of the dimer and an extensive hydrogen-bonding network of sidechain–sidechain, sidechain–backbone, and water-bridged contacts that coordinate residues across the TREX1 dimer interface (8, 20). TREX1 catalytic function depends on the dimeric structure, with residues from one protomer contributing to DNA binding and degradation in the opposing protomer (21, 22). Some TREX1 disease-causing mutants exhibit complete loss of catalytic function, whereas others exhibit altered cellular localization (8, 10). A subset of TREX1 catalytic mutants at amino acid positions Asp-18 and Asp-200 exhibit selectively dysfunctional activities on dsDNA. These mutations cause autosomal-dominant disease by retaining DNA-binding proficiency and blocking access to DNA 3′ termini for degradation by TREX1 WT enzyme (21, 23, 24). The TREX1 catalytic sites accommodate four nucleotides of ssDNA, and additional structural elements are positioned adjacent to the active sites for potential DNA polynucleotide interactions.The connection between failure to degrade DNA by TREX1 and immune activation was first made in the TREX1 null mouse that showed a dramatically reduced survival associated with inflammatory myocarditis (25). However, the origin and nature of the disease-driving DNA polynucleotides resulting from TREX1 deficiency have not been clearly established. One model posits that TREX1 acts in the SET complex to degrade genomic dsDNA during granzyme A-mediated cell death by rapidly degrading DNA from the 3′ ends generated by the NM23-H1 endonuclease (26). Two additional models propose that TREX1 prevents immune activation by degrading ssDNA, but these models differ on the possible source of offending DNA polynucleotide. In TREX1-deficient cells there is an accumulation of ssDNA fragments within the cytoplasm proposed, in one model, to be generated from failed processing of aberrant replication intermediates that result in chronic activation of the DNA damage response pathway (27, 28). Another model proposes the source of accumulating ssDNA in TREX1-deficient cells to be derived from unrestrained endogenous retroelement replication, leading to activation of the cytosolic DNA-sensing cGAS–STING pathway (29–33). This concept is also supported by the participation of TREX1 in degradation of HIV-derived cytosolic DNA (34). Thus, disparate concepts on the DNA polynucleotide-driving immune activation in TREX1 deficiency have been proposed, and it is possible that the robust TREX1 exonuclease participates in multiple DNA degradation pathways. We present here structural and in vivo data supporting the concept that TREX1 degradation of dsDNA is critical to prevent immune activation.