A toolbox for generating single‐stranded DNA in optical tweezers experiments

Essential genomic transactions such as DNA‐damage repair and DNA replication take place on single‐stranded DNA (ssDNA) or require specific single‐stranded/double‐stranded DNA (ssDNA/dsDNA) junctions (SDSJ). A significant challenge in single‐molecule studies of DNA–protein interactions using optical trapping is the design and generation of appropriate DNA templates. In contrast to dsDNA, only a limited toolbox is available for the generation of ssDNA constructs for optical tweezers experiments. Here, we present several kinds of DNA templates suitable for single‐molecule experiments requiring segments of ssDNA of several kilobases in length. These different biotinylated dsDNA templates can be tethered between optically trapped microspheres and can, by the subsequent use of force‐induced DNA melting, be converted into partial or complete ssDNA molecules. We systematically investigated the time scale and efficiency of force‐induced melting at different ionic strengths for DNA molecules of different sequences and lengths. Furthermore, we quantified the impact of microspheres of different sizes on the lifetime of ssDNA tethers in optical tweezers experiments. Together, these experiments provide deeper insights into the variables that impact the production of ssDNA for single molecules studies and represent a starting point for further optimization of DNA templates that permit the investigation of protein binding and kinetics on ssDNA. © 2013 Wiley Periodicals, Inc. Biopolymers 99:611–620, 2013.     

A Dimer of Escherichia coli UvrD is the active form of the helicase in vitro

The Escherichia coli UvrD protein is a 3' to 5' SF1 DNA helicase involved in methyl-directed mismatch repair and nucleotide excision repair of DNA. We have characterized in vitro UvrD-catalyzed unwinding of a series of 18 bp duplex DNA substrates with 3' single-stranded DNA (ssDNA) tails ranging in length from two to 40 nt. Single turnover DNA-unwinding experiments were performed using chemical quenched flow methods, as a function of both [UvrD] and [DNA] under conditions such that UvrD-DNA binding is stoichiometric. Although a single UvrD monomer binds tightly to the single-stranded/double-stranded DNA (dsDNA) junction if the 3' ssDNA tail is at least four nt, no unwinding was observed for DNA substrates with tail-lengths /=12 nt, and the unwinding amplitude displays a sigmoidal dependence on [UvrD(tot)]/[DNA(tot)]. Quantitative analysis of these data indicates that a single UvrD monomer bound at the ssDNA/dsDNA junction of any DNA substrate, independent of 3' ssDNA tail length, is not competent to fully unwind even a short 18 bp duplex DNA, and that two UvrD monomers must bind the DNA substrate in order to form a complex that is able to unwind short DNA substrates in vitro. Other proteins, including a mutant UvrD with no ATPase activity as well as a monomer of the structurally homologous E.coli Rep helicase, cannot substitute for the second UvrD monomer, suggesting a specific interaction between two UvrD monomers and that both must be able to hydrolyze ATP. Initiation of DNA unwinding in vitro appears to require a dimeric UvrD complex in which one subunit is bound to the ssDNA/dsDNA junction, while the second subunit is bound to the 3' ssDNA tail.     

Cellular pathways controlling integron cassette site folding

By mobilizing small DNA units, integrons have a major function in the dissemination of antibiotic resistance among bacteria. The acquisition of gene cassettes occurs by recombination between the attI and attC sites catalysed by the IntI1 integron integrase. These recombination reactions use an unconventional mechanism involving a folded single‐stranded attC site. We show that cellular bacterial processes delivering ssDNA, such as conjugation and replication, favour proper folding of the attC site. By developing a very sensitive in vivo assay, we also provide evidence that attC sites can recombine as cruciform structures by extrusion from double‐stranded DNA. Moreover, we show an influence of DNA superhelicity on attC site extrusion in vitro and in vivo. We show that the proper folding of the attC site depends on both the propensity to form non‐recombinogenic structures and the length of their variable terminal structures. These results draw the network of cell processes that regulate integron recombination.     

The solution structure of full‐length dodecameric MCM by SANS and molecular modeling

The solution structure of the full‐length DNA helicase minichromosome maintenance protein from Methanothermobacter thermautotrophicus was determined by small‐angle neutron scattering (SANS) data together with all‐atom molecular modeling. The data were fit best with a dodecamer (dimer of hexamers). The 12 monomers were linked together by the B/C domains, and the adenosine triphosphatase (AAA+) catalytic regions were found to be freely movable in the full‐length dodecamer both in the presence and absence of Mg²⁺ and 50‐meric single‐stranded DNA (ssDNA). In particular, the SANS data and molecular modeling indicate that all 12 AAA+ domains in the dodecamer lie approximately the same distance from the axis of the molecule, but the positions of the helix–turn–helix region at the C‐terminus of each monomer differ. In addition, the A domain at the N‐terminus of each monomer is tucked up next to the AAA+ domain for all 12 monomers of the dodecamer. Finally, binding of ssDNA does not lock the AAA+ domains in any specific position, which leaves them with the flexibility to move both for helicase function and for binding along the ssDNA. Proteins 2014; 82:2364–2374. © 2014 Wiley Periodicals, Inc.     

RADX interacts with single‐stranded DNA to promote replication fork stability

Single‐stranded DNA (ssDNA) regions form as an intermediate in many DNA‐associated transactions. Multiple cellular proteins interact with ssDNA via the oligonucleotide/oligosaccharide‐binding (OB) fold domain. The heterotrimeric, multi‐OB fold domain‐containing Replication Protein A (RPA) complex has an essential genome maintenance role, protecting ssDNA regions from nucleolytic degradation and providing a recruitment platform for proteins involved in responses to replication stress and DNA damage. Here, we identify the uncharacterized protein RADX (CXorf57) as an ssDNA‐binding factor in human cells. RADX binds ssDNA via an N‐terminal OB fold cluster, which mediates its recruitment to sites of replication stress. Deregulation of RADX expression and ssDNA binding leads to enhanced replication fork stalling and degradation, and we provide evidence that a balanced interplay between RADX and RPA ssDNA‐binding activities is critical for avoiding these defects. Our findings establish RADX as an important component of cellular pathways that promote DNA replication integrity under basal and stressful conditions by means of multiple ssDNA‐binding proteins.     

The SOSS1 single‐stranded DNA binding complex promotes DNA end resection in concert with Exo1

The human SSB homologue 1 (hSSB1) has been shown to facilitate homologous recombination and double‐strand break signalling in human cells. Here, we compare the DNA‐binding properties of the SOSS1 complex, containing SSB1, with Replication Protein A (RPA), the primary single‐strand DNA (ssDNA) binding complex in eukaryotes. Ensemble and single‐molecule approaches show that SOSS1 binds ssDNA with lower affinity compared to RPA, and exhibits less stable interactions with DNA substrates. Nevertheless, the SOSS1 complex is uniquely capable of promoting interaction of human Exo1 with double‐strand DNA ends and stimulates its activity independently of the MRN complex in vitro. Both MRN and SOSS1 also act to mitigate the inhibitory action of the Ku70/80 heterodimer on Exo1 activity in vitro. These results may explain why SOSS complexes do not localize with RPA to replication sites in human cells, yet have a strong effect on double‐strand break resection and homologous recombination.     

Robustness and modularity properties of a non-covalent DNA catalytic reaction

The biophysics of nucleic acid hybridization and strand displacement have been used for the rational design of a number of nanoscale structures and functions. Recently, molecular amplification methods have been developed in the form of non-covalent DNA catalytic reactions, in which single-stranded DNA (ssDNA) molecules catalyze the release of ssDNA product molecules from multi-stranded complexes. Here, we characterize the robustness and specificity of one such strand displacement-based catalytic reaction. We show that the designed reaction is simultaneously sensitive to sequence mutations in the catalyst and robust to a variety of impurities and molecular noise. These properties facilitate the incorporation of strand displacement-based DNA components in synthetic chemical and biological reaction networks.     

A label‐free DNAzyme‐cleaving fluorescence method for the determination of trace Pb2+ based on catalysis of AuPd nanoalloy on the reduction of rhodamine 6G

The substrate chain of double‐stranded DNA (dsDNA) could be specifically cleaved by Pb²⁺ to release single‐stranded DNA (ssDNA) that adsorbs onto the AuPd nanoalloy (AuPdNP) to form a stable AuPdNP–ssDNA complex, but the dsDNA can not protect AuPdNPs in large AuPdNP aggregates (AuPdNPA) under the action of NaCl. AuPdNP–ssDNA and large AuPdNPA could be separated by centrifugation. On increasing the concentration of Pb²⁺, the amount of released ssDNA increased; AuPdNP–ssDNA increased in the centrifugation solution exhibiting a catalytic effect on the slow reaction of rhodamine 6G (Rh6G) and NaH₂PO₂, which led to fluorescence quenching at 552 nm. The decrease in fluorescence intensity (ΔF) was linear to the concentration of Pb²⁺ within the range 0.33–8.00 nmol/L, with a detection limit of 0.21 nmol/L. The proposed method was applied to detect Pb²⁺ in water samples, with satisfactory results. Copyright © 2014 John Wiley & Sons, Ltd.     

Probing tethered targets of a single biomolecular complex with atomic force microscopy

DNA origami shows tremendous promise as templates for the assembly of nano‐components and detection of molecular recognition events. So far, the method of choice for evaluating these structures has been atomic force microscopy (AFM), a powerful tool for imaging nanoscale objects. In most cases, tethered targets on DNA origami have proven to be highly effective samples for investigation. Still, while maximal assembly of the nanostructures might benefit from the greatest flexibility in the tether, AFM imaging requires a sufficient stability of the adsorbed components. The balance between the tether flexibility and sample stability is a major, poorly understood, concern in such studies. Here, we investigated the dependence of the tethering length on molecular capture events monitored by AFM. In our experiments, single biotin molecules were attached to DNA origami templates with various linker lengths of thymidine nucleotides, and their interaction with streptavidin was observed with AFM. Our results show that the streptavidin‐biotin complexes are easily detected with short tethered lengths, and that their morphological features clearly change with the tethering length. We identify the functionally useful tether lengths for these investigations, which are also expected to prove useful in the construction and further application of DNA origami in bio‐nanotechnology studies. Copyright © 2013 John Wiley & Sons, Ltd.     

Surface shapes and surrounding environment analysis of single‐ and double‐stranded DNA‐binding proteins in protein‐DNA interface

Protein‐DNA bindings are critical to many biological processes. However, the structural mechanisms underlying these interactions are not fully understood. Here, we analyzed the residues shape (peak, flat, or valley) and the surrounding environment of double‐stranded DNA‐binding proteins (DSBs) and single‐stranded DNA‐binding proteins (SSBs) in protein‐DNA interfaces. In the results, we found that the interface shapes, hydrogen bonds, and the surrounding environment present significant differences between the two kinds of proteins. Built on the investigation results, we constructed a random forest (RF) classifier to distinguish DSBs and SSBs with satisfying performance. In conclusion, we present a novel methodology to characterize protein interfaces, which will deepen our understanding of the specificity of proteins binding to ssDNA (single‐stranded DNA) or dsDNA (double‐stranded DNA). Proteins 2016; 84:979–989. © 2016 Wiley Periodicals, Inc.     

Influence of the single‐strand linker composition on the structural/dynamical properties of a truncated octahedral DNA nano‐cage family

The structural/dynamical properties of three truncated octahedral DNA nano‐cages composed by identical double helices but single strand linkers with different composition, namely 7 thymidines, 7 adenines, and 7 alternated thymidines and adenines, have been investigated through classical molecular dynamics simulations. Trajectories have been analyzed to investigate the role of the linkers in defining nano‐cages stability and flexibility, including possible influence on the internal cages motions. The data indicate that the cages behavior is almost identical and that the structural/dynamical parameters measured along the trajectories are not particularly affected by the presence of different bases. These results demonstrate that the constraints imposed by the nano‐structure geometry are the main factor in modulating these properties. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 992–999, 2014.     

Using measures of single‐cell physiology and physiological state to understand organismic aging

Genetically identical organisms in homogeneous environments have different lifespans and healthspans. These differences are often attributed to stochastic events, such as mutations and ‘epimutations’, changes in DNA methylation and chromatin that change gene function and expression. But work in the last 10 years has revealed differences in lifespan‐ and health‐related phenotypes that are not caused by lasting changes in DNA or identified by modifications to DNA or chromatin. This work has demonstrated persistent differences in single‐cell and whole‐organism physiological states operationally defined by values of reporter gene signals in living cells. While some single‐cell states, for example, responses to oxygen deprivation, were defined previously, others, such as a generally heightened ability to make proteins, were, revealed by direct experiment only recently, and are not well understood. Here, we review technical progress that promises to greatly increase the number of these measurable single‐cell physiological variables and measureable states. We discuss concepts that facilitate use of single‐cell measurements to provide insight into physiological states and state transitions. We assert that researchers will use this information to relate cell level physiological readouts to whole‐organism outcomes, to stratify aging populations into groups based on different physiologies, to define biomarkers predictive of outcomes, and to shed light on the molecular processes that bring about different individual physiologies. For these reasons, quantitative study of single‐cell physiological variables and state transitions should provide a valuable complement to genetic and molecular explanations of how organisms age.     

Probing ATR Activation with Model DNA Templates

The ATR kinase is a critical upstream component of a checkpoint pathway that responds to many forms of damaged and incompletely replicated DNA. Cellular processes such as DNA replication and repair are thought to convert these DNA lesions into a common DNA intermediate that activates this signaling pathway. Indeed, numerous studies have shown that two DNA structures formed during these processes – single-stranded DNA (ssDNA) and junctions between double-stranded DNA (dsDNA) and ssDNA – are important components of the ATR-activating structure. However, an unanswered question is whether primed ssDNA is sufficient for activation of the ATR response. We recently demonstrated that primed ssDNA is sufficient to induce a bona fide checkpoint response in Xenopus egg extracts. This is the first well-defined DNA structure capable of eliciting ATR activation. Using this structure, we examined the contribution of ds/ssDNA junctions and ssDNA to checkpoint activation. Our results indicate the context in which the checkpoint-activating structure is generated may contribute significantly to its signaling properties. Here we discuss the implications of our findings, in the context of other recent work in the field, on our understanding of checkpoint signaling.     

The intrinsically disordered linker of E. coli SSB is critical for the release from single‐stranded DNA

The Escherichia coli single stranded DNA binding protein (SSB) is crucial for DNA replication, recombination and repair. Within each process, it has two seemingly disparate roles: it stabilizes single‐stranded DNA (ssDNA) intermediates generated during DNA processing and, forms complexes with a group of proteins known as the SSB‐interactome. Key to both roles is the C‐terminal, one‐third of the protein, in particular the intrinsically disordered linker (IDL). Previously, they have shown using a series of linker deletion mutants that the IDL links both ssDNA and target protein binding by mediating interactions with the oligosaccharide/oligonucleotide binding fold in the target. In this study, they examine the role of the linker region in SSB function in a variety of DNA metabolic processes in vitro. Using the same linker mutants, the results show that in addition to association reactions (either DNA or protein), the IDL is critical for the release of SSB from DNA. This release can be under conditions of ssDNA competition or active displacement by a DNA helicase or recombinase. Consistent with their previous work these results indicate that SSB linker mutants are defective for SSB–SSB interactions, and when the IDL is removed a terminal SSB–DNA complex results. Formation of this complex inhibits downstream processing of DNA by helicases such as RecG or PriA as well as recombination, mediated by RecA. A model, based on the evidence herein, is presented to explain how the IDL acts in SSB function.     

Stretching DNA and RNA to probe their interactions with proteins

When interacting with a single stretched DNA, many proteins modify its end-to-end distance. This distance can be monitored in real time using various micromanipulation techniques that were initially used to determine the elastic properties of bare nucleic acids and their mechanically induced structural transitions. These methods are currently being applied to the study of DNA enzymes such as DNA and RNA polymerases, topoisomerases and structural proteins such as RecA. They permit the measurement of the probability distributions of the rate, processivity, on-time, affinity and efficiency for a large variety of DNA-based molecular motors.     

Interaction of double-stranded T4 DNA with cationic gel of poly(diallyldimethylammonium chloride)

Interaction between duplex T4 DNA and a slightly cross-linked cationic gel of poly(diallyldimethylammonium chloride) in aqueous media was studied by fluorescent microscopy. While short DNA chains such as plasmid DNAs penetrate into the gel and form a phase of polyelectrolyte complex with the cationic network, the genomic giant DNA chains of T4 phages form complexes only on local areas of the gel surface. The DNA/gel complex exhibited different characteristic morphologies depending on the conditions for preparing the complex, such as the DNA concentration, flux of the solution, and surface geometry of the gel: (1) In the interaction with the flat surface of film-type gel, compact round objects, which reflected a condensed state of single DNA chains, were observed. (2) In the interaction with partly dried gel, a characteristic pattern similar to propagating waves was formed on the gel surface. (3) When flux is generated for a concentrated DNA solution, long oriented fiberlike structures were formed, which consisted of ensembles of chains. The interaction with small pieces of mechanically decomposed gel leads to complete covering of their surface by the DNA.     

DNA binding properties of the adenovirus DNA replication priming protein pTP

The precursor terminal protein pTP is the primer for the initiation of adenovirus (Ad) DNA replication and forms a heterodimer with Ad DNA polymerase (pol). Pol can couple dCTP to pTP directed by the fourth nucleotide of the viral genome template strand in the absence of other replication proteins, which suggests that pTP/pol binding destabilizes the origin or stabilizes an unwound state. We analyzed the contribution of pTP to pTP/pol origin binding using various DNA oligonucleotides. We show that two pTP molecules bind cooperatively to short DNA duplexes, while longer DNA fragments are bound by single pTP molecules as well. Cooperative binding to short duplexes is DNA sequence independent and most likely mediated by protein/protein contacts. Furthermore, we observed that pTP binds single-stranded (ss)DNA with a minimal length of approximately 35 nt and that random ssDNA competed 25-fold more efficiently than random duplex DNA for origin binding by pTP. Remarkably, short DNA fragments with two opposing single strands supported monomeric pTP binding. pTP did not stimulate, but inhibited strand displacement by the Ad DNA binding and unwinding protein DBP. These observations suggest a mechanism in which the ssDNA affinity of pTP stabilizes Ad pol on partially unwound origin DNA.     

Design and evaluation of single-stranded DNA carrier molecules for DNA-directed assembly

Due to the exceptional molecular recognition properties of nucleic acids, the computational design of DNA sequence motifs is of paramount interest for a wide variety of applications, ranging from DNA-based nanotechnology and DNA computing to the broad field of DNA microarray technologies. These applications rely on the specificity of Watson-Crick base-pairing, and thus, are highly sensitive to non-specific interactions and the formation of any undesired secondary structures, which contradict an efficient intermolecular hybridization. Here we report on the in silico design and in vitro evaluation of single-stranded DNA (ssDNA) carrier strands for the directional DNA-based positioning of streptavidin (STV) conjugates covalently tagged with short ssDNA oligonucleotides. Each such carrier strand consists of four hybridization sites complementary to the conjugate DNA strands. The high and homogeneous hybridization efficiency measured in vitro by microarray hybridization assays confirms the quality of our in silico sequence design method. Hybridization efficiency of DNA-STV-conjugates depends on the position of the hybridization site in the carrier sequence, where the positions nearest to and farthest from the microarray surface proved to be most favorable.