Complex DNA Structures Research Articles

Recent Advances in Understanding Werner Syndrome.

Aging, the universal phenomenon, affects human health and is the primary risk factor for major disease pathologies. Progeroid diseases, which mimic aging at an accelerated rate, have provided cues in understanding the hallmarks of aging. Mutations in DNA repair genes as well as in telomerase subunits are known to cause progeroid syndromes. Werner syndrome (WS), which is characterized by accelerated aging, is an autosomal-recessive genetic disorder. Hallmarks that define the aging process include genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulation of nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication. WS recapitulates these hallmarks of aging and shows increased incidence and early onset of specific cancers. Genome integrity and stability ensure the normal functioning of the cell and are mainly guarded by the DNA repair machinery and telomeres. WRN, being a RecQ helicase, protects genome stability by regulating DNA repair pathways and telomeres. Recent advances in WS research have elucidated WRN’s role in DNA repair pathway choice regulation, telomere maintenance, resolution of complex DNA structures, epigenetic regulation, and stem cell maintenance.

Multiscale Visualization and Scale-Adaptive Modification of DNA Nanostructures.

We present an approach to represent DNA nanostructures in varying forms of semantic abstraction, describe ways to smoothly transition between them, and thus create a continuous multiscale visualization and interaction space for applications in DNA nanotechnology. This new way of observing, interacting with, and creating DNA nanostructures enables domain experts to approach their work in any of the semantic abstraction levels, supporting both low-level manipulations and high-level visualization and modifications. Our approach allows them to deal with the increasingly complex DNA objects that they are designing, to improve their features, and to add novel functions in a way that no existing single-scale approach offers today. For this purpose we collaborated with DNA nanotechnology experts to design a set of ten semantic scales. These scales take the DNA's chemical and structural behavior into account and depict it from atoms to the targeted architecture with increasing levels of abstraction. To create coherence between the discrete scales, we seamlessly transition between them in a well-defined manner. We use special encodings to allow experts to estimate the nanoscale object's stability. We also add scale-adaptive interactions that facilitate the intuitive modification of complex structures at multiple scales. We demonstrate the applicability of our approach on an experimental use case. Moreover, feedback from our collaborating domain experts confirmed an increased time efficiency and certainty for analysis and modification tasks on complex DNA structures. Our method thus offers exciting new opportunities with promising applications in medicine and biotechnology.

An ultrasensitive electrochemical biosensor for the detection of mecA gene in methicillin-resistant Staphylococcus aureus

Electrochemical DNA biosensor has unique advantages for on-site pathogenic microorganism detection, yet the detection of long DNA towards genome DNA (gDNA) analysis remains challenge. In this work, we report a novel electrochemical biosensor for the ultrasensitive analysis of mecA DNA on methicillin-resistant Staphylococcus aureus (MRSA) genome, using a multi-signal probes (MSP) system. The MSP consists of 7 biotin-labelled signal probes that will combine to the target DNA in a prehybridization step, and then the complex will be captured by a DNA tetrahedron structure probe (TSP) on the electrode surface. Then, after the introduction of the streptavidin-labelled HRP enzyme, a catalysis current signal is detected that is found to be corresponding to the concentration of the target DNA. MSP in this work plays a critical role not only for the signal amplification through bringing 7 biotins, but also dramatically improves the accessibility of the target sequence embedded in the double-strand DNA molecules and complex second structures. The 3-D DNA TSP here provides steady support and optimized surface density for the very "large" complex of MSP system and gDNA, as a base of the capture probe. Finally, as low as 10fM synthetic target DNA was successfully detected, which is at least 3 magnitudes lower than that using single signal probe. Most importantly, we demonstrated the practicability of our analysis method by analyzing a 57fM MRSA gDNA sample showing excellent selectivity, and the reliability of the analysis was also demonstrated by digital PCR.

Twisting of DNA Origami from Intercalators

DNA nanostructures represent the confluence of materials science, computer science, biology, and engineering. As functional assemblies, they are capable of performing mechanical and chemical work. In this study, we demonstrate global twisting of DNA nanorails made from two DNA origami six-helix bundles. Twisting was controlled using ethidium bromide or SYBR Green I as model intercalators. Our findings demonstrate that DNA nanorails: (i) twist when subjected to intercalators and the amount of twisting is concentration dependent, and (ii) twisting saturates at elevated concentrations. This study provides insight into how complex DNA structures undergo conformational changes when exposed to intercalators and may be of relevance when exploring how intercalating drugs interact with condensed biological structures such as chromatin and chromosomes, as well as chromatin analogous gene expression devices.

Structure and dynamics of polymyxin-resistance-associated response regulator PmrA in complex with promoter DNA.

PmrA, an OmpR/PhoB family response regulator, manages genes for antibiotic resistance. Phosphorylation of OmpR/PhoB response regulator induces the formation of a symmetric dimer in the N-terminal receiver domain (REC), promoting two C-terminal DNA-binding domains (DBDs) to recognize promoter DNA to elicit adaptive responses. Recently, determination of the KdpE–DNA complex structure revealed an REC–DBD interface in the upstream protomer that may be necessary for transcription activation. Here, we report the 3.2-Å-resolution crystal structure of the PmrA–DNA complex, which reveals a similar yet different REC–DBD interface. However, NMR studies show that in the DNA-bound state, two domains tumble separately and an REC–DBD interaction is transiently populated in solution. Reporter gene analyses of PmrA variants with altered interface residues suggest that the interface is not crucial for supporting gene expression. We propose that REC–DBD interdomain dynamics and the DBD–DBD interface help PmrA interact with RNA polymerase holoenzyme to activate downstream gene transcription.

Thermodynamics of interaction and structure of DNA complexes with phenacylimidazo[5,1-a]isoquinoline derivatives

Interaction of native calf thymus DNA (ctDNA) with phenacylimidazo[5,1- a]isoquinoline derivatives was studied by the methods of spectrophotometry, viscometry, isothermal titration calorimetry (ITC) and dynamic birefringence. It was found that both of investigated compounds form complexes with the DNA molecule, the structure of compounds affects the mode of binding these ligands to DNA. The primary binding mode can not be described by the classical models of groove binding or intercalation. It has been suggested that the primary mode of binding is "partial intercalation".

Nanotechnology: Pathfinder for DNA constructs.

Representations of 3D surfaces used in computer graphics have been adopted as templates in an efficient method for making nanoscale objects from DNA, lowering the barriers to applications of DNA nanotechnology. See Letter p.441 Constraints arising from the inherent properties of DNA impose important restrictions on how it can be assembled, so that procedures such as DNA origami still require considerable manual adjustment if complex structures are to be achieved. Here Erik Benson and colleagues present a general method — using single helices rather than bundles of closely packed helices as construction elements — which makes it possible to design and produce complex DNA structures that would have been very difficult to achieve using existing approaches. The resulting nanostructures are more stable than conventional DNA origami under conditions typically encountered in biological assays. And because the overall design process is readily amenable to a high level of automation, it brings the prospect of practical nanoscale three-dimensional printing a step closer.

Self-Assembly of Peptide Amphiphiles: Molecularly Engineered Bionanomaterials

Molecular self-assembly is ubiquitous in nature and has now emerged as a new approach in chemical synthesis, engineering, nanotechnology, polymer science, and materials. Molecular self-assembly has been attracting increasing interest from the scientific community in the recent years due to its importance in understanding biology and a variety of diseases at the molecular level. In the last few years, considerable advances have been made in the use of peptides as building blocks to produce biological materials for wide range of applications, including fabricating novel supra-molecular structures and scaffolding for tissue repair. Today, the study of biological self-assembly systems represent a significant advance in the molecular engineering and is a rapidly growing scientific and engineering field that crosses the boundaries of existing disciplines. Many self-assembling systems are range from bi-and tri-block copolymers to complex DNA structures as well as simple and complex proteins and peptides. The attractiveness of such bottom-up processes lies in their capability to build uniform, functional units or arrays and the possibility to exploit such structures at meso-and macroscopic scale for life and non-life science applications.

Crystal Structure of the Vaccinia Virus Uracil-DNA Glycosylase in Complex with DNA

Vaccinia virus polymerase holoenzyme is composed of the DNA polymerase catalytic subunit E9 associated with its heterodimeric co-factor A20·D4 required for processive genome synthesis. Although A20 has no known enzymatic activity, D4 is an active uracil-DNA glycosylase (UNG). The presence of a repair enzyme as a component of the viral replication machinery suggests that, for poxviruses, DNA synthesis and base excision repair is coupled. We present the 2.7 Å crystal structure of the complex formed by D4 and the first 50 amino acids of A20 (D4·A201-50) bound to a 10-mer DNA duplex containing an abasic site resulting from the cleavage of a uracil base. Comparison of the viral complex with its human counterpart revealed major divergences in the contacts between protein and DNA and in the enzyme orientation on the DNA. However, the conformation of the dsDNA within both structures is very similar, suggesting a dominant role of the DNA conformation for UNG function. In contrast to human UNG, D4 appears rigid, and we do not observe a conformational change upon DNA binding. We also studied the interaction of D4·A201-50 with different DNA oligomers by surface plasmon resonance. D4 binds weakly to nonspecific DNA and to uracil-containing substrates but binds abasic sites with a Kd of <1.4 μm. This second DNA complex structure of a family I UNG gives new insight into the role of D4 as a co-factor of vaccinia virus DNA polymerase and allows a better understanding of the structural determinants required for UNG action.

Human RECQ1 helicase-driven DNA unwinding, annealing, and branch migration: Insights from DNA complex structures

RecQ helicases are a widely conserved family of ATP-dependent motors with diverse roles in nearly every aspect of bacterial and eukaryotic genome maintenance. However, the physical mechanisms by which RecQ helicases recognize and process specific DNA replication and repair intermediates are largely unknown. Here, we solved crystal structures of the human RECQ1 helicase in complexes with tailed-duplex DNA and ssDNA. The structures map the interactions of the ssDNA tail and the branch point along the helicase and Zn-binding domains, which, together with reported structures of other helicases, define the catalytic stages of helicase action. We also identify a strand-separating pin, which (uniquely in RECQ1) is buttressed by the protein dimer interface. A duplex DNA-binding surface on the C-terminal domain is shown to play a role in DNA unwinding, strand annealing, and Holliday junction (HJ) branch migration. We have combined EM and analytical ultracentrifugation approaches to show that RECQ1 can form what appears to be a flat, homotetrameric complex and propose that RECQ1 tetramers are involved in HJ recognition. This tetrameric arrangement suggests a platform for coordinated activity at the advancing and receding duplexes of an HJ during branch migration.

Effect of Peptide Charge Distribution on the Structure and Kinetics of DNA Complex

The complexes formed by DNA or siRNA interacting with polycations showed great potential as nonviral vectors for gene delivery. The physicochemical properties of the DNA/siRNA complexes, which could be tuned by adjusting the characteristics of polycations, were directly related to their performance in gene delivery. Using 21 bp double-stranded oligonucleotide (ds-oligo) and two icosapeptides (with the repeating units being KKGG and KGKG, respectively) of the same charge density as model molecules, we investigated the effect of charge distribution on the kinetics of complexation and the structure of the final complexes. Even though the distribution of the charged groups in peptides was only adjusted by one position, the complexes formed by (KKGG)5 and ds-oligo were larger in size and easier to precipitate than those formed by (KGKG)5. Counterintuitively, it was not the charged groups but the hydrophilic neutral spacers that determined the kinetics and the structure of the complex. We attributed such an effect to the water-mediated disproportionation process. The hydrophilic spacers next to each other were better than that in the separated pattern in holding water molecules after forming the complex. The water-rich domains in the complex functioned as a lubricant and facilitated the relaxation of the polyelectrolyte, resulting in a fast complexation process. The resulting complex was thus larger in size and lower in surface energy.

Role of DNA conformation & energetic insights in Msx-1-DNA recognition as revealed by molecular dynamics studies on specific and nonspecific complexes

In most of homeodomain–DNA complexes, glutamine or lysine is present at 50th position and interacts with 5th and 6th nucleotide of core recognition region. Molecular dynamics simulations of Msx-1–DNA complex (Q50-TG) and its variant complexes, that is specific (Q50K-CC), nonspecific (Q50-CC) having mutation in DNA and (Q50K-TG) in protein, have been carried out. Analysis of protein–DNA interactions and structure of DNA in specific and nonspecific complexes show that amino acid residues use sequence-dependent shape of DNA to interact. The binding free energies of all four complexes were analysed to define role of amino acid residue at 50th position in terms of binding strength considering the variation in DNA on stability of protein–DNA complexes. The order of stability of protein–DNA complexes shows that specific complexes are more stable than nonspecific ones. Decomposition analysis shows that N-terminal amino acid residues have been found to contribute maximally in binding free energy of protein–DNA complexes. Among specific protein–DNA complexes, K50 contributes more as compared to Q50 towards binding free energy in respective complexes. The sequence dependence of local conformation of DNA enables Q50/Q50K to make hydrogen bond with nucleotide(s) of DNA. The changes in amino acid sequence of protein are accommodated and stabilized around TAAT core region of DNA having variation in nucleotides.

Revealing Structure and Dynamics of Telomere Maintenance Proteins on DNA: One Molecule at a Time

Telomeres play important roles in maintaining the stability of linear chromosomes. A specialized protein complex, called shelterin or telosome, binds to and protects telomeres at chromosome ends. Telomere maintenance involves dynamic actions of multiple proteins interacting with long repetitive sequences and complex dynamic DNA structures, such as T-loops. Furthermore, it was shown recently that in contrast to cohesion along chromosome arms, sister telomere association is a specialized process requiring a tighter association provided by the cohesin subunit SA1 in conjunction with specific proteins from the shelterin complex. To better understand the telomere maintenance pathways, we established complementary single-molecule imaging platforms: a newly developed Dual-Resonance-frequency-Enhanced Electrostatic force Microscopy (DREEM) technique capable of revealing DNA paths in protein-DNA complexes, fluorescence imaging of quantum dot-labeled proteins for tracking dynamics of proteins on DNA tightropes, and a nanochannel based imaging platform for studying protein-mediated DNA-DNA pairing/looping in real time. I will highlight our recent results on: 1) Revealing DNA paths inside TRF2 complexes during DNA compaction through DREEM. 2) Cohesin SA1 and shelterin protein TRF1 mediated sister telomere cohesion. 3) Dynamics of SA1 and TRF1 mediated DNA-DNA pairing inside nanochannels.

Functional Implications of the RecQ Helicase - Topoisomerase III - SSB Complex: Insights from Single Molecule Measurements

RecQ Helicases are a highly conserved class of ATP-dependent DNA helicases that perform multifunctional roles in genome maintenance. In E. coli, RecQ is known to physically and functionally interact with Topoisomerase III, a type IA topoisomerase. The coupling of helicase activity and topoisomerase activity that results from this interaction is responsible for resolving complex DNA structures such as double Holliday junctions. Similar interactions have been demonstrated in homologous proteins in other organisms, including the human RecQ helicase BLM and TopoIIIα, and the yeast helicase SgsI and TopoIII. There is also evidence that single-stranded DNA binding protein (SSB) interacts with RecQ and is a necessary component of this complex in vivo. We sought to explore the mechanism by which RecQ stimulates TopoIII activity, and by which SSB stimulates RecQ activity, as well as the individual contributions of these proteins to topological changes in DNA using single molecule experiments. To investigate the roles of RecQ, TopoIII, and SSB, individually and in a ternary complex, we measured their effect on DNA hairpin unwinding and refolding using magnetic tweezers. We also conducted experiments on over- and under-wound double-stranded DNA to probe the roles of RecQ and SSB in activation of TopoIII relaxation of supercoiled DNA. This technique allows us to probe changes in DNA topology as a consequence of enzyme activity in real time. Our findings demonstrate a complex set of interactions and provide a framework for understanding the mechanism of the resolvase activity of the RecQ-TopoIII- SSB complex.

Functionalization of nc-Si/SiO2 semiconductor quantum dots by oligonucleotides

nc-Si/SiO2 crystalline semiconductor quantum dots are very attractive as fluorescent labels for developing biosensors integrated with biomedical materials due to their unique physical properties in the visible region of the spectrum. We report on the functionalization of such nanostructures by single-strand short oligonucleotides using the d(20G,20T) system (d is deoxyribonucleotide, G is guanine, and T is thymine) as an example. Oligonucleotides are obtained by chemical synthesis using the solid-phase phosphoramidite technique. Studies using developed methods of Raman spectroscopy of high spectral and spatial resolution are performed on such complexes. The previously unpredicted phenomenon of multiband selective resonant light scattering by isolated molecular groups, caused by the nonradiative transfer of photoexcited electrons, is observed using a system of nc-Si/SiO2 quantum dots functionalized by d(20G,20T) oligonucleotides as an example. The results obtained suggest that the developed approach can be used to study the molecular structure of semiconductor quantum-dot and DNA complexes.

The human oncoprotein and chromatin architectural factor DEK counteracts DNA replication stress.

DNA replication stress is a major source of DNA strand breaks and genomic instability, and a hallmark of precancerous lesions. In these hyperproliferative tissues, activation of the DNA damage response results in apoptosis or senescence preventing or delaying their development to full malignancy. In cells, in which this antitumor barrier is disabled by mutations (for example, in p53), viability and further uncontrolled proliferation depend on factors that help to cope with replication-associated DNA damage. Replication problems preferentially arise in chromatin regions harboring complex DNA structures. DEK is a unique chromatin architectural factor which binds to non-B-form DNA structures, such as cruciform DNA or four-way junctions. It regulates DNA topology and chromatin organization, and is essential for the maintenance of heterochromatin integrity. Since its isolation as part of an oncogenic fusion in a subtype of AML, DEK has been consistently associated with tumor progression and chemoresistance. How DEK promotes cancer, however, is poorly understood. Here we show that DEK facilitates cellular proliferation under conditions of DNA replication stress by promoting replication fork progression. DEK also protects from the transmission of DNA damage to the daughter cell generation. We propose that DEK counteracts replication stress and ensures proliferative advantage by resolving problematic DNA and/or chromatin structures at the replication fork.

Structural studies of AraR from B. thetaiotaomicron

The NrtR family of bacterial transcription factors is characterized by an N-terminal Nudix hydrolase-like effector binding domain and a C-terminal DNA binding domain. A bioinformatics analysis of the NrtR family represented by uncharacterized protein BT0354 in Bacteroides thetaiotaomicron suggests that these regulators control the catabolic pathways for L-arabinose. Many bacteria use L-arabinose as the sole source of carbon energy. The L-arabinose utilization pathway and its transcriptional regulation have been studied for a long time in several model microorganisms. Here we provide biochemical and structural characterization of the novel arabinose-responsive regulator of NrtR family protein BT0354, L-arabinose regulator from B. thetaiotaomicron (BtAraR). The BtAraR DNA binding and the role of effector molecule L-arabinose were confirmed using electrophoretic mobility shift assays. We have solved the crystal structures of BtAraR for two apo forms, and complexes with L-arabinose and double-stranded DNA target. The apo-1 form was solved as two dimers/AU in the R3 space group at 2.35 Å, while the apo-2 form was solved as one monomer/AU in the I213 space group at 2.56 Å resolution. The L-arabinose and DNA complex structures were solved as a dimer/AU in the P21 space group at 1.95 Å resolution and the P23 space group at 3.05 Å resolution, respectively. The biological unit of this protein is a dimer while the N-terminal ligand binding domain of the monomer adopts a Nudix hydrolase-like fold and the C-terminal DNA binding domain is a winged helix-turn-helix. The DNA binding-releasing mechanism can be rationalized through the comparison and analyses of these structures. The apo and DNA bound structures are more similar compared to the L-arabinose-bound structure. The r.m.s. deviation for the apo and DNA bound structures is 1.13 Å, while that for apo and the L-arabinose-bound structures is 4.54 Å. Details about the DNA binding mode, L-arabinose binding and L-arabinose induced structural change will be presented. This work was supported by National Institutes of Health grant GM094585 and by the U. S. Department of Energy, Office of Biological and Environmental Research, under contract DE-AC02-06CH11357.

Low-Resolution Refinement of the DNA and Protein Complex Structures

Macromolecular complexes of protein and DNA are often resolved in a low resolution structure (3.0 angstroms or lower). Because nucleic acids suffer radiation damage more than amino acids, the resulting temperature factors for DNA are generally higher than those for protein. Recognition of DNA-specific interactions with protein is a challenge at low resolution. The use of low-resolution refinement ([1]) or the reference high resolution model could improve DNA densities. A number of DNA/protein and nucleosome complexes (i.e. RAGE-DNA [2], CENP-C-NCP[3]) that we have recently refined demonstrated the validation of these methods.

DNA bridging and looping by HMO1 provides a mechanism for stabilizing nucleosome-free chromatin.

The regulation of chromatin structure in eukaryotic cells involves abundant architectural factors such as high mobility group B (HMGB) proteins. It is not understood how these factors control the interplay between genome accessibility and compaction. In vivo, HMO1 binds the promoter and coding regions of most ribosomal RNA genes, facilitating transcription and possibly stabilizing chromatin in the absence of histones. To understand how HMO1 performs these functions, we combine single molecule stretching and atomic force microscopy (AFM). By stretching HMO1-bound DNA, we demonstrate a hierarchical organization of interactions, in which HMO1 initially compacts DNA on a timescale of seconds, followed by bridge formation and stabilization of DNA loops on a timescale of minutes. AFM experiments demonstrate DNA bridging between strands as well as looping by HMO1. Our results support a model in which HMO1 maintains the stability of nucleosome-free chromatin regions by forming complex and dynamic DNA structures mediated by protein–protein interactions.

Structure of catabolite activator protein with cobalt(II) and sulfate.

The crystal structure of cyclic AMP-catabolite activator protein (CAP) from Escherichia coli containing cobalt(II) chloride and ammonium sulfate is reported at 1.97 Å resolution. Each of the two CAP subunits in the asymmetric unit binds one cobalt(II) ion, in each case coordinated by N-terminal domain residues His19, His21 and Glu96 plus an additional acidic residue contributed via a crystal contact. The three identified N-terminal domain cobalt-binding residues are part of a region of CAP that is important for transcription activation at class II CAP-dependent promoters. Sulfate anions mediate additional crystal lattice contacts and occupy sites corresponding to DNA backbone phosphate positions in CAP-DNA complex structures.