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At least 667 records · Page 37
All sourcesNASA NTRSDOE OSTI
DOE OSTI2015

Protein search for multiple targets on DNA

Protein-DNA interactions are crucial for all biological processes. One of the most important fundamental aspects of these interactions is the process of protein searching and recognizing specific binding sites on DNA. A large number of experimental and theoretical investigations have been devoted to uncovering the molecular description of these phenomena, but many aspects of the mechanisms of protein search for the targets on DNA remain not well understood. One of the most intriguing problems is the role of multiple targets in protein search dynamics. Using a recently developed theoretical framework we analyze this question in detail. Our method is based on a discrete-state stochastic approach that takes into account most relevant physical-chemical processes and leads to fully analytical description of all dynamic properties. Specifically, systems with two and three targets have been explicitly investigated. It is found that multiple targets in most cases accelerate the search in comparison with a single target situation. However, the acceleration is not always proportional to the number of targets. Surprisingly, there are even situations when it takes longer to find one of the multiple targets in comparison with the single target. It depends on the spatial position of the targets, distances between them, average scanning lengths of protein molecules on DNA, and the total DNA lengths. Physical-chemical explanations of observed results are presented. Our predictions are compared with experimental observations as well as with results from a continuum theory for the protein search. Extensive Monte Carlo computer simulations fully support our theoretical calculations.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2015

Rupture of DNA aptamer: New insights from simulations

Base-pockets (non-complementary base-pairs) in a double-stranded DNA play a crucial role in biological processes. Because of thermal fluctuations, it can lower the stability of DNA, whereas, in case of DNA aptamer, small molecules, e.g., adenosinemonophosphate and adenosinetriphosphate, form additional hydrogen bonds with base-pockets termed as “binding-pockets,” which enhance the stability. Using the Langevin dynamics simulations of coarse grained model of DNA followed by atomistic simulations, we investigated the influence of base-pocket and binding-pocket on the stability of DNA aptamer. Striking differences have been reported here for the separation induced by temperature and force, which require further investigation by single molecule experiments.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2015

The influences of geometric shape of cross section on equilibrium configuration of DNA in elastic rod model

The equilibrium configuration equations of DNA chain with elastic rod model are derived in detail by the variation of the free energy functional, which depends on the curvature, torsion, twisting angle and its derivative with respect to the arc-length of central axis curve of rod. With the different shapes of rod, we obtain the equilibrium equations of DNA with circular and noncircular cross sections, which provide an approach to describe the physical behaviors of A-, B-, Z-DNA. The results show that the elastic rod model with circular cross section can accurately characterize the equilibrium configurations of A-, B-DNA, while the model with elliptical cross sections is more suitable for Z-DNA.

71 CLASSICAL AND QUANTUM MECHANICS, GENERAL PHYSIC↗
DOE OSTI2015

DNA-mediated engineering of multicomponent enzyme crystals

The ability to predictably control the coassembly of multiple nanoscale building blocks, especially those with disparate chemical and physical properties such as biomolecules and inorganic nanoparticles, has far-reaching implications in catalysis, sensing, and photonics, but a generalizable strategy for engineering specific contacts between these particles is an outstanding challenge. This is especially true in the case of proteins, where the types of possible interparticle interactions are numerous, diverse, and complex. In this paper, we explore the concept of trading protein–protein interactions for DNA–DNA interactions to direct the assembly of two nucleic-acid–functionalized proteins with distinct surface chemistries into six unique lattices composed of catalytically active proteins, or of a combination of proteins and DNA-modified gold nanoparticles. The programmable nature of DNA–DNA interactions used in this strategy allows us to control the lattice symmetries and unit cell constants, as well as the compositions and habit, of the resulting crystals. Finally, this study provides a potentially generalizable strategy for constructing a unique class of materials that take advantage of the diverse morphologies, surface chemistries, and functionalities of proteins for assembling functional crystalline materials.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Electrostatic confinement and manipulation of DNA molecules for genome analysis

Very large DNA molecules enable comprehensive analysis of complex genomes, such as human, cancer, and plants because they span across sequence repeats and complex somatic events. When physically manipulated, or analyzed as single molecules, long polyelectrolytes are problematic because of mechanical considerations that include shear-mediated breakage, dealing with the massive size of these coils, or the length of stretched DNAs using common experimental techniques and fluidic devices. Accordingly, we harness analyte “issues” as exploitable advantages by our invention and characterization of the “molecular gate,” which controls and synchronizes formation of stretched DNA molecules as DNA dumbbells within nanoslit geometries. Molecular gate geometries comprise micro- and nanoscale features designed to synergize very low ionic strength conditions in ways we show effectively create an “electrostatic bottle.” This effect greatly enhances molecular confinement within large slit geometries and supports facile, synchronized electrokinetic loading of nanoslits, even without dumbbell formation. Device geometries were considered at the molecular and continuum scales through computer simulations, which also guided our efforts to optimize design and functionalities. In addition, we show that the molecular gate may govern DNA separations because DNA molecules can be electrokinetically triggered, by varying applied voltage, to enter slits in a size-dependent manner. Lastly, mapping the Mesoplasma florum genome, via synchronized dumbbell formation, validates our nascent approach as a viable starting point for advanced development that will build an integrated system capable of large-scale genome analysis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Microchemomechanical devices using DNA hybridization

Significance With simple DNA origami lever arms arranged in hinges and accordion structures, we amplify the nanometer displacements from DNA hairpin zippers to 4-μm motion, easily observable and quantified in real space and real time with conventional optical microscopy. Mechanically pulling a bead tethered on the accordion end, we measure high-energy recovery and retraction speeds up to 50 μm/s. On longer time scales, we have also opened and closed the hinges with light and heat. DNA nanotechnology, and particularly DNA origami, combined with colloids and emulsions can provide powerful architectures. The present study is a step toward activating such colloidal/cellular scale devices using DNA as a power source/fuel. We envision artificial active flagella, cilia, micropumps, and other cellular scale devices.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2017

Insights into Rad3 kinase recruitment from the crystal structure of the DNA damage checkpoint protein Rad26

Metabolic products and environmental factors constantly damage DNA. To protect against these insults and maintain genome integrity, cells have evolved mechanisms to repair DNA lesions. One such mechanism involves Rad3, a master kinase coordinating the DNA damage response. Rad26 is a functional subunit of the Rad3-Rad26 complex and is responsible for bringing the kinase to sites of DNA damage. Here, I present the crystal structure of Rad26 and identify the elements important for recruiting Rad3. The structure suggests that Rad26 is a dimer with a conserved interface in the N-terminal part of the protein. Biochemical data showed that Rad26 uses its C-terminal domain and the flanking kinase-docking motif to bind specific HEAT repeats in Rad3. Analysis of the reconstituted Rad3-Rad26 heterotetrameric complex with electron microscopy enabled me to propose a structural model for its quaternary structure. Lastly, these results suggest that Rad26 exists as a dimer and provide crucial insight into how Rad3 is recruited and incorporated into the Rad3-Rad26 DNA repair complex.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Sub-10 nm patterning with DNA nanostructures: a short perspective

DNA is the hereditary material that contains our unique genetic code. Since the first demonstration of two-dimensional (2D) nanopatterns by using designed DNA origami ~10 years ago, DNA has evolved into a novel technique for 2D and 3D nanopatterning. It is now being used as a template for the creation of sub-10 nm structures via either 'top-down' or 'bottom-up' approaches for various applications spanning from nanoelectronics, plasmonic sensing, and nanophotonics. This paper starts with an histroric overview and discusses the current state-of-the-art in DNA nanolithography. Finally, emphasis is put on the challenges and prospects of DNA nanolithography as the next generation nanomanufacturing technique.

77 NANOSCIENCE AND NANOTECHNOLOGY↗
DOE OSTI2015

Structural analysis of DNA binding by C.Csp231I, a member of a novel class of R-M controller proteins regulating gene expression

The structure of the new class of controller proteins (exemplified by C.Csp231I) in complex with its 21 bp DNA-recognition sequence is presented, and the molecular basis of sequence recognition in this class of proteins is discussed. An unusual extended spacer between the dimer binding sites suggests a novel interaction between the two C-protein dimers. In a wide variety of bacterial restriction–modification systems, a regulatory ‘controller’ protein (or C-protein) is required for effective transcription of its own gene and for transcription of the endonuclease gene found on the same operon. We have recently turned our attention to a new class of controller proteins (exemplified by C.Csp231I) that have quite novel features, including a much larger DNA-binding site with an 18 bp (∼60 Å) spacer between the two palindromic DNA-binding sequences and a very different recognition sequence from the canonical GACT/AGTC. Using X-ray crystallography, the structure of the protein in complex with its 21 bp DNA-recognition sequence was solved to 1.8 Å resolution, and the molecular basis of sequence recognition in this class of proteins was elucidated. An unusual aspect of the promoter sequence is the extended spacer between the dimer binding sites, suggesting a novel interaction between the two C-protein dimers when bound to both recognition sites correctly spaced on the DNA. A U-bend model is proposed for this tetrameric complex, based on the results of gel-mobility assays, hydrodynamic analysis and the observation of key contacts at the interface between dimers in the crystal.

75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND↗
DOE OSTI2017

Sequencing and functional validation of the JGI Brachypodium distachyon T-DNA collection

Due to a large and growing collection of genomic and experimental resources, Brachypodium distachyon has emerged as a powerful experimental model for the grasses. To add to these resources we sequenced 21 165 T-DNA lines, 15 569 of which were produced in this study. This increased the number of unique insertion sites in the T-DNA collection by 21 078, bringing the overall total to 26 112. Thirty-seven per cent (9754) of these insertion sites are within genes (including untranslated regions and introns) and 28% (7217) are within 500 bp of a gene. Approximately 31% of the genes in the v.2.1 annotation have been tagged in this population. To demonstrate the utility of this collection, we phenotypically characterized six T-DNA lines with insertions in genes previously shown in other systems to be involved in cellulose biosynthesis, hemicellulose biosynthesis, secondary cell wall development, DNA damage repair, wax biosynthesis and chloroplast synthesis. In all cases, the phenotypes observed supported previous studies, demonstrating the utility of this collection for plant functional genomics. The Brachypodium T-DNA collection can be accessed at http://jgi.doe.gov/our-science/science-programs/plant-genomics/brachypodium/brachypodium-t-dna-collection/.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

RecJ3/4-aRNase J form a Ubl-associated nuclease complex functioning in survival against DNA damage in Haloferax volcanii

Nucleases are strictly regulated and often localized in the cell to avoid the uncontrolled degradation of DNA and RNA. Here, a new type of nuclease complex, composed of RecJ3, RecJ4, and aRNase J, was identified through its ATP-dependent association with the ubiquitin-like SAMP1 and AAA-ATPase Cdc48a. The complex was discovered in Haloferax volcanii , an archaeon lacking an RNA exosome. Genetic analysis revealed aRNase J to be essential and RecJ3, RecJ4, and Cdc48a to function in the recovery from DNA damage including genotoxic agents that generate double-strand breaks. The RecJ3:RecJ4:aRNase J complex (isolated in 2:2:1 stoichiometry) functioned primarily as a 3′−5′ exonuclease in hydrolyzing RNA and ssDNA, with the mechanism non-processive for ssDNA. aRNase J could also be purified as a homodimer that catalyzed endoribonuclease activity and, thus, was not restricted to the 5′−3′ exonuclease activity typical of aRNase J homologs. Moreover, RecJ3 and RecJ4 could be purified as a 560-kDa subcomplex in equimolar subunit ratio with nuclease activities mirroring the full RecJ3/4-aRNase J complex. These findings prompted reconstitution assays that suggested RecJ3/4 could suppress, alter, and/or outcompete the nuclease activities of aRNase J. Based on the phenotypic results, this control mechanism of aRNase J by RecJ3/4 is not necessary for cell growth but instead appears important for DNA repair. IMPORTANCE Nucleases are critical for various cellular processes including DNA replication and repair. Here, a dynamic type of nuclease complex is newly identifiedidentifiedidentifiedin the archaeon Haloferax volcanii , which is missing the canonical RNA exosome. The complex, composed of RecJ3, RecJ4, and aRNase J, functions primarily as a 3′−5′ exonuclease and was discovered through its ATP-dependent association with the ubiquitin-like SAMP1 and Cdc48a. aRNase J alone forms a homodimer that has endonuclease function and, thus, is not restricted to 5′−3′ exonuclease activity typical of other aRNase J enzymes. RecJ3/4 appears to suppress, alter, and/or outcompete the nuclease activities of aRNase J. While aRNase J is essential for growth, RecJ3/4, Cdc48a, and SAMPs are important for recovery against DNA damage. These biological distinctions may correlate with the regulated nuclease activity of aRNase J in the RecJ3/4-aRNaseJ complex.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Marine DNA methylation patterns are associated with microbial community composition and inform virus-host dynamics

Background: DNA methylation in prokaryotes is involved in many different cellular processes including cell cycle regulation and defense against viruses. To date, most prokaryotic methylation systems have been studied in culturable microorganisms, resulting in a limited understanding of DNA methylation from a microbial ecology perspective. Here, we analyze the distribution patterns of several microbial epigenetics marks in the ocean microbiome through genome-centric metagenomics across all domains of life. Results: We reconstructed 15,056 viral, 252 prokaryotic, 56 giant viral, and 6 eukaryotic metagenome-assembled genomes from northwest Pacific Ocean seawater samples using short- and long-read sequencing approaches. These metagenome-derived genomes mostly represented novel taxa, and recruited a majority of reads. Thanks to single-molecule real-time (SMRT) sequencing technology, base modification could also be detected for these genomes. This showed that DNA methylation can readily be detected across dominant oceanic bacterial, archaeal, and viral populations, and microbial epigenetic changes correlate with population differentiation. Furthermore, our genome-wide epigenetic analysis of Pelagibacter suggests that GANTC, a DNA methyltransferase target motif, is related to the cell cycle and is affected by environmental conditions. Yet, the presence of this motif also partitions the phylogeny of the Pelagibacter phages, possibly hinting at a competitive co-evolutionary history and multiple effects of a single methylation mark. Conclusions: Overall, this study elucidates that DNA methylation patterns are associated with ecological changes and virus-host dynamics in the ocean microbiome.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Atypical Response Regulator ChxR from Chlamydia trachomatis Is Structurally Poised for DNA Binding

ChxR is an atypical two-component signal transduction response regulator (RR) of the OmpR/PhoB subfamily encoded by the obligate intracellular bacterial pathogen Chlamydia trachomatis. Despite structural homology within both receiver and effector domains to prototypical subfamily members, ChxR does not require phosphorylation for dimer formation, DNA binding or transcriptional activation. Thus, we hypothesized that ChxR is in a conformation optimal for DNA binding with limited interdomain interactions. To address this hypothesis, the NMR solution structure of the ChxR effector domain was determined and used in combination with the previously reported ChxR receiver domain structure to generate a full-length dimer model based upon SAXS analysis. Small-angle scattering of ChxR supported a dimer with minimal interdomain interactions and effector domains in a conformation that appears to require only subtle reorientation for optimal major/ minor groove DNA interactions. SAXS modeling also supported that the effector domains were in a head-to-tail conformation, consistent with ChxR recognizing tandem DNA repeats. The effector domain structure was leveraged to identify key residues that were critical for maintaining protein - nucleic acid interactions. In combination with prior analysis of the essential location of specific nucleotides for ChxR recognition of DNA, a model of the full-length ChxR dimer bound to its cognate cis-acting element was generated.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Heavy-Duty Vehicle Activity Updates for MOVES Using NREL Fleet DNA and CE-CERT Data

The U.S. Environmental Protection Agency's (EPA's) Motor Vehicle Emission Simulator (MOVES) is a publicly available tool used by researchers and policymakers to help understand motor vehicle emission sources at a national, county, and project level. Estimates of heavy-duty activity in the most recent version of the model at the time this work was conducted, MOVES2014, was identified as an area in need of improvement. The start activity in MOVES2014 is based on a limited and dated data set. In addition, MOVES2014 relies on drive cycles that represent on-network activity but do not account for idling activity that occurs on off-network roads, such as at a distribution center, while the truck is queuing or during loading and unloading. As a result, MOVES2014 may currently underestimate the number of starts and idle and soak time for heavy-duty trucks in real-world operation. The National Renewable Energy Laboratory (NREL) has previously leveraged its expansive Fleet DNA database of heavy-duty vehicles to idle and start activity for six of the nine heavy-duty vehicle source types of classes in the MOVES model. The data available in Fleet DNA from 416 conventional, diesel-powered vehicles provided activity estimates from more than 120,000 hours of operation throughout 14,682 vehicle days between October 2006 and January 2016. NREL calculated start fraction, starts per day, soak fraction, and idle fraction by hour of the day for each vehicle type, state, and vocation, and provided results in .CSV files that can be translated to MOVES table inputs. The idle and start activity from this initial analysis of Fleet DNA data was used to develop default idle and start data for heavy-duty vehicles in MOVES3. Satisfied with the results from the Fleet DNA data used for MOVES3, the EPA asked NREL to extend this start/soak/idle analysis using additional data from a larger number of vehicles for a potential future update to the MOVES model. Such a data set was achieved from a project led by the University of California at Riverside, College of Engineering, Center for Environmental Research & Technology (CE-CERT) and funded by California Air Resources Board. Specifically, this data set consists of 90 heavy-duty vehicles operated mainly in California, which can be separated into five of the nine heavy-duty vehicle classes in the MOVES model. In addition, the heavy-duty activity database collected by CE-CERT provided activity estimates from more than 44,000 hours of operation throughout 4,724 vehicle days between November 2014 and September 2016. This report details the analysis of the heavy-duty activity database collected from the University of California at Riverside by providing graphical analysis and context for the start, soak, and idle distributions. The comparison of the related results from both the Fleet DNA and CE-CERT data sets are documented as well.

33 ADVANCED PROPULSION SYSTEMS↗
DOE OSTI2024

DNA Strand Displacement Driven Molecular Additive Manufacturing (DSD-MAM)

The goal of this project was to validate two-dimensional molecular printers, initially selfassembled from DNA and then actuated by externally driven cycles of DNA strand displacement, as prototype integrated nanosystems for molecular additive manufacturing. Novel functionalities of these nanomachines were explored during this project, including the following: nanometer-precision positioning mechanisms based on DNA strand displacement with multivalent interactions for discrete stepping or else diffusive capture; integration of independently moving layers of DNA origami to achieve 2D controllable motion; integration of spatial positioning with deposition functionality. The principal importance of this project was to provide an essential step in the development of a new technology for atomically precise manufacturing. Our first generation molecular 2D printer offers several advantages over conventional DNA-origami patterning, such as faster prototyping, faster dynamic rearrangement of patterns, and the ability to respond with feedback. We anticipate that our first-generation molecular printers may inspire future generations of molecular printers with iterative improvements in robustness and throughput. Potential applications of atomically precise manufacturing include the following: photovoltaics; photosynthetic and fuel cells; thermoelectrics and anisotropic heat spreaders; solid-state lighting; molecular electronic and plasmonic circuits; selectively preamble membranes; self-repairing materials with high strength-to-weight and fracture resistance.

36 MATERIALS SCIENCE↗
DOE OSTI2023

Evaluation of DNA Vaccine Candidates against Foot-and-Mouth Disease Virus in Cattle

We evaluated four DNA vaccine candidates for their ability to produce virus-like particles (VLPs) and elicit a protective immune response against Foot-and-mouth disease virus (FMDV) in cattle. Two traditional DNA plasmids and two DNA minicircle constructs were evaluated. Both the pTarget O1P1-3C plasmid and O1P1-3C minicircle encoded a wild-type FMDV 3C protease to process the P1-2A polypeptide, whereas the O1P1-HIV-3CT minicircle used an HIV-1 ribosomal frameshift to down-regulate expression of a mutant 3C protease. A modified pTarget plasmid with a reduced backbone size, mpTarget O1P1-3C LT , used a 3C protease containing two mutations reported to enhance expression. All constructs produced mature FMDV P1 cleavage products in transfected cells, as seen by western blot analysis. Three constructs, O1P1-3C minicircles, pTarget O1P1-3C, and mpTarget O1P1-3C LT plasmids, produced intracellular VLP crystalline arrays detected by electron microscopy. Despite VLP formation in vitro, none of the DNA vaccine candidates elicited protection from clinical disease when administered independently. Administration of pTarget O1P1-3C plasmid enhanced neutralizing antibody titers when used as a priming dose prior to administration of a conditionally licensed adenovirus-vectored FMD vaccine. Further work is needed to develop these DNA plasmid-based constructs into standalone FMD vaccines in cattle.

75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND↗
DOE OSTI2014

Regulatory interplay between TFIID’s conformational transitions and its modular interaction with core promoter DNA

Recent structural and biochemical studies of human TFIID have significantly increased our understanding of the mechanisms underlying the recruitment of TFIID to promoter DNA and its role in transcription initiation. Structural studies using cryoEM revealed that modular interactions underlie TFIID’s ability to bind simultaneously multiple promoter motifs and to define a DNA state that will facilitate transcription initiation. Here we propose a general model of promoter binding by TFIID, where co-activators, activators, and histone modifications promote and/or stabilize a conformational state of TFIID that results in core promoter engagement. Within this high affinity conformation, we propose that TFIID’s extensive interaction with promoter DNA leads to topological changes in the DNA that facilitate the eventual loading of RNAP II. While more work is required to dissect the individual contributions of activators and repressors to TFIID’s DNA binding, the recent cryoEM studies provide a physical framework to guide future structural, biophysical, and biochemical experiments.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1994

Amino acid racemization in amber-entombed insects: implications for DNA preservation

DNA depurination and amino acid racemization take place at similar rates in aqueous solution at neutral pH. This relationship suggests that amino acid racemization may be useful in accessing the extent of DNA chain breakage in ancient biological remains. To test this suggestion, we have investigated the amino acids in insects entombed in fossilized tree resins ranging in age from <100 years to 130 million years. The amino acids present in 40 to 130 million year old amber-entombed insects resemble those in a modern fly and are probably the most ancient, unaltered amino acids found so far on Earth. In comparison to other geochemical environments on the surface of the Earth, the amino acid racemization rate in amber insect inclusions is retarded by a factor of >10(4). These results suggest that in amber insect inclusions DNA depurination rates would also likely be retarded in comparison to aqueous solution measurements, and thus DNA fragments containing many hundreds of base pairs should be preserved. This conclusion is consistent with the reported successful retrieval of DNA sequences from amber-entombed organisms.

NASA Discipline Exobiology↗