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At least 739 records · Page 41
All sourcesNASA NTRSDOE OSTI
DOE OSTI2015

Mechanism of CRISPR-RNA guided recognition of DNA targets in Escherichia coli

In bacteria and archaea, short fragments of foreign DNA are integrated into Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) loci, providing a molecular memory of previous encounters with foreign genetic elements. In Escherichia coli, short CRISPR-derived RNAs are incorporated into a multi-subunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Recent structures of Cascade capture snapshots of this seahorse-shaped RNA-guided surveillance complex before and after binding to a DNA target. Here we determine a 3.2 Å x-ray crystal structure of Cascade in a new crystal form that provides insight into the mechanism of double-stranded DNA binding. Molecular dynamic simulations performed using available structures reveal functional roles for residues in the tail, backbone and belly subunits of Cascade that are critical for binding double-stranded DNA. Structural comparisons are used to make functional predictions and these predictions are tested in vivo and in vitro. Collectively, the results in this study reveal underlying mechanisms involved in target-induced conformational changes and highlight residues important in DNA binding and protospacer adjacent motif recognition.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Structural basis of damage recognition by thymine DNA glycosylase: Key roles for N-terminal residues

Thymine DNA Glycosylase (TDG) is a base excision repair enzyme functioning in DNA repair and epigenetic regulation. TDG removes thymine from mutagenic G·T mispairs arising from deamination of 5-methylcytosine (mC), and it processes other deamination-derived lesions including uracil (U). Essential for DNA demethylation, TDG excises 5- formylcytosine and 5-carboxylcytosine, derivatives of mC generated by Tet (ten-eleven translocation) enzymes. Here, we report structural and functional studies of TDG82-308, a new construct containing 29 more N-terminal residues than TDG111-308, the construct used for previous structures of DNAbound TDG. Crystal structures and NMR experiments demonstrate that most of these N-terminal residues are disordered, for substrate- or productbound TDG82-308. Nevertheless, G·T substrate affinity and glycosylase activity of TDG82-308 greatly exceeds that of TDG111-308 and is equivalent to full-length TDG. We report the first high-resolution structures of TDG in an enzyme-substrate complex, for G·U bound to TDG82-308 (1.54 A) and TDG ' 111-308 (1.71 A), revealing ' new enzyme-substrate contacts, direct and watermediated. We also report a structure of the TDG82-308 product complex (1.70 A). TDG ' 82-308 forms unique enzyme–DNA interactions, supporting its value for structure-function studies. The results advance understanding of how TDG recognizes and removes modified bases from DNA, particularly those resulting from deamination.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Activator Protein-1: redox switch controlling structure and DNA-binding

The transcription factor, activator protein-1 (AP-1), binds to cognate DNA under redox control; yet, the underlying mechanism has remained enigmatic. A series of crystal structures of the AP-1 FosB/JunD bZIP domains reveal ordered DNA-binding regions in both FosB and JunD even in absence DNA. However, while JunD is competent to bind DNA, the FosB bZIP domain must undergo a large conformational rearrangement that is controlled by a ‘redox switch’ centered on an inter-molecular disulfide bond. Solution studies confirm that FosB/JunD cannot undergo structural transition and bind DNA when the redox-switch is in the ‘OFF’ state, and show that the mid-point redox potential of the redox switch affords it sensitivity to cellular redox homeostasis. The molecular and structural studies presented here thus reveal the mechanism underlying redox-regulation of AP-1 Fos/Jun transcription factors and provide structural insight for therapeutic interventions targeting AP-1 proteins.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

A DNA G-quadruplex/i-motif hybrid

Abstract DNA can form many structures beyond the canonical Watson–Crick double helix. It is now clear that noncanonical structures are present in genomic DNA and have biological functions. G-rich G-quadruplexes and C-rich i-motifs are the most well-characterized noncanonical DNA motifs that have been detected in vivo with either proscribed or postulated biological roles. Because of their independent sequence requirements, these structures have largely been considered distinct types of quadruplexes. Here, we describe the crystal structure of the DNA oligonucleotide, d(CCAGGCTGCAA), that self-associates to form a quadruplex structure containing two central antiparallel G-tetrads and six i-motif C–C+ base pairs. Solution studies suggest a robust structural motif capable of assembling as a tetramer of individual strands or as a dimer when composed of tandem repeats. This hybrid structure highlights the growing structural diversity of DNA and suggests that biological systems may harbor many functionally important non-duplex structures.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Structure of HhaI endonuclease with cognate DNA at an atomic resolution of 1.0 Å

HhaI, a Type II restriction endonuclease, recognizes the symmetric sequence 5'-GCG↓C-3' in duplex DNA and cleaves (‘↓’) to produce fragments with 2-base, 3'-overhangs. We determined the structure of HhaI in complex with cognate DNA at an ultra-high atomic resolution of 1.0 Å. Most restriction enzymes act as dimers with two catalytic sites, and cleave the two strands of duplex DNA simultaneously, in a single binding event. HhaI, in contrast, acts as a monomer with only one catalytic site, and cleaves the DNA strands sequentially, one after the other. HhaI comprises three domains, each consisting of a mixed five-stranded β sheet with a defined function. The first domain contains the catalytic-site; the second contains residues for sequence recognition; and the third contributes to non-specific DNA binding. The active-site belongs to the ‘PD-D/EXK’ superfamily of nucleases and contains the motif SD-X11-EAK. The first two domains are similar in structure to two other monomeric restriction enzymes, HinP1I (G↓CGC) and MspI (C↓CGG), which produce fragments with 5'-overhangs. The third domain, present only in HhaI, shifts the positions of the recognition residues relative to the catalytic site enabling this enzyme to cleave the recognition sequence at a different position. The structure of M.HhaI, the biological methyltransferase partner of HhaI, was determined earlier. Together, these two structures represent the first natural pair of restriction-modification enzymes to be characterized in atomic detail.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Structural basis for preferential binding of human TCF4 to DNA containing 5-carboxylcytosine

The psychiatric risk-associated transcription factor 4 (TCF4) is linked to schizophrenia. Rare TCF4 coding variants are found in individuals with Pitt-Hopkins syndrome—an intellectual disability and autism spectrum disorder. TCF4 contains a C-terminal basic-helix-loop-helix (bHLH) DNA binding domain which recognizes the enhancer-box (E-box) element 5'-CANNTG-3' (where N = any nucleotide). A subset of the TCF4-occupancy sites have the expanded consensus binding specificity 5'-C(A/G)-CANNTG-3', with an added outer Cp(A/G) dinucleotide; for example in the promoter for CNIH3, a gene involved in opioid dependence. In mammalian genomes, particularly brain, the CpG and CpA dinucleotides can be methylated at the 5-position of cytosine (5mC), and then may undergo successive oxidations to the 5-hydroxymethyl (5hmC), 5-formyl (5fC), and 5-carboxyl (5caC) forms. We find that, in the context of 5'- 0 CG- 1 CA- 2 CG- 3 TG-3'(where the numbers indicate successive dinucleotides), modification of the central E-box 2 CG has very little effect on TCF4 binding, E-box 1 CA modification has a negative influence on binding, while modification of the flanking 0 CG, particularly carboxylation, has a strong positive impact on TCF4 binding to DNA. Crystallization of TCF4 in complex with unmodified or 5caC-modified oligonucleotides revealed that the basic region of bHLH domain adopts multiple conformations, including an extended loop going through the DNA minor groove, or the N-terminal portion of a long helix binding in the DNA major groove. The different protein conformations enable arginine 576 (R576) to interact, respectively, with a thymine in the minor groove, a phosphate group of DNA backbone, or 5caC in the major groove. The Pitt-Hopkins syndrome mutations affect five arginine residues in the basic region, two of them (R569 and R576) involved in 5caC recognition. Our analyses indicate, and suggest a structural basis for, the preferential recognition of 5caC by a transcription factor centrally important in brain development.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

DNA-mediated assembly of Au bipyramids into anisotropic light emitting kagome superlattices

Colloidal crystal engineering with DNA allows one to design diverse superlattices with tunable lattice symmetry, composition, and spacing. Most of these structures follow the complementary contact model, maximizing DNA hybridization on building blocks and producing relatively close-packed lattices. Here, low-symmetry kagome superlattices are assembled from DNA-modified gold bipyramids that can engage only in partial DNA surface matching. The bipyramid dimensions and DNA length can be engineered for two different superlattices with rhombohedral unit cells, including one composed of a periodic stacking of kagome lattices. Enabled by the partial facet alignment, the kagome lattices exhibit lattice distortion, bipyramid twisting, and planar chirality. When conjugated with Cy-5 dyes, the kagome lattices serve as cavities with high-density optical states and large Purcell factors along lateral directions, leading to strong dipole radiation along thezaxis and facet-dependent light emission. Such complex optical properties make these materials attractive for lasers, displays, and quantum sensing constructs.

Science & Technology - Other Topics↗
DOE OSTI2019

Hachimoji DNA and RNA: A genetic system with eight building blocks

We report DNA- and RNA-like systems built from eight nucleotide “letters” (hence the name “hachimoji”) that form four orthogonal pairs. These synthetic systems meet the structural requirements needed to support Darwinian evolution, including a polyelectrolyte backbone, predictable thermodynamic stability, and stereoregular building blocks that fit a Schrödinger aperiodic crystal. Measured thermodynamic parameters predict the stability of hachimoji duplexes, allowing hachimoji DNA to increase the information density of natural terran DNA. Three crystal structures show that the synthetic building blocks do not perturb the aperiodic crystal seen in the DNA double helix. Hachimoji DNA was then transcribed to give hachimoji RNA in the form of a functioning fluorescent hachimoji aptamer. These results expand the scope of molecular structures that might support life, including life throughout the cosmos.

Hoshika, Shuichi (ORCID:0000000191281183)↗
DOE OSTI2023

Trivalent rare earth metal cofactors confer rapid NP-DNA polymerase activity

Here, a DNA polymerase with a single mutation and a divalent calcium cofactor catalyzes the synthesis of unnatural N3'→P5' phosphoramidate (NP) bonds to form NP-DNA. However, this template-directed phosphoryl transfer activity remains orders of magnitude slower than native phosphodiester synthesis. Here, we used time-resolved x-ray crystallography to show that NP-DNA synthesis proceeds with a single detectable calcium ion in the active site. Using insights from isotopic and elemental effects, we propose that one-metal-ion electrophilic substrate activation is inferior to the native two-metal-ion mechanism. We found that this deficiency in divalent activation could be ameliorated by trivalent rare earth and post–transition metal cations, substantially enhancing NP-DNA synthesis. Scandium(III), in particular, confers highly specific NP activity with kinetics enhanced by more than 100-fold over calcium(II), yielding NP-DNA strands up to 100 nucleotides in length.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Horizontal transfer of chromosomal DNA mediated by an integrative and conjugative element generates frequent localized recombination in Novosphingobium aromaticivorans

Horizontal gene transfer is an important evolutionary process by which DNA is exchanged between cells that are physically co-located but not direct evolutionary descendants. Horizontal transfer of highly divergent DNA is relatively easy to detect and can produce major phenotypic changes, exemplified by the acquisition of antibiotic resistance determinants. However, transfer of high-identity DNA, for example, between strains of the same species, is likely to be more frequent, harder to detect, and highly impactful in aggregate. In this work, we demonstrate that soil isolates of the alphaproteobacterium Novosphingobium aromaticivorans can exchange chromosomal DNA, leading to multiple unselected recombination events spanning approximately 10% of the chromosome. Chromosomal recombination was directional and more efficient near an integrative and conjugative element (ICE), and required a relaxase found in the ICE. Recombination could not be observed in strains from closely related Novosphingobium species. In combination, these results suggest that ICE-mediated recombination can efficiently recombine DNA within N. aromaticivorans, increasing the adaptive potential of the species while also enforcing species boundaries through preferential intraspecific recombination.

Allemann, Marco [ORNL]↗
DOE OSTI2014

Facile, High Quality Sequencing of Bacterial Genomes from Small Amounts of DNA

Sequencing bacterial genomes has traditionally required large amounts of genomic DNA (~1 μg).There have been few studies to determine the effects of the input DNA amount or library preparation method on the quality of sequencing data. Several new commercially available library preparation methods enable shotgun sequencing from as little as 1 ng of input DNA. In this study, we evaluated the NEBNext Ultra library preparation reagents for sequencing bacterial genomes. We have evaluated the utility of NEBNext Ultra for resequencing and de novo assembly of four bacterial genomes and compared its performance with the TruSeq library preparation kit. The NEBNext Ultra reagents enable high quality resequencing and de novo assembly of a variety of bacterial genomes when using 100 ng of input genomic DNA. For the two most challenging genomes (Burkholderia spp.), which have the highest GC content and are the longest, we also show that the quality of both resequencing and de novo assembly is not decreased when only 10 ng of input genomic DNA is used.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

DNA Persistence in a Sink Drain Environment

Biofilms are organized structures composed mainly of cells and extracellular polymeric substances produced by the constituent microorganisms. Ubiquitous in nature, biofilms have an innate ability to capture and retain passing material and may therefore act as natural collectors of contaminants or signatures of upstream activities. To determine the persistence and detectability of DNA passing through a sink drain environment, Bacillus anthracis strain Ames35 was cultured (6.35 x 10 7 CFU/mL), sterilized, and disposed of by addition to a sink drain apparatus with an established biofilm. The sink drain apparatus was sampled before and for several days after the addition of the sterilized B. anthracis culture to detect the presence of B. anthracis DNA. Multiple PCR primer pairs were used to screen for chromosomal and plasmid DNA with primers targeting shorter sequences showing greater amplification efficiency and success. PCR amplification and detection of target sequences indicate persistence of chromosomal DNA and plasmid DNA in the biofilm for 5 or more and 14 or more days, respectively.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

RNA and DNA Targeting by a Reconstituted Thermus thermophilus Type III-A CRISPR-Cas System

CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) systems are RNA-guided adaptive immunity pathways used by bacteria and archaea to defend against phages and plasmids. Type III-A systems use a multi-subunit interference complex called Csm, containing Cas proteins and a CRISPR RNA (crRNA) to target cognate nucleic acids. The Csm complex is intriguing in that it mediates RNA-guided targeting of both RNA and transcriptionally active DNA, but the mechanism is not well understood. Here, we overexpressed the five components of the Thermus thermophilus (T. thermophilus) Type III-A Csm complex (TthCsm) with a defined crRNA sequence, and purified intact TthCsm complexes from E. coli cells. The complexes were thermophilic, targeting complementary ssRNA more efficiently at 65°C than at 37°C. Sequence-independent, endonucleolytic cleavage of single-stranded DNA (ssDNA) by TthCsm was triggered by recognition of a complementary ssRNA, and required a lack of complementarity between the first 8 nucleotides (50 tag) of the crRNA and the 30 flanking region of the ssRNA. Mutation of the histidine-aspartate (HD) nuclease domain of the TthCsm subunit, Cas10/Csm1, abolished DNA cleavage. Activation of DNA cleavage was dependent on RNA binding but not cleavage. This leads to a model in which binding of an ssRNA target to the Csm complex would stimulate cleavage of exposed ssDNA in the cell, such as could occur when the RNA polymerase unwinds double-stranded DNA (dsDNA) during transcription. Our findings establish an amenable, thermostable system for more in-depth investigation of the targeting mechanism using structural biology methods, such as cryoelectron microscopy and x-ray crystallography.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Tobacco-specific nitrosamine 1-(N-methyl-N-nitrosamino)-1-(3-pyridinyl)-4-butanal (NNA) causes DNA damage and impaired replication/transcription in human lung cells

Thirdhand smoke (THS) is a newly described health hazard composed of toxicants, mutagens and carcinogens, including nicotine-derived tobacco specific nitrosamines (TSNAs), one of which is 1-(N-methyl-N-nitrosamino)-1-(3-pyridinyl)-4-butanal (NNA). Although TSNAs are generally potent carcinogens, the risk of NNA, which is specific to THS, is poorly understood. We recently reported that THS exposure-induced adverse impact on DNA replication and transcription with implications in the development of cancer and other diseases. Here, we investigated the role of NNA in THS exposure-induced harmful effects on fundamental cellular processes. We exposed cultured human lung epithelial BEAS-2B cells to NNA. The formation of DNA base damages was assessed by Long Amplicon QPCR (LA-QPCR); DNA double-strand breaks (DSBs) and NNA effects on replication and transcription by immunofluorescence (IF); and genomic instability by micronuclei (MN) formation. We found increased accumulation of oxidative DNA damage and DSBs as well as activation of DNA damage response pathway, after exposure of cells to NNA. Impaired S phase progression was also evident. Consistent with these results, we found increased MN formation, a marker of genomic instability, in NNA-exposed cells. Furthermore, ongoing RNA synthesis was significantly reduced by NNA exposure, however, RNA synthesis resumed fully after a 24h recovery period only in wild-type cells but not in those deficient in transcription-coupled nucleotide excision repair (TC-NER). Importantly, these cellular effects are common with the THS-exposure induced effects. Our findings suggest that NNA in THS could be a contributing factor for THS exposure-induced adverse health effect.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Building an ab initio solvated DNA model using Euclidean neural networks

Accurately modeling large biomolecules such as DNA from first principles is fundamentally challenging due to the steep computational scaling of ab initio quantum chemistry methods. This limitation becomes even more prominent when modeling biomolecules in solution due to the need to include large numbers of solvent molecules. We present a machine-learned electron density model based on a Euclidean neural network framework that includes a built-in understanding of equivariance to model explicitly solvated double-stranded DNA. By training the machine learning model using molecular fragments that sample the key DNA and solvent interactions, we show that the model predicts electron densities of arbitrary systems of solvated DNA accurately, resolves polarization effects that are neglected by classical force fields, and captures the physics of the DNA-solvent interaction at the ab initio level.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Structures of ISCth4 transpososomes reveal the role of asymmetry in copy-out/paste-in DNA transposition

Copy-out/paste-in transposition is a major bacterial DNA mobility pathway. It contributes significantly to the emergence of antibiotic resistance, often by upregulating expression of downstream genes upon integration. Unlike other transposition pathways, it requires both asymmetric and symmetric strand transfer steps. Here, we report the first structural study of a copy-out/paste-in transposase and demonstrate its ability to catalyze all pathway steps in vitro. X-ray structures of ISCth4 transposase, a member of the IS256 family of insertion sequences, bound to DNA substrates corresponding to three sequential steps in the reaction reveal an unusual asymmetric dimeric transpososome. During transposition, an array of N-terminal domains binds a single transposon end while the catalytic domain moves to accommodate the varying substrates. These conformational changes control the path of DNA flanking the transposon end and the generation of DNA-binding sites. Our results explain the asymmetric outcome of the initial strand transfer and show how DNA binding is modulated by the asymmetric transposase to allow the capture of a second transposon end and to integrate a circular intermediate.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Systems Biology Model of Interactions Between Tissue Growth Factors and DNA Damage Pathways: Low Dose Response and Cross-Talk in TGFbeta and ATM Signaling

The etiology of radiation carcinogenesis has been described in terms of aberrant changes that span several levels of biological organization. Growth factors regulate many important cellular and tissue functions including apoptosis, differentiation and proliferation. A variety of genetic and epigenetic changes of growth factors have been shown to contribute to cancer initiation and progression. It is known that cellular and tissue damage to ionizing radiation is in part initiated by the production of reactive oxygen species, which can activate cytokine signaling, and the DNA damage response pathways, most notably the ATM signaling pathway. Recently the transforming growth factor β (TGFβ) pathway has been shown to regulate or directly interact with the ATM pathway in the response to radiation. The relevance of this interaction with the ATM pathway is not known although p53 becomes phosphorylated and DNA damage responses are involved. However, growth factor interactions with DNA damage responses have not been elucidated particularly at low doses and further characterization of their relationship to cancer processes is warranted. Our goal will be to use a systems biology approach to mathematically and experimentally describe the low dose responses and cross-talk between the ATM and TGFβ pathways initiated by low and high LET radiation. We will characterize ATM and TGFβ signaling in epithelial and fibroblast cells using 2D models and ultimately extending to 3D organotypic cell culture models to begin to elucidate possible differences that may occur for different cell types and/or inter-cellular communication. We will investigate the roles of the Smad and Activating transcription factor 2 (ATF2) proteins as the potential major contributors to cross- talk between the TGFβ and ATM pathways, and links to cell cycle control and/or the DNA damage response, and potential differences in their responses at low and high doses. We have developed various experimental approaches to apply to these problems using confocal microscopy and flow cytometry to detail changes at low dose/dose-rate in order to understand individual cell responses, and will establish our mathematical models based on the experimental findings resulting from changes in DNA repair, apoptosis and proliferation.

O'Neill, Peter↗
DOE OSTI2019

In Situ Loading and Delivery of Short Single- and Double-Stranded DNA by Supramolecular Organic Frameworks

Short DNA represents an important class of biomacromolecules that are widely applied in gene therapy, editing, and modulation. However, the development of simple and reliable methods for their intracellular delivery remains a challenge. Herein, we describe that seven water-soluble, homogeneous supramolecular organic frameworks (SOFs) with a well-defined pore size and high stability in water that can accomplish in situ inclusion of single-stranded (ss) and double-stranded (ds) DNA (21, 23, and 58 nt) and effective intracellular delivery (including two noncancerous and six cancerous cell lines). Fluorescence quenching experiments for single and double end-labeled ss- and ds-DNA support that the DNA sequences can be completely enveloped by the SOFs. Confocal laser scanning microscopy and flow cytometry reveal that five of the SOFs exhibit excellent delivery efficiencies that, in most of the studied cases, outperform the commercial standard Lipo2000, even at low SOF–nucleic acid ratios. In addition to high delivery efficiencies, the water-soluble, self-assembled SOF carriers have a variety of advantages, including convenient preparation, high stability, and in situ DNA inclusion, which are all critical for practical applications in nucleic acid delivery.

59 BASIC BIOLOGICAL SCIENCES↗