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252 records · Page 14
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Conformational Control of DNA Origami by DNA Oligomers, Intercalators and UV Light

DNA origami has garnered great attention due to its excellent programmability and precision. It offers a powerful means to create complex nanostructures which may not be possible by other methods. The macromolecular structures may be used as static templates for arranging proteins and other molecules. They are also capable of undergoing structural transformation in response to external signals, which may be exploited for sensing and actuation at the nanoscale. Such on-demand reconfigurations are executed mostly by DNA oligomers through base-pairing and/or strand displacement, demonstrating drastic shape changes between two different states, for example, open and close. Recent studies have developed new mechanisms to modulate the origami conformation in a controllable, progressive manner. Here we present several methods for conformational control of DNA origami nanostructures including chemical adducts and UV light as well as widely applied DNA oligomers. The detailed methods should be useful for beginners in the field of DNA nanotechnology.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Toward Revealing Microcystin Distribution in Mouse Liver Tissue Using MALDI-MS Imaging

Cyanotoxins can be found in water and air during cyanobacterial harmful algal blooms (cHABs) in lakes and rivers. Therefore, it is very important to monitor their potential uptake by animals and humans as well as their health effects and distribution in affected organs. Herein, the distribution of hepatotoxic peptide microcystin-LR (MC-LR) is investigated in liver tissues of mice gavaged with this most common MC congener. Preliminary matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) imaging experiments performed using a non-automated MALDI matrix deposition device and a MALDI-time-of-flight (TOF) mass spectrometer yielded ambiguous results in terms of MC-LR distribution in liver samples obtained from MC-LR-gavaged mice. The tissue preparation for MALDI-MS imaging was improved by using an automated sprayer for matrix deposition, and liver sections were imaged using an Nd:YAG MALDI laser coupled to a 15 Tesla Fourier-transform ion cyclotron resonance (FT-ICR)-mass spectrometer. MALDI-FT-ICR-MS imaging provided unambiguous detection of protonated MC-LR (calculated m/z 995.5560, z = +1) and the sodium adduct of MC-LR (m/z 1017.5380, z = +1) in liver sections from gavaged mice with great mass accuracy and ultra-high mass resolution. Since both covalently bound and free MC-LR can be found in liver of mice exposed to this toxin, the present results indicate that the distribution of free microcystins in tissue sections from affected organs, such as liver, can be monitored with high-resolution MALDI-MS imaging.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Aristolochic acid-induced genotoxicity and toxicogenomic changes in rodents

Aristolochic acid (AA) is a group of structurally related nitrophenanthrene carboxylic acids found in many plants that are widely used by many cultures as traditional herbal medicines. AA is a causative agent for Chinese herbs nephropathy, a term replaced later by AA nephropathy. Evidence indicates that AA is nephrotoxic, genotoxic, and carcinogenic in humans; and it also induces tumors in the forestomach, kidney, renal pelvis, urinary bladder, and lung of rats and mice. Therefore, plants containing AA have been classified as carcinogenic to humans (Group 1) by the International Agency for Research on Cancer. In our laboratories, we have conducted a series of genotoxicity and toxicogenomic studies in the rats exposed to AA of 0.1–10 mg/kg for 12 weeks. Our results demonstrated that AA treatments induced DNA adducts and mutations in the kidney, liver, and spleen of rats, as well as significant alteration of gene expression in both its target and nontarget tissues. AA treatments altered mutagenesis- or carcinogenesis-related microRNA expression in rat kidney and resulted in significant changes in protein expression profiling. We also applied benchmark dose (BMD) modeling to the 3-month AA-induced genotoxicity data. The obtained BMDL10 (the lower 95% confidence interval of the BMD 10 that is a 10% increase over the background level) for AA-induced mutations in the kidney of rats was about 7 μg/kg body weight per day. This review constitutes an overview of our investigations on AA-induced genotoxicity and toxicogenomic changes including gene expression, microRNA expression, and proteomics; and presents updated information focused on AA-induced genotoxicity in rodents.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Cloud response to co-condensation of water and organic vapors over the boreal forest

Abstract. Accounting for the condensation of organic vapors along with water vapor (co-condensation) has been shown in adiabatic cloud parcel model (CPM) simulations to enhance the number of aerosol particles that activate to form cloud droplets. The boreal forest is an important source of biogenic organic vapors, but the role of these vapors in co-condensation has not been systematically investigated. In this work, the environmental conditions under which strong co-condensation-driven cloud droplet number enhancements would be expected over the boreal biome are identified. Recent measurement technology, specifically the Filter Inlet for Gases and AEROsols (FIGAERO) coupled to an iodide-adduct chemical ionization mass spectrometer (I-CIMS), is utilized to construct volatility distributions of the boreal atmospheric organics. Then, a suite of CPM simulations initialized with a comprehensive set of concurrent aerosol observations collected in the boreal forest of Finland during spring 2014 is performed. The degree to which co-condensation impacts droplet formation in the model is shown to be dependent on the initialization of temperature, relative humidity, updraft velocity, aerosol size distribution, organic vapor concentration, and the volatility distribution. The predicted median enhancements in cloud droplet number concentration (CDNC) due to accounting for the co-condensation of water and organics fall on average between 16 % and 22 %. This corresponds to activating particles 10–16 nm smaller in dry diameter that would otherwise remain as interstitial aerosol. The highest CDNC enhancements (ΔCDNC) are predicted in the presence of a nascent ultrafine aerosol mode with a geometric mean diameter of ∼ 40 nm and no clear Hoppel minimum, indicative of pristine environments with a source of ultrafine particles (e.g., via new particle formation processes). Such aerosol size distributions are observed 30 %–40 % of the time in the studied boreal forest environment in spring and fall when new particle formation frequency is the highest. To evaluate the frequencies with which such distributions are experienced by an Earth system model over the whole boreal biome, 5 years of UK Earth System Model (UKESM1) simulations are further used. The frequencies are substantially lower than those observed at the boreal forest measurement site (< 6 % of the time), and the positive values, peaking in spring, are modeled only over Fennoscandia and the western parts of Siberia. Overall, the similarities in the size distributions between observed and modeled (UKESM1) are limited, which would limit the ability of this model, or any model with a similar aerosol representation, to project the climate relevance of co-condensation over the boreal forest. For the critical aerosol size distribution regime, ΔCDNC is shown to be sensitive to the concentrations of semi-volatile and some intermediate-volatility organic compounds (SVOCs and IVOCs), especially when the overall particle surface area is low. The magnitudes of ΔCDNC remain less affected by the more volatile vapors such as formic acid and extremely low- and low-volatility organic compounds (ELVOCs and LVOCs). The reasons for this are that most volatile organic vapors condense inefficiently due to their high volatility below the cloud base, and the concentrations of LVOCs and ELVOCs are too low to gain significant concentrations of soluble mass to reduce the critical supersaturations enough for droplet activation to occur. A reduction in the critical supersaturation caused by organic condensation emerges as the main driver of the modeled ΔCDNC. The results highlight the potential significance of co-condensation in pristine boreal environments close to sources of fresh ultrafine particles. For accurate predictions of co-condensation effects on CDNC, also in larger-scale models, an accurate representation of the aerosol size distribution is critical. Further studies targeted at finding observational evidence and constraints for co-condensation in the field are encouraged.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2020

Facilitated Transport Membranes with Tunable Amine-CO 2 Chemistry for CO 2 /H 2 Separation

CO 2 -selective, amine-containing facilitated transport membranes are of great interest for syngas purification since high-pressure H 2 can be retained upon CO 2 removal. A variety of amine-containing polymers have shown decent chemical and thermal stability at aggressive conditions, but their CO 2 /H 2 separation properties are largely limited by the hydrostatic compaction and severe carrier saturation associated with the high syngas pressure. Herein, we report a new approach to enhance the CO 2 permeability by manipulating the steric hindrance of the amine carriers. A series of α-aminoacids with different alkyl or hydroxyethyl substituents are deprotonated by 2-(1-piperazinyl)ethylamine, leading to nonvolatile amine carriers with different degrees of steric hindrance. For hosting the low MW amine carriers, a highly crosslinked poly(vinyl acetal) is synthesized as a water-swellable polymer network. In order to avoid membrane compaction, perforated graphene oxide mono-sheets are dispersed as reinforcement fillers. In the presence of moisture, a bulkier alkyl substituent to the amino site (increasing steric hindrance) destabilizes the carbamate adduct and thus drastically increases the chemisorption of CO 2 , while the incorporation of ethylene oxide groups provides additional physisorption of CO 2 . The enhanced CO 2 solubility significantly mitigates the carrier saturation behavior, and an unprecedented CO 2 /H 2 selectivity greater than 100 is demonstrated at 107 °C and 12.5 atm of CO 2 partial pressure. As the CO 2 partial pressure reduces to 0.4 atm, a less hindered amine yields a higher reactive diffusivity of CO 2 , resulting in a CO 2 permeance of 435 GPU with a selectivity greater than 500. These reaction-mediated polymeric membranes are well above the theoretical upper bound, and they are of great interest for designing a highly-selective membrane process for syngas purification.

08 HYDROGEN↗
DOE OSTI2021

Methods for electropolishing and coating aluminum on air and/or moisture sensitive substrates

Methods for electropolishing and coating aluminum on a surface of an air and/or moisture sensitive substrate, including: in a vessel, submerging the substrate in a first molten salt bath and applying an anodizing current to the substrate at a first temperature to electropolish the surface of the substrate; wherein the first molten salt bath includes one of a first organic salt bath and first inorganic salt bath; wherein, when used, the first organic salt bath includes one of (a) aluminum halide and ionic liquid, (b) a combination of an aluminum halide and halogenatedmethylphenylsulfone (C 6 (H 5−y ,X y )SO 2 CX 3 , where y is a number from 0-5), (c) a combination of an aluminum halide, an ionic liquid, and halogenatedmethylphenylsulfone (C 6 (H 5−y ,X y )SO 2 CX 3 ), and (d) AlF 3 -organofluoride-hydrofluoric acid adduct; wherein, when used, the first inorganic salt bath includes aluminum halide and alkali metal halide; and wherein the anodizing current is 10-30 mA/cm 2 .

Freiderich, John W.↗
DOE OSTI2022

Formation of high quality alane

Methods for forming alane are described. The method includes addition of toluene at a temperature above the crystallization temperature of alane to a lower temperature solution that includes alane adduct, ether, and toluene. Upon the addition, a crystallization mixture is formed that is at or near the crystallization temperature of alane. The alane of the mixture crystallizes over a period of time to form a high purity alane polymorph.

Zidan, Ragaiy↗
DOE OSTI2022

Methods for electropolishing and coating aluminum on air and/or moisture sensitive substrates

Methods for electropolishing and coating aluminum on a surface of an air and/or moisture sensitive substrate, including: in a vessel, submerging the substrate in a first molten salt bath and applying an anodizing current to the substrate at a first temperature to electropolish the surface of the substrate; wherein the first molten salt bath includes one of a first organic salt bath and first inorganic salt bath; wherein, when used, the first organic salt bath includes one of (a) aluminum halide and ionic liquid, (b) a combination of an aluminum halide and halogenatedmethylphenylsulfone (C 6 (H 5-y ,X y )SO 2 CX 3 , where y is a number from 0-5), (c) a combination of an aluminum halide, an ionic liquid, and halogenatedmethylphenylsulfone (C 6 (H 5-y ,X y )SO 2 CX 3 ), and (d) AlF 3 -organofluoride-hydrofluoric acid adduct; wherein, when used, the first inorganic salt bath includes aluminum halide and alkali metal halide; and wherein the anodizing current is 10-30 mA/cm 2 .

Freiderich, John W.↗
DOE OSTI2020

Design and Development of a Robotic Exoskeleton Device for use in Glove Boxes - 20374

Glovebox operators in nuclear facilities often operate under radioactive environment and typically perform monotonous, dull and repetitive work. This can result in muscle fatigue leading to increased levels of accidents. Current research work aims in assisting the glovebox operators by designing a novel upper limb exoskeleton device to reduce injuries and increase worker efficiency. Almost none of the commercially available hand and upper body exoskeletons are directly suitable for use in gloveboxes. This explains the novelty of the present work in developing a wearable powered robotic exoskeleton device. The device consists of electronics, motors and spring systems mounted on adjustable plates and is supported by a stationary frame behind the worker. The device is intended to be portable and adjustable according to the operator height. The elbow has a flexion movement assisted with motor control by way of a Bowden cable system. The system is floated on a spring and pulley combination that allows for extra safety and smoothness of operation. The end points are fixed near the pivot points to help minimize unwanted movement/torque. The extension movement of the upper and lower arm are assisted by gravity and a counter spring. The shoulder and upper arm movement are held in a neutral position with a spring system for counter balance allowing small movements within the glovebox. This initial concept considers only the vertical movement of the arm. The abduction and adduction movement will not be assisted or impeded by the over shoulder plate or at the forearm plate allowing for full range of motion. Several glovebox aspects such as the level of task assistance, ergonomic design, minimum hindrance in dexterity/operator movements and tear resistance for hazardous safety reasons will be considered in the design of the exoskeleton. Computer models will be simulated to test the load levels and stress conditions on the exoskeleton to enhance the designs. The exoskeleton focusses on major muscular involvement reduction through weight offset and active assistance with minimal parts and hence the device is derived with several joint links and contains both passive and active joints. The paper details the various stages in the design development and prototype testing of the exoskeleton device. Upon successful prototype device development, human testing will be conducted in a replica of the SRS-235F hot cell facility constructed at FIU-ARC. The final stage of the project will be potential testing/deployment of the exoskeleton device in one of the glovebox facilities in Savannah River Nuclear Waste site. (authors)

12 MANAGEMENT OF RADIOACTIVE AND NON-RADIOACTIVE W↗
DOE OSTI2025

Treyson Ricks - Intern Showcase Poster

Quinone-based sorbents offer a tunable, energy-efficient route to electrochemical CO2 capture, but systematic guidance for molecular design is lacking. Here, we report a high-throughput computational workflow that combines density functional theory (DFT) screening with machine-learning (ML) modeling to evaluate CO2 binding thermodynamics across several quinone derivatives, spanning benzoquinones, naphthoquinones, and anthraquinones. In addition to using solvents to stabilize the quinone anion and dianion, we studied the effect of ion-pairing on the reduction potentials and the CO2 binding energy. Automated Python scripts handled geometry optimizations and adduct-formation energies on an HPC cluster, reducing manual effort significantly. This integrated platform can uncover structure–property relationships and enables rapid in silico evaluation of untested candidates. We present one example from our workflow to showcase the capability of using quinones with ion-pairing to effectively capture CO2. Our approach paves the way for the rational selection of optimal quinone sorbents and can be extended with experimental thermochemical and kinetic data, alternative redox cycles, and stability assessments to accelerate development of next-generation electrochemical CO2 capture materials.

37 - INORGANIC, ORGANIC, PHYSICAL AND ANALYTICAL C↗
DOE OSTI2021

Synthesis and Characterization of Divalent Samarium and Thulium N , N -Dimethylaminodiboranates

Here, the syntheses and molecular structures of new Sm II and Tm II N,N-dimethylaminodiboranate (DMADB) complexes are described. Treating SmI 2 (THF) 2 with Na(H 3 BNMe 2 BH 3 ) in THF results in the formation of Sm(H 3 BNMe 2 BH 3 ) 2 (THF) 3 (1), which can be readily converted to Sm(H 3 BNMe 2 BH 3 ) 2 (DME) 2 (DME = 1,2-dimethoxyethane) or Sm(H 3 BNMe 2 BH 3 ) 2 (diglyme) by exchange with the corresponding ether. We also show that Sm(H 3 BNMe 2 BH 3 ) 2 (THF) 3 can be prepared by reduction of the SmIII compound Sm(H 3 BNMe 2 BH 3 ) 3 (THF) with KC 8 and that addition of 18-crown-6 to this reaction mixture results in the formation of the Sm II compound Sm(H 3 BNMe 2 BH 3 ) 2 (18-crown-6). In a similar fashion, two new Tm II complexes have been synthesized: treatment of TmI 2 in THF with Na(H 3 BNMe 2 BH 3 ) results in the formation of Tm(H 3 BNMe 2 BH 3 ) 2 (THF) 2 and Tm(H 3 BNMe 2 BH 3 ) 2 (THF) 3 , which form a cocrystal. IR data and elemental analyses are reported for all the new compounds, as are their crystal structures. 1 H and 11 B NMR data are provided where available.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Dinickel-Catalyzed N═N Bond Rotation

Azoarenes function as molecular switches that can be triggered by external stimuli, such as heat, light, and electrochemical potential. Here, in this work, we show that a dinickel catalyst can induce cis/trans isomerization in azoarenes through a N═N bond rotation mechanism. Catalytic intermediates containing azoarenes bound in both the cis and trans forms are characterized. Solid-state structures reveal the importance of π-back-bonding interactions from the dinickel active site in lowering the N═N bond order and accelerating bond rotation. The scope of the catalytic isomerization includes high-performance acyclic, cyclic, and polymeric azoarene switches.

adducts↗
DOE OSTI2025

Mechanisms and Energetics of CO 2 Chemisorption in Choline-Based Eutectic Solvents

Understanding the influences of proton sharing and hydrogen transfer in amine and hydroxyl functionalized eutectic solvents is important for tuning CO 2 binding as they relate to the chemisorption capacity and energetics of solvent regeneration in carbon capture. In this study, we examine how the CO 2 binding mechanism and energetics vary in eutectic solvents composed of choline-based hydrogen-bond acceptors (HBAs) with amine and hydroxyl moieties and ethylene glycol (EG), propylene glycol (PG), 1,2-butanediol (BD), and monoethanolamine (MEA)-type hydrogen-bond donors (HBDs), through absorption–desorption experiments and spectral analysis, supported with density functional theory (DFT) calculations. While the HBD-CO 2 complex is the major and the thermodynamically more favored product of absorption in all of the examined eutectic solvents, this complexation is only possible proceeding a proton transfer from HBD to the HBA in diol-based systems (EG, PG, BD). The interplay between HBA-CO 2 and HBD-CO 2 speciation is governed by the HBD concentration, basicity, and steric effects, with increased HBD-CO 2 interactions in the presence of smaller HBDs (size: BD > PG > EG). On the other hand, in MEA-based systems, CO 2 readily binds to the amine followed by a proton transfer from the amine to the HBA as confirmed by DFT calculations. In this way, the MEA-CO 2 chemisorption capacities exceeded that of the conventional amine (up to 0.93 mol of CO 2 per mol of MEA versus 0.5). Furthermore, the increase in CO 2 capacities and enhanced binding in eutectics including MEA or the more basic HBAs also resulted in regeneration temperatures of 70 to 60 °C with calculated reaction energies of −82 to −57 kJ/mol, compared to 50 °C for others with weaker binding, following CO 2 absorption at 25 °C.

absorption↗
DOE OSTI2020

Effects of Noncovalent Interactions on High-Spin Fe(IV)–Oxido Complexes

High-valent nonheme Fe IV –oxido species are key intermediates in biological oxidation, and their properties are proposed to be influenced by the unique microenvironments present in protein active sites. Microenvironments are regulated by noncovalent interactions, such as hydrogen bonds (H-bonds) and electrostatic interactions; however, there is little quantitative information about how these interactions affect crucial properties of high valent metal–oxido complexes. To address this knowledge gap, we introduced a series of Fe IV –oxido complexes that have the same S = 2 spin ground state as those found in nature and then systematically probed the effects of noncovalent interactions on their electronic, structural, and vibrational properties. The key design feature that provides access to these complexes is the new tripodal ligand [poat] 3– , which contains phosphinic amido groups. An important structural aspect of [Fe IV poat(O)] - is the inclusion of an auxiliary site capable of binding a Lewis acid (LA II ); we used this unique feature to further modulate the electrostatic environment around the Fe–oxido unit. Experimentally, studies confirmed that H-bonds and LA II s can interact directly with the oxido ligand in Fe IV –oxido complexes, which weakens the Fe=O bond and has an impact on the electronic structure. Additionally, we found that relatively large vibrational changes in the Fe–oxido unit correlate with small structural changes that could be difficult to measure, especially within a protein active site. Our work demonstrates the important role of noncovalent interactions on the properties of metal complexes, and that these interactions need to be considered when developing effective oxidants.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Structural Insights into the Mechanism of a Polyketide Synthase Thiocysteine Lyase Domain

Polyketide synthases (PKSs) are renowned for the structural diversity of the polyketide natural products they produce, but sulfur-containing functionalities are rarely installed by PKSs. We previously characterized thiocysteine lyase (SH) domains involved in the biosynthesis of the leinamycin (LNM) family of natural products, exemplified by LnmJ-SH and guangnanmycin (GnmT-SH). Here we report a detailed investigation into the PLP-dependent reaction catalyzed by the SH domains, guided by a 1.8 Å resolution crystal structure of GnmT-SH. A series of elaborate substrate mimics were synthesized to answer specific questions garnered from the crystal structure and from the biosynthetic logic of the LNM family of natural products. Here, through a combination of bioinformatics, molecular modeling, in vitro assays, and mutagenesis, we have developed a detailed model of acyl carrier protein (ACP)-tethered substrate-SH, and interdomain interactions, that contribute to the observed substrate specificity. Comparison of the GnmT-SH structure with archetypical PLP-dependent enzyme structures revealed how Nature, via evolution, has modified a common protein structural motif to accommodate an ACP-tethered substrate, which is significantly larger than any of those previously characterized. Overall, this study demonstrates how PLP-dependent chemistry can be incorporated into the context of PKS assembly lines and sets the stage for engineering PKSs to produce sulfur-containing polyketides.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Bidentate Lewis Base Ligand-Mediated Surface Stabilization and Modulation of the Electronic Structure of CsPbBr 3 Perovskite Nanocrystals

The desorption of conventional ligands from the surface of halide perovskite nanocrystals (NCs) often causes their structural instability and the deterioration of the optoelectronic properties. To address this challenge, we present an approach of using a bidentate Lewis base ligand, namely, 1,4-bis(diphenylphosphino)butane (DBPP), for the synthesis of CsPbBr 3 NCs. The phosphine group of DBPP has a strong interaction with PbBr 2 precursor, forming a highly crystalline intermediate complex during the reaction. In the presence of oleic acid, the uncoordinated phosphine group of DBPP is converted into the phosphonium cation, which strongly binds with the surface bromide of the formed CsPbBr 3 NCs through hydrogen bonding. Density functional theory calculations suggest that DBPP can strongly bind to the undercoordinated lead and surface bromide ions of CsPbBr 3 NCs through its unprotonated and protonated phosphine groups, respectively. The robust binding of DBPP with the surface of perovskite NCs helps to preserve their structural integrity under various environmental stresses. Moreover, the electron density and energy levels are regulated in DBPP-capped CsPbBr 3 NCs via the efficient transfer of electrons from the ligands to the NCs, resulting in their improved photocatalytic CO 2 reduction performance. Furthermore, our study highlights the potential of using bidentate ligands to stabilize the surface of perovskite NCs and modulate their optical and electronic properties.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Dimerization-dependent serine protease activity of FAM111A prevents replication fork stalling at topoisomerase 1 cleavage complexes

FAM111A, a serine protease, plays roles in DNA replication and antiviral defense. Missense mutations in the catalytic domain cause hyper-autocleavage and are associated with genetic disorders with developmental defects. Despite the enzyme’s biological significance, the molecular architecture of the FAM111A serine protease domain (SPD) is unknown. Here, we show that FAM111A is a dimerization-dependent protease containing a narrow, recessed active site that cleaves substrates with a chymotrypsin-like specificity. X-ray crystal structures and mutagenesis studies reveal that FAM111A dimerizes via the N-terminal helix within the SPD. This dimerization induces an activation cascade from the dimerization sensor loop to the oxyanion hole through disorder-to-order transitions. Dimerization is essential for proteolytic activity in vitro and for facilitating DNA replication at DNA-protein crosslink obstacles in cells, while it is dispensable for autocleavage. These findings underscore the role of dimerization in FAM111A’s function and highlight the distinction in its dimerization dependency between substrate cleavage and autocleavage.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Watson-Crick Base-Pairing Requirements for ssDNA Recognition and Processing in Replication-Initiating HUH Endonucleases

Replication-initiating HUH endonucleases (Reps) are sequence-specific nucleases that cleave and rejoin single-stranded DNA (ssDNA) during rolling-circle replication. These functions are mediated by covalent linkage of the Rep to its substrate post cleavage. Here, we describe the structures of the endonuclease domain from the Muscovy duck circovirus Rep in complex with its cognate ssDNA 10-mer with and without manganese in the active site. Structural and functional analyses demonstrate that divalent cations play both catalytic and structural roles in Reps by polarizing and positioning their substrate. Further structural comparisons highlight the importance of an intramolecular substrate Watson-Crick (WC) base pairing between the -4 and +1 positions. Subsequent kinetic and functional analyses demonstrate a functional dependency on WC base pairing between these positions regardless of the pair’s identity (i.e., A·T, T·A, G·C, or C·G), highlighting a structural specificity for substrate interaction. Finally, considering how well WC swaps were tolerated in vitro, we sought to determine to what extent the canonical -4T·+1A pairing is conserved in circular Rep-encoding single-stranded DNA viruses and found evidence of noncanonical pairings in a minority of these genomes. Altogether, our data suggest that substrate intramolecular WC base pairing is a universal requirement for separation and reunion of ssDNA in Reps.

59 BASIC BIOLOGICAL SCIENCES↗