Engineering Papers⌕ Search

SEARCH · Engineering Papers

Results for “DNA REPLICATION”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 397 records · Page 22
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Evaluation of the Deletion of the African Swine Fever Virus Gene O174L from the Genome of the Georgia Isolate

African swine fever virus (ASFV) is a structurally complex, double-stranded DNA virus, which causes African swine fever (ASF), a contagious disease affecting swine. ASF is currently affecting pork production in a large geographical region, including Eurasia and the Caribbean. ASFV has a large genome, which harbors more than 160 genes, but most of these genes’ functions have not been experimentally characterized. One of these genes is the O174L gene which has been experimentally shown to function as a small DNA polymerase. Here, we demonstrate that the deletion of the O174L gene from the genome of the virulent strain ASFV Georgia2010 (ASFV-G) does not significantly affect virus replication in vitro or in vivo. A recombinant virus, having deleted the O174L gene, ASFV-G-ΔO174L, was developed to study the effect of the O174L protein in replication in swine macrophages cultures in vitro and disease production when inoculated in pigs. The results demonstrated that ASFV-G-ΔO174L has similar replication kinetics to parental ASFV-G in swine macrophage cultures. In addition, animals intramuscularly inoculated with 10 2 HAD 50 of ASFV-G-ΔO174L presented a clinical form of the disease that is indistinguishable from that induced by the parental virulent strain ASFV-G. All animals developed a lethal disease, being euthanized around day 7 post-infection. Therefore, although O174L is a well-characterized DNA polymerase, its function is apparently not critical for the process of virus replication, both in vitro and in vivo, or for disease production in domestic pigs.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1974

The prebiotic synthesis of oligonucleotides

This paper is primarily a review of recent developments in the abiotic synthesis of nucleotides, short chain oligonucleotides, and their mode of replication in solution. It also presents preliminary results from this laboratory on the prebiotic synthesis of thymidine oligodeoxynucleotides. A discussion, based on the physicochemical properties of RNA and DNA oligomers, relevant to the molecular evolution of these compounds leads to the tentative hypothesis that oligodeoxyribonucleotides of about 12 units may have been of sufficient length to initiate a self replicating coding system. Two models are suggested to account for the synthesis of high molecular weight oligomers using short chain templates and primers.

Oro, J.↗
DOE OSTI2024

Identification and characterization of small molecule inhibitors of the LINE-1 retrotransposon endonuclease

The long interspersed nuclear element-1 (LINE-1 or L1) retrotransposon is the only active autonomously replicating retrotransposon in the human genome. L1 harms the cell by inserting new copies, generating DNA damage, and triggering inflammation. Therefore, L1 inhibition could be used to treat many diseases associated with these processes. Previous research has focused on inhibition of the L1 reverse transcriptase due to the prevalence of well-characterized inhibitors of related viral enzymes. Here we present the L1 endonuclease as another target for reducing L1 activity. We characterize structurally diverse small molecule endonuclease inhibitors using computational, biochemical, and biophysical methods. We also show that these inhibitors reduce L1 retrotransposition, L1-induced DNA damage, and inflammation reinforced by L1 in senescent cells. These inhibitors could be used for further pharmacological development and as tools to better understand the life cycle of this element and its impact on disease processes.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

The DnaK Chaperone Uses Different Mechanisms To Promote and Inhibit Replication of Vibrio cholerae Chromosome 2

Replication of Vibrio cholerae chromosome 2 (Chr2) depends on molecular chaperone DnaK to facilitate binding of the initiator (RctB) to the replication origin. The binding occurs at two kinds of site, 12-mers and 39-mers, which promote and inhibit replication, respectively. Here we show that DnaK employs different mechanisms to enhance the two kinds of binding. We found that mutations in rctB that reduce DnaK binding also reduce 12-mer binding and initiation. The initiation defect is suppressed by second-site mutations that increase 12-mer binding only marginally. Instead, they reduce replication inhibitory mechanisms: RctB dimerization and 39-mer binding. One suppressing change was in a dimerization domain which is folded similarly to the initiator of an iteron plasmid—the presumed progenitor of Chr2. In plasmids, DnaK promotes initiation by reducing dimerization. A different mutation was in the 39-mer binding domain of RctB and inactivated it, indicating an alternative suppression mechanism. Paradoxically, although DnaK increases 39-mer binding, the increase was also achieved by inactivating the DnaK binding site of RctB. This result suggests that the site inhibits the 39-mer binding domain (via autoinhibition) when prevented from binding DnaK. Taken together, our results reveal an important feature of the transition from plasmid to chromosome: the Chr2 initiator retains the plasmid-like dimerization domain and its control by chaperones but uses the chaperones in an unprecedented way to control the inhibitory 39-mer binding.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

The Nucleoid-Associated Protein GapR Uses Conserved Structural Elements To Oligomerize and Bind DNA

Nucleoid-associated proteins (NAPs) are DNA binding proteins critical for the organization and function of the bacterial chromosome. A newly discovered NAP in Caulobacter crescentus, GapR, is thought to facilitate the movement of the replication and transcription machines along the chromosome by stimulating type II topoisomerases to remove positive supercoiling. Here, utilizing genetic, biochemical, and biophysical studies of GapR in light of a recently published DNA-bound crystal structure of GapR, we identified the structural elements involved in oligomerization and DNA binding. Moreover, we show that GapR is maintained as a tetramer upon its dissociation from DNA and that tetrameric GapR is capable of binding DNA molecules in vitro. Analysis of protein chimeras revealed that two helices of GapR are functionally conserved in H-NS, demonstrating that two evolutionarily distant NAPs with distinct mechanisms of action utilize conserved structural elements to oligomerize and bind DNA.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

CUT&RUN identifies centromeric DNA regions of Rhodotorula toruloides IFO0880

ABSTRACT Rhodotorula toruloides has been increasingly explored as a host for bioproduction of lipids, fatty acid derivatives and terpenoids. Various genetic tools have been developed, but neither a centromere nor an autonomously replicating sequence (ARS), both necessary elements for stable episomal plasmid maintenance, has yet been reported. In this study, cleavage under targets and release using nuclease (CUT&RUN), a method used for genome-wide mapping of DNA–protein interactions, was used to identify R. toruloides IFO0880 genomic regions associated with the centromeric histone H3 protein Cse4, a marker of centromeric DNA. Fifteen putative centromeres ranging from 8 to 19 kb in length were identified and analyzed, and four were tested for, but did not show, ARS activity. These centromeric sequences contained below average GC content, corresponded to transcriptional cold spots, were primarily nonrepetitive and shared some vestigial transposon-related sequences but otherwise did not show significant sequence conservation. Future efforts to identify an ARS in this yeast can utilize these centromeric DNA sequences to improve the stability of episomal plasmids derived from putative ARS elements.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

An Innovative High Throughput Genome Releaser for Rapid and Efficient PCR Screening

High-throughput PCR screening is vital in synthetic biology and metabolic engineering as it allows researchers to rapidly analyze and detect numerous targeted genetic mutation in the genome. Current challenges for high-throughput PCR screening in synthetic biology include efficiently preparing genomic DNA, optimizing protocols for diverse sample types, managing contamination risks, and effectively analyzing the large volumes of data generated while ensuring consistent and accurate results. In this study, we present the development of a High Throughput Genome Releaser (HTGR), an innovative device addressing common challenges in screening PCR. This genome DNA releaser is designed based on a squash method for rapid, cost-effective, and efficient DNA release, optimized for subsequent PCR reactions. After experimenting with various synthetic materials, we selected a plastic that closely replicates the smooth surface and compression properties of microscope slides, ensuring reliable performance. We engineered a device featuring a 96-Well Plate and a shear applicator, operable both manually and automatically, and compatible with standard liquid-handling robot platform. This compatibility enhances ease of use in high-throughput PCR workflows. Additionally, we developed software to support its automatic functions. Our results demonstrated that the specially engineered 96-Well Plate and HTGR can effectively squash fungal spores , which release enough genome DNA for PCR screening. The genome releaser facilitates the preparation of PCR-amplifiable genomic DNA substrate from 96 samples within minutes, eliminates the need for extraction buffers, and is adaptable to a wide range of microorganisms and cells, which could significantly advance biomanufacturing processes.

Yuan, Guoliang [BATTELLE (PACIFIC NW LAB)]↗
DOE OSTI2019

Experimental Evolution of Extreme Resistance to Ionizing Radiation in Escherichia coli after 50 Cycles of Selection

In prior work (D. R. Harris et al., J Bacteriol 191:5240–5252, 2009, https://doi.org/10.1128/JB.00502-09; B. T. Byrne et al., Elife 3:e01322, 2014, https://doi.org/10.7554/eLife.01322), we demonstrated that Escherichia coli could acquire substantial levels of resistance to ionizing radiation (IR) via directed evolution. Major phenotypic contributions involved adaptation of organic systems for DNA repair. We have currently undertaken an extended effort to generate E. coli populations that are as resistant to IR as Deinococcus radiodurans . After an initial 50 cycles of selection using high-energy electron beam IR, four replicate populations exhibit major increases in IR resistance but have not yet reached IR resistance equivalent to D. radiodurans . Regular deep sequencing reveals complex evolutionary patterns with abundant clonal interference. Prominent IR resistance mechanisms involve novel adaptations to DNA repair systems and alterations in RNA polymerase. Adaptation is highly specialized to resist IR exposure, since isolates from the evolved populations exhibit highly variable patterns of resistance to other forms of DNA damage. Sequenced isolates from the populations possess between 184 and 280 mutations. IR resistance in one isolate, IR9-50-1, is derived largely from four novel mutations affecting DNA and RNA metabolism: RecD A90E, RecN K429Q, and RpoB S72N/RpoC K1172I. Additional mechanisms of IR resistance are evident. IMPORTANCE Some bacterial species exhibit astonishing resistance to ionizing radiation, with Deinococcus radiodurans being the archetype. As natural IR sources rarely exceed mGy levels, the capacity of Deinococcus to survive 5,000 Gy has been attributed to desiccation resistance. To understand the molecular basis of true extreme IR resistance, we are using experimental evolution to generate strains of Escherichia coli with IR resistance levels comparable to Deinococcus . Experimental evolution has previously generated moderate radioresistance for multiple bacterial species. Yet, these efforts could not take advantage of modern genomic sequencing technologies. In this report, we examine four replicate bacterial populations after 50 selection cycles. Genomic sequencing allows us to follow the genesis of mutations in populations throughout selection. Novel mutations affecting genes encoding DNA repair proteins and RNA polymerase enhance radioresistance. However, more contributors are apparent.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Mechanism of Error-Free Bypass of the Environmental Carcinogen N -(2'-Deoxyguanosin-8-yl)-3-aminobenzanthrone Adduct by Human DNA Polymerase η

The environmental pollutant 3-nitrobenzanthrone produces bulky aminobenzanthrone (ABA) DNA adducts with both guanine and adenine nucleobases. A major product occurs at the C8 position of guanine (C8-dG-ABA). These adducts present a strong block to replicative polymerases but, remarkably, can be bypassed in a largely error-free manner by the human Y-family polymerase η (hPol η). Here, we report the crystal structure of a ternary Pol•DNA•dCTP complex between a C8-dG-ABA-containing template:primer duplex and hPol η. The complex was captured at the insertion stage and provides crucial insight into the mechanism of error-free bypass of this bulky lesion. Specifically, bypass involves accommodation of the ABA moiety inside a hydrophobic cleft to the side of the enzyme active site and formation of an intra-nucleotide hydrogen bond between the phosphate and ABA amino moiety, allowing the adducted guanine to form a standard Watson–Crick pair with the incoming dCTP.

Patra, Amritraj↗
DOE OSTI2024

A compact stem-loop DNA aptamer targets a uracil-binding pocket in the SARS-CoV-2 nucleocapsid RNA-binding domain

Abstract SARS-CoV-2 nucleocapsid (N) protein is a structural component of the virus with essential roles in the replication and packaging of the viral RNA genome. The N protein is also an important target of COVID-19 antigen tests and a promising vaccine candidate along with the spike protein. Here, we report a compact stem-loop DNA aptamer that binds tightly to the N-terminal RNA-binding domain of SARS-CoV-2 N protein. Crystallographic analysis shows that a hexanucleotide DNA motif (5′-TCGGAT-3′) of the aptamer fits into a positively charged concave surface of N-NTD and engages essential RNA-binding residues including Tyr109, which mediates a sequence-specific interaction in a uracil-binding pocket. Avid binding of the DNA aptamer allows isolation and sensitive detection of full-length N protein from crude cell lysates, demonstrating its selectivity and utility in biochemical applications. We further designed a chemically modified DNA aptamer and used it as a probe to examine the interaction of N-NTD with various RNA motifs, which revealed a strong preference for uridine-rich sequences. Our studies provide a high-affinity chemical probe for the SARS-CoV-2 N protein RNA-binding domain, which may be useful for diagnostic applications and investigating novel antiviral agents.

Biochemistry & Molecular Biology↗
DOE OSTI2026

Extracellular DNA Alters Detection of Subtle Bacterial Responses to Soil Rewetting

Microbial communities are often characterized using DNA-based sequencing, but these approaches also capture extracellular DNA (exDNA) released from dead cells, potentially altering inference about microbial responses to environmental change. This may be especially important during pulse disturbances, such as soil drying–rewetting, which can increase microbial mortality and transient necromass pools. We assessed whether exDNA altered inference about bacterial responses to drying–rewetting (an 80 mm simulated rainfall event following a 28-day drought) in conventionally tilled corn and perennial switchgrass soils. We quantified bacterial abundance (16 S rRNA gene copies), alpha diversity, and community composition in paired soil samples with exDNA included (+ exDNA) and in samples treated with propidium monoazide (PMAxx) to reduce amplification of exDNA (− exDNA). At our level of replication (n = 4), PMAxx treatment did not significantly alter overall temporal response patterns (i.e., no significant main effect of DNA treatment or DNA × time interaction). However, PMAxx treatment increased sensitivity to detect some pairwise temporal changes in bacterial abundance and community composition in corn soils following rewetting. exDNA pools were proportionally highest immediately after rewetting in corn soils, suggesting transient extracellular DNA may contribute to masking during disturbance recovery. In contrast, PMAxx treatment had comparatively small effects in switchgrass soils, which exhibited weaker temporal responses overall. Inclusion of exDNA also changed which taxa appeared most responsive to rewetting. Together, our results suggest that exDNA does not uniformly bias soil microbial inference, but may reduce detectability of subtle disturbance-driven shifts in certain soils. Future studies should advance knowledge of microbial turnover and necromass dynamics, particularly using multiple complementary methods, to help predict when exDNA is most likely to influence ecological inference.

drying-rewetting↗
NASA NTRS1998

Formation of oligonucleotide-PNA-chimeras by template-directed ligation

DNA sequences have previously been reported to act as templates for the synthesis of PNA, and vice versa. A continuous evolutionary transition from an informational replicating system based on one polymer to a system based on the other would be facilitated if it were possible to form chimeras, that is molecules that contain monomers of both types. Here we show that ligation to form chimeras proceeds efficiently both on PNA and on DNA templates. The efficiency of ligation is primarily determined by the number of backbone bonds at the ligation site and the relative orientation of template and substrate strands. The most efficient reactions result in the formation of chimeras with ligation junctions resembling the structures of the backbones of PNA and DNA and with antiparallel alignment of both components of the chimera with the template, that is, ligations involving formation of 3'-phosphoramidate and 5'-ester bonds. However, double helices involving PNA are stable both with antiparallel and parallel orientation of the two strands. Ligation on PNA but not on DNA templates is, therefore, sometimes possible on templates with reversed orientation. The relevance of these findings to discussions of possible transitions between genetic systems is discussed.

NASA Discipline Exobiology↗
DOE OSTI2021

Moisture modulates soil reservoirs of active DNA and RNA viruses

Abstract Soil is known to harbor viruses, but the majority are uncharacterized and their responses to environmental changes are unknown. Here, we used a multi-omics approach (metagenomics, metatranscriptomics and metaproteomics) to detect active DNA viruses and RNA viruses in a native prairie soil and to determine their responses to extremes in soil moisture. The majority of transcribed DNA viruses were bacteriophage, but some were assigned to eukaryotic hosts, mainly insects. We also demonstrated that higher soil moisture increased transcription of a subset of DNA viruses. Metaproteome data validated that the specific viral transcripts were translated into proteins, including chaperonins known to be essential for virion replication and assembly. The soil viral chaperonins were phylogenetically distinct from previously described marine viral chaperonins. The soil also had a high abundance of RNA viruses, with highest representation of Reoviridae . Leviviridae were the most diverse RNA viruses in the samples, with higher amounts in wet soil. This study demonstrates that extreme shifts in soil moisture have dramatic impacts on the composition, activity and potential functions of both DNA and RNA soil viruses.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

A comparison of cost and quality of three methods for estimating density for wild pig (Sus scrofa)

A critical element in efective wildlife management is monitoring the status of wildlife populations; however, resources to monitor wildlife populations are typically limited. We compared cost efectiveness of three common population estimation methods (i.e. non-invasive DNA sampling, camera sampling, and sampling from trapping) by applying them to wild pigs (Sus scrofa) across three habitats in South Carolina, U.S.A where they are invasive. We used mark-recapture analyses for fecal DNA sampling data, spatially-explicit capture-recapture analyses for camera sampling data, and a removal analysis for removal sampling from trap data. Density estimates were similar across methods. Camera sampling was the least expensive, but had large variances. Fecal DNA sampling was the most expensive, although this technique generally performed well. We examined how reductions in efort by method related to increases in relative bias or imprecision. For removal sampling, the largest cost savings while maintaining unbiased density estimates was from reducing the number of traps. For fecal DNA sampling, a reduction in efort only minimally reduced costs due to the need for increased lab replicates while maintaining high quality estimates. For camera sampling, efort could only be marginally reduced before inducing bias. We provide a decision tree for researchers to help make monitoring decisions.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2026

Next-Generation Sequencing Data from a CUT&RUN Study of R. toruloides IFO0880 Cse4 and Orc1 Binding Sites

Rhodotorula toruloides has been increasingly explored as a host for bioproduction of lipids, fatty acid derivatives and terpenoids. Various genetic tools have been developed, but neither a centromere nor an autonomously replicating sequence (ARS), both necessary elements for stable episomal plasmid maintenance, has yet been reported. In this study, cleavage under targets and release using nuclease (CUT&RUN), a method used for genome-wide mapping of DNA–protein interactions, was used to identify R. toruloides IFO0880 genomic regions associated with the centromeric histone H3 protein Cse4, a marker of centromeric DNA. Fifteen putative centromeres ranging from 8 to 19 kb in length were identified and analyzed, and four were tested for, but did not show, ARS activity. These centromeric sequences contained below average GC content, corresponded to transcriptional cold spots, were primarily nonrepetitive and shared some vestigial transposon-related sequences but otherwise did not show significant sequence conservation. Future efforts to identify an ARS in this yeast can utilize these centromeric DNA sequences to improve the stability of episomal plasmids derived from putative ARS elements.

Genome Engineering↗
DOE OSTI2014

Crystal structure of a Fanconi anemia-associated nuclease homolog bound to 5' flap DNA: basis of interstrand cross-link repair by FAN1

Fanconi anemia (FA) is an autosomal recessive genetic disorder caused by defects in any of 15 FA genes responsible for processing DNA interstrand cross-links (ICLs). The ultimate outcome of the FA pathway is resolution of cross-links, which requires structure-selective nucleases. FA-associated nuclease 1 (FAN1) is believed to be recruited to lesions by a monoubiquitinated FANCI–FANCD2 (ID) complex and participates in ICL repair. Here, we determined the crystal structure of Pseudomonas aeruginosa FAN1 (PaFAN1) lacking the UBZ (ubiquitin-binding zinc) domain in complex with 5' flap DNA. All four domains of the right-hand-shaped PaFAN1 are involved in DNA recognition, with each domain playing a specific role in bending DNA at the nick. The six-helix bundle that binds the junction connects to the catalytic viral replication and repair (VRR) nuclease (VRR nuc) domain, enabling FAN1 to incise the scissile phosphate a few bases distant from the junction. The six-helix bundle also inhibits the cleavage of intact Holliday junctions. PaFAN1 shares several conserved features with other flap structure-selective nucleases despite structural differences. A clamping motion of the domains around the wedge helix, which acts as a pivot, facilitates nucleolytic cleavage. The PaFAN1 structure provides insights into how archaeal Holliday junction resolvases evolved to incise 5' flap substrates and how FAN1 integrates with the FA complex to participate in ICL repair.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

The mobility of packaged phage genome controls ejection dynamics

The cell decision between lytic and lysogenic infection is strongly influenced by dynamics of DNA injection into a cell from a phage population, as phages compete for limited resources and progeny. However, what controls the timing of viral DNA ejection events was not understood. This in vitro study reveals that DNA ejection dynamics for phages can be synchronized (occurring within seconds) or desynchronized (displaying minutes-long delays in initiation) based on mobility of encapsidated DNA, which in turn is regulated by environmental factors, such as temperature and extra-cellular ionic conditions. This mechano-regulation of ejection dynamics is suggested to influence viral replication where the cell’s decision between lytic and latent infection is associated with synchronized or desynchronized delayed ejection events from phage population adsorbed to a cell. Our findings are of significant importance for understanding regulatory mechanisms of latency in phage and Herpesviruses, where encapsidated DNA undergoes a similar mechanical transition.

Evilevitch, Alex↗