Search NASA⌕ Search

SEARCH · Search NASA

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 73 records · Page 4
All sourcesNASA NTRSDOE OSTI
NASA NTRS1986

Thioredoxins in evolutionarily primitive organisms

Thioredoxins are low molecular weight redox proteins, alternating between the S-S (oxidized) and SH (reduced) states, that function in a number of biochemical processes, including DNA synthesis, DNA replication, and enzyme regulation. Until recently, reduced ferredoxin was known to serve as the source of reducing power for the reduction of thioredoxins only in oxygenic photosynthetic cells. In all other organisms, the source of hydrogen (electrons) for thioredoxin reduction was considered to be NADPH. It was found that Clostridium pasteurianum, an anaerobic organism normally living in the soil unexposed to light, resembles photosynthetic cells in using ferredoxin for the reduction of thioredoxin. The results reveal the existence of a pathway in which ferredoxin, provides the reducing power for the reduction of thioredoxin via the flavoprotein enzyme, ferredoxinthioredoxin reductase. In related studies, it was found that Chromatium vinosum, an anaerobic photosynthetic purple sulfur bacterium, resembles evolutionarily more advanced micro-organisms in having an NADP-thioredoxin system composed of a single thioredoxin which is reduced by NADPH via NADP-thioredoxin reductase. The adoption of the NADP-thioredoxin system by Chromatium seems appropriate in view of evidence tha the organi sm utilizes ATP-driven reverse electron transport. Finally, results of research directed towards the identification of target enzymes of the ferredoxin/thioredoxin system in a cyanobacterium (Nostoc muscorum), show that thioredoxin-linked photosynthetic enzymes of cyanobateria are similar to those of chloroplasts. It now seems that the ferredoxin/thioredoxin system functions in regulating CO2 assimilation via the reductive pentose phosphate cycle in oxygenic but not anoxygenic photosynthetic cells.

Buchanan, B. B.↗
NASA NTRS1990

Polyamines in plant physiology

The diamine putrescine, the triamine spermidine, and the tetramine spermine are ubiquitous in plant cells, while other polyamines are of more limited occurrence. Their chemistry and pathways of biosynthesis and metabolism are well characterized. They occur in the free form as cations, but are often conjugated to small molecules like phenolic acids and also to various macromolecules. Their titer varies from approximately micromolar to more than millimolar, and depends greatly on environmental conditions, especially stress. In cereals, the activity of one of the major polyamine biosynthetic enzymes, arginine decarboxylase, is rapidly and dramatically increased by almost every studied external stress, leading to 50-fold or greater increases in putrescine titer within a few hours. The physiological significance of this increase is not yet clear, although most recent work suggests an adaptive, protective role. Polyamines produced through the action of ornithine decarboxylase, by contrast, seem essential for DNA replication and cell division. The application of exogenous polyamines produces effects on patterns of senescence and morphogenesis, suggesting but not proving a regulatory role for polyamines in these processes. The evidence for such a regulatory role is growing.

Review↗
NASA NTRS1997

Calbindin and parvalbumin are early markers of non-mitotically regenerating hair cells in the bullfrog vestibular otolith organs

Earlier studies have demonstrated hair cell regeneration in the absence of cell proliferation, and suggested that supporting cells could phenotypically convert into hair cells following hair cell loss. Because calcium-binding proteins are involved in gene up-regulation, cell growth, and cell differentiation, we wished to determine if these proteins were up-regulated in scar formations and regenerating hair cells following gentamicin treatment. Calbindin and parvalbumin immunolabeling was examined in control or gentamicin-treated (GT) bullfrog saccular and utricular explants cultured for 3 days in amphibian culture medium or amphibian culture medium supplemented with aphidicolin, a blocker of nuclear DNA replication in eukaryotic cells. In control cultures, calbindin and parvalbumin immunolabeled the hair bundles and, less intensely, the cell bodies of mature hair cells. In GT or mitotically-blocked GT (MBGT) cultures, calbindin and parvalbumin immunolabeling was also seen in the hair bundles, cuticular plates, and cell bodies of hair cells with immature hair bundles. Thus, these antigens were useful markers for both normal and regenerating hair cells. Supporting cell immunolabeling was not seen in control cultures nor in the majority of supporting cells in GT cultures. In MBGT cultures, calbindin and parvalbumin immunolabeling was up-regulated in the cytosol of single supporting cells participating in scar formations and in supporting cells with hair cell-like characteristics. These data provide further evidence that non-mitotic hair cell regeneration in cultures can be accomplished by the conversion of supporting cells into hair cells.

Non-NASA Center↗
NASA NTRS1994

Radiation-induced genomic instability

Quantitative assessment of the heritable somatic effects of ionizing radiation exposures has relied upon the assumption that radiation-induced lesions were 'fixed' in the DNA prior to the first postirradiation mitosis. Lesion conversion was thought to occur during the initial round of DNA replication or as a consequence of error-prone enzymatic processing of lesions. The standard experimental protocols for the assessment of a variety of radiation-induced endpoints (cell death, specific locus mutations, neoplastic transformation and chromosome aberrations) evaluate these various endpoints at a single snapshot in time. In contrast with the aforementioned approaches, some studies have specifically assessed radiation effects as a function of time following exposure. Evidence has accumulated in support of the hypothesis that radiation exposure induces a persistent destabilization of the genome. This instability has been observed as a delayed expression of lethal mutations, as an enhanced rate of accumulation of non-lethal heritable alterations, and as a progressive intraclonal chromosomal heterogeneity. The genetic controls and biochemical mechanisms underlying radiation-induced genomic instability have not yet been delineated. The aim is to integrate the accumulated evidence that suggests that radiation exposure has a persistent effect on the stability of the mammalian genome.

NASA Discipline Radiation Health↗
NASA NTRS2012

Adaptation of Organisms by Resonance of RNA Transcription with the Cellular Redox Cycle

Sequence variation in organisms differs across the genome and the majority of mutations are caused by oxidation, yet its origin is not fully understood. It has also been shown that the reduction-oxidation reaction cycle is the fundamental biochemical cycle that coordinates the timing of all biochemical processes in that cell, including energy production, DNA replication, and RNA transcription. It is shown that the temporal resonance of transcriptome biosynthesis with the oscillating binary state of the reduction-oxidation reaction cycle serves as a basis for non-random sequence variation at specific genome-wide coordinates that change faster than by accumulation of chance mutations. This work demonstrates evidence for a universal, persistent and iterative feedback mechanism between the environment and heredity, whereby acquired variation between cell divisions can outweigh inherited variation.

Stolc, Viktor↗
NASA NTRS2016

Electromagnetic Basis of Metabolism and Heredity

Living organisms control their cellular biological clocks to maintain functional oscillation of the redox cycle, also called the "metabolic cycle" or "respiratory cycle". Organization of cellular processes requires parallel processing on a synchronized time-base. These clocks coordinate the timing of all biochemical processes in the cell, including energy production, DNA replication, and RNA transcription. When this universal time keeping function is perturbed by exogenous induction of reactive oxygen species (ROS), the rate of metabolism changes. This causes oxidative stress, aging and mutations. Therefore, good temporal coordination of the redox cycle not only actively prevents chemical conflict between the reductive and oxidative partial reactions; it also maintains genome integrity and lifespan. Moreover, this universal biochemical rhythm can be disrupted by ROS induction in vivo. This in turn can be achieved by blocking the electron transport chain either endogenously or exogenously by various metabolites, e.g. hydrogen sulfide (H2S), highly diffusible drugs, and carbon monoxide (CO). Alternatively, the electron transport in vivo can be attenuated via a coherent or interfering transfer of energy from exogenous ultralow frequency (ULF) and extremely low frequency (ELF) electromagnetic (EM) fields, suggesting that-on Earth-such ambient fields are an omnipresent (and probably crucially important) factor for the time-setting basis of universal biochemical reactions in living cells. Our work demonstrated previously un-described evidence for quantum effects in biology by electromagnetic coupling below thermal noise at the universal electron transport chain (ETC) in vivo.

Electromagnetic↗
NASA NTRS2015

Induction of Oxidation in Living Cells by Time-Varying Electromagnetic Fields

We are studying how biological systems can harness quantum effects of time varying electromagnetic (EM) waves as the time-setting basis for universal biochemical organization via the redox cycle. The effects of extremely weak EM field on the biochemical redox cycle can be monitored through real-time detection of oxidation-induced light emissions of reporter molecules in living cells. It has been shown that EM fields can also induce changes in fluid transport rates through capillaries (approximately 300 microns inner diameter) by generating annular proton gradients. This effect may be relevant to understanding cardiovascular dis-function in spaceflight, beyond the ionosphere. Importantly, we show that these EM effects can be attenuated using an active EM field cancellation device. Central for NASA's Human Research Program is the fact that the absence of ambient EM field in spaceflight can also have a detrimental influence, namely via increased oxidative damage, on DNA replication, which controls heredity.

oxidation↗
DOE OSTI2026

EEPD1 evolved a unique DNA clamping dimer protecting reversed replication forks

Exonuclease/endonuclease/phosphatase (EEP)-fold hydrolases are canonically monomeric phosphodiesterases exemplified by APE1, DNase I, and TDP2 nucleases. While EEP family domain containing protein 1 (EEPD1) acts in DNA stress responses, its proposed nuclease activities are enigmatic. Here, we integrate hybrid structural methods, evolution, biochemistry, cancer genomics, plus molecular and cell biology to define EEPD1 structure, assembly, and function at stalled DNA replication forks. Results imply EEPD1 surprisingly requires both unique EEP domain dimer and distinctive tandem Helix-hairpin-Helix [(HhH) 2 ] domains to clamp double-stranded (ds) DNA at reversed DNA replication forks for fork protection. Small-angle X-ray Scattering (SAXS), crystal, and cryo-EM structures unveil an unprecedented tryptophan handshake dimer, conserved interface di-Trp-Pro pocket, and adjustable “wrist” enabling an open-closed conformational switch. EEPD1 dimer cooperatively binds complex dsDNA replication fork intermediates but alone lacks nuclease activity due to loss of key EEP catalytic residues during Metazoan evolution and atmospheric oxygen buildup. Instead, EEPD1 prevents nucleolytic degradation of reversed replication forks by MRE11. Furthermore, cancer bioinformatics support oxidative damage-dependent EEPD1 association as a significant modulator of overall patient survival. Collective findings uncover unexpected EEP dimer and fork protection function in clamping, not cleaving, reversed replication forks for metazoan oxidative stress responses controlling genome stability and cancer outcomes.

Shen, Runze [Univ. of Texas, Houston, TX (United S↗
DOE OSTI2021

Decoding Cancer Variants of Unknown Significance for Helicase–Nuclease–RPA Complexes Orchestrating DNA Repair During Transcription and Replication

All tumors have DNA mutations, and a predictive understanding of those mutations could inform clinical treatments. However, 40% of the mutations are variants of unknown significance (VUS), with the challenge being to objectively predict whether a VUS is pathogenic and supports the tumor or whether it is benign. To objectively decode VUS, we mapped cancer sequence data and evolutionary trace (ET) scores onto crystallography and cryo-electron microscopy structures with variant impacts quantitated by evolutionary action (EA) measures. As tumors depend on helicases and nucleases to deal with transcription/replication stress, we targeted helicase–nuclease–RPA complexes: (1) XPB-XPD (within TFIIH), XPF-ERCC1, XPG, and RPA for transcription and nucleotide excision repair pathways and (2) BLM, EXO5, and RPA plus DNA2 for stalled replication fork restart. As validation, EA scoring predicts severe effects for most disease mutations, but disease mutants with low ET scores not only are likely destabilizing but also disrupt sophisticated allosteric mechanisms. For sites of disease mutations and VUS predicted to be severe, we found strong co-localization to ordered regions. Rare discrepancies highlighted the different survival requirements between disease and tumor mutations, as well as the value of examining proteins within complexes. In a genome-wide analysis of 33 cancer types, we found correlation between the number of mutations in each tumor and which pathways or functional processes in which the mutations occur, revealing different mutagenic routes to tumorigenesis. We also found upregulation of ancient genes including BLM, which supports a non-random and concerted cancer process: reversion to a unicellular, proliferation-uncontrolled, status by breaking multicellular constraints on cell division. Together, these genes and global analyses challenge the binary “driver” and “passenger” mutation paradigm, support a gradient impact as revealed by EA scoring from moderate to severe at a single gene level, and indicate reduced regulation as well as activity. The objective quantitative assessment of VUS scoring and gene overexpression in the context of functional interactions and pathways provides insights for biology, oncology, and precision medicine.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

In crystallo observation of three metal ion promoted DNA polymerase misincorporation

Error-free replication of DNA is essential for life. Despite the proofreading capability of several polymerases, intrinsic polymerase fidelity is in general much higher than what base-pairing energies can provide. Although researchers have investigated this long-standing question with kinetics, structural determination, and computational simulations, the structural factors that dictate polymerase fidelity are not fully resolved. Time-resolved crystallography has elucidated correct nucleotide incorporation and established a three-metal-ion-dependent catalytic mechanism for polymerases. Using X-ray time-resolved crystallography, we visualize the complete DNA misincorporation process catalyzed by DNA polymerase η. The resulting molecular snapshots suggest primer 3´-OH alignment mediated by A-site metal ion binding is the key step in substrate discrimination. Moreover, we observe that C-site metal ion binding preceded the nucleotidyl transfer reaction and demonstrate that the C-site metal ion is strictly required for misincorporation. Our results highlight the essential but separate roles of the three metal ions in DNA synthesis.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1983

DNA fusion product of phage P2 with plasmid pBR322 - A new phasmid

The chromosome of the temperate bacteriophage P2 and that of the plasmid pBR322 have been joined in vitro after treatment with restriction endonuclease EcoRI. The fusion product - a phasmid - can behave as a plasmid, as a phage and as a prophage. It can replicate its DNA under the control of either the specific replication mechanism of the parent phage in a polA mutant or that of the parent plasmid in a rep mutant. Several interesting interactions between the two replication modes are indicated. In particular, phage particles may be produced even when the phage mode of DNA replication is blocked, and this throws new light on the involvement of the early gene A in the regulation of late gene expression in phage P2.

Nicoletti, M.↗
NASA NTRS1993

B-DNA to Z-DNA structural transitions in the SV40 enhancer: stabilization of Z-DNA in negatively supercoiled DNA minicircles

During replication and transcription, the SV40 control region is subjected to significant levels of DNA unwinding. There are three, alternating purine-pyrimidine tracts within this region that can adopt the Z-DNA conformation in response to negative superhelix density: a single copy of ACACACAT and two copies of ATGCATGC. Since the control region is essential for both efficient transcription and replication, B-DNA to Z-DNA transitions in these vital sequence tracts may have significant biological consequences. We have synthesized DNA minicircles to detect B-DNA to Z-DNA transitions in the SV40 enhancer, and to determine the negative superhelix density required to stabilize the Z-DNA. A variety of DNA sequences, including the entire SV40 enhancer and the two segments of the enhancer with alternating purine-pyrimidine tracts, were incorporated into topologically relaxed minicircles. Negative supercoils were generated, and the resulting topoisomers were resolved by electrophoresis. Using an anti-Z-DNA Fab and an electrophoretic mobility shift assay, Z-DNA was detected in the enhancer-containing minicircles at a superhelix density of -0.05. Fab saturation binding experiments demonstrated that three, independent Z-DNA tracts were stabilized in the supercoiled minicircles. Two other minicircles, each with one of the two alternating purine-pyrimidine tracts, also contained single Z-DNA sites. These results confirm the identities of the Z-DNA-forming sequences within the control region. Moreover, the B-DNA to Z-DNA transitions were detected at superhelix densities observed during normal replication and transcription processes in the SV40 life cycle.

Non-NASA Center↗
DOE OSTI2024

A goldilocks computational protocol for inhibitor discovery targeting DNA damage responses including replication-repair functions

While many researchers can design knockdown and knockout methodologies to remove a gene product, this is mainly untrue for new chemical inhibitor designs that empower multifunctional DNA Damage Response (DDR) networks. Here, we present a robust Goldilocks (GL) computational discovery protocol to efficiently innovate inhibitor tools and preclinical drug candidates for cellular and structural biologists without requiring extensive virtual screen (VS) and chemical synthesis expertise. By computationally targeting DDR replication and repair proteins, we exemplify the identification of DDR target sites and compounds to probe cancer biology. Our GL pipeline integrates experimental and predicted structures to efficiently discover leads, allowing early-structure and early-testing (ESET) experiments by many laboratories. By employing an efficient VS protocol to examine protein-protein interfaces (PPIs) and allosteric interactions, we identify ligand binding sites beyond active sites, leveraging in silico advances for molecular docking and modeling to screen PPIs and multiple targets. A diverse 3,174 compound ESET library combines Diamond Light Source DSI-poised, Protein Data Bank fragments, and FDA-approved drugs to span relevant chemotypes and facilitate downstream hit evaluation efficiency for academic laboratories. Two VS per library and multiple ranked ligand binding poses enable target testing for several DDR targets. This GL library and protocol can thus strategically probe multiple DDR network targets and identify readily available compounds for early structural and activity testing to overcome bottlenecks that can limit timely breakthrough drug discoveries. By testing accessible compounds to dissect multi-functional DDRs and suggesting inhibitor mechanisms from initial docking, the GL approach may enable more groups to help accelerate discovery, suggest new sites and compounds for challenging targets including emerging biothreats and advance cancer biology for future precision medicine clinical trials.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1997

Gene-Based Detection of Microorganisms in Environmental Samples Using PCR

Contaminating microorganisms pose a serious potential risk to the crew's well being and water system integrity aboard the International Space Station (ISS). We are developing a gene-based microbial monitor that functions by replicating specific segments of DNA as much as 10(exp 12) x. Thus a single molecule of DNA can be replicated to detectable levels, and the kinetics of that molecule's accumulation can be used to determine the original concentration of specific microorganisms in a sample. Referred to as the polymerase chain reaction (PCR), this enzymatic amplification of specific segments of the DNA or RNA from contaminating microbes offers the promise of rapid, sensitive, quantitative detection and identification of bacteria, fungi, viruses, and parasites. We envision a small instrument capable of assaying an ISS water sample for 48 different microbes in a 24 hour period. We will report on both the developments in the chemistry necessary for the PCR assays to detect microbial contaminants in ISS water, and on progress towards the miniaturization and automation of the instrumentation.

Glass, John I.↗
NASA NTRS1998

Osteoblastic phenotype of rat marrow stromal cells cultured in the presence of dexamethasone, beta-glycerolphosphate, and L-ascorbic acid

We investigated the effects of the time course of addition of osteogenic supplements dexamethasone, beta-glycerolphosphate, and L-ascorbic acid to rat marrow stromal cells, and the exposure time on the proliferation and differentiation of the cells. It was the goal of these experiments to determine the time point for supplement addition to optimize marrow stromal cell proliferation and osteoblastic differentiation. To determine this, two studies were performed; one study was based on the age of the cells from harvest, and the other study was based on the duration of exposure to supplemented medium. Cells were seen to proliferate rapidly at early time points in the presence and absence of osteogenic supplements as determined by 3H-thymidine incorporation into the DNA of replicating cells. These results were supported by cell counts ascertained through total DNA analysis. Alkaline phosphatase (ALP) activity and osteocalcin production at 21 days were highest for both experimental designs when the cells were exposed to supplemented medium immediately upon harvest. The ALP levels at 21 days were six times greater for cells maintained in supplements throughout than for control cells cultured in the absence of supplements for both studies, reaching an absolute value of 75 x 10(-7) micromole/min/cell. Osteocalcin production reached 20 x 10(-6) ng/cell at 21 days in both studies for cells maintained in supplemented medium throughout the study, whereas the control cells produced an insignificant amount of osteocalcin. These results suggest that the addition of osteogenic supplements to marrow-derived cells early in the culture period did not inhibit proliferation and greatly enhanced the osteoblastic phenotype of cells in a rat model.

Non-NASA Center↗