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At least 1,225 records · Page 68
All sourcesNASA NTRSDOE OSTI
DOE OSTI2014

Diffusion-controlled reactions modeling in Geant4-DNA

Context Under irradiation, a biological system undergoes a cascade of chemical reactions that can lead to an alteration of its normal operation. There are different types of radiation and many competing reactions. As a result the kinetics of chemical species is extremely complex. The simulation becomes then a powerful tool which, by describing the basic principles of chemical reactions, can reveal the dynamics of the macroscopic system. To understand the dynamics of biological systems under radiation, since the 80s there have been on-going efforts carried out by several research groups to establish a mechanistic model that consists in describing all the physical, chemical and biological phenomena following the irradiation of single cells. This approach is generally divided into a succession of stages that follow each other in time: (1) the physical stage, where the ionizing particles interact directly with the biological material; (2) the physico-chemical stage, where the targeted molecules release their energy by dissociating, creating new chemical species; (3) the chemical stage, where the new chemical species interact with each other or with the biomolecules; (4) the biological stage, where the repairing mechanisms of the cell come into play. This article focuses on the modeling of the chemical stage. Method This article presents a general method of speeding-up chemical reaction simulations in fluids based on the Smoluchowski equation and Monte-Carlo methods, where all molecules are explicitly simulated and the solvent is treated as a continuum. The model describes diffusion-controlled reactions. This method has been implemented in Geant4-DNA. The keys to the new algorithm include: (1) the combination of a method to compute time steps dynamically with a Brownian bridge process to account for chemical reactions, which avoids costly fixed time step simulations; (2) a k–d tree data structure for quickly locating, for a given molecule, its closest reactants. The performance advantage is presented in terms of complexity, and the accuracy of the new algorithm is demonstrated by simulating radiation chemistry in the context of the Geant4-DNA project. Application The time-dependent radiolytic yields of the main chemical species formed after irradiation are computed for incident protons at different energies (from 50 MeV to 500 keV). Both the time-evolution and energy dependency of the yields are discussed. The evolution, at one microsecond, of the yields of hydroxyls and solvated electrons with respect to the linear energy transfer is compared to theoretical and experimental data. According to our results, at high linear energy transfer, modeling radiation chemistry in the trading compartment representation might be adopted.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2013

Evolution of I-SceI Homing Endonucleases with Increased DNA Recognition Site Specificity

Elucidating how homing endonucleases undergo changes in recognition site specificity will facilitate efforts to engineer proteins for gene therapy applications. I-SceI is a monomeric homing endonuclease that recognizes and cleaves within an 18-bp target. It tolerates limited degeneracy in its target sequence, including substitution of a C:G{sub +4} base pair for the wild-type A:T{sub +4} base pair. Libraries encoding randomized amino acids at I-SceI residue positions that contact or are proximal to A:T{sub +4} were used in conjunction with a bacterial one-hybrid system to select I-SceI derivatives that bind to recognition sites containing either the A:T{sub +4} or the C:G{sub +4} base pairs. As expected, isolates encoding wild-type residues at the randomized positions were selected using either target sequence. All I-SceI proteins isolated using the C:G{sub +4} recognition site included small side-chain substitutions at G100 and either contained (K86R/G100T, K86R/G100S and K86R/G100C) or lacked (G100A, G100T) a K86R substitution. Interestingly, the binding affinities of the selected variants for the wild-type A:T{sub +4} target are 4- to 11-fold lower than that of wild-type I-SceI, whereas those for the C:G{sub +4} target are similar. The increased specificity of the mutant proteins is also evident in binding experiments in vivo. These differences in binding affinities account for the observed -36-fold difference in target preference between the K86R/G100T and wild-type proteins in DNA cleavage assays. An X-ray crystal structure of the K86R/G100T mutant protein bound to a DNA duplex containing the C:G{sub +4} substitution suggests how sequence specificity of a homing enzyme can increase. This biochemical and structural analysis defines one pathway by which site specificity is augmented for a homing endonuclease.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

MSH1 Is a Plant Organellar DNA Binding and Thylakoid Protein under Precise Spatial Regulation to Alter Development

As metabolic centers, plant organelles participate in maintenance, defense, and signaling. MSH1 is a plant-specific protein involved in organellar genome stability in mitochondria and plastids. Plastid depletion of MSH1 causes heritable, non-genetic changes in development and DNA methylation. We investigated the msh1 phenotype using hemi-complementation mutants and transgene-null segregants from RNAi suppression lines to sub-compartmentalize MSH1 effects. We show that MSH1 expression is spatially regulated, specifically localizing to plastids within the epidermis and vascular parenchyma. The protein binds DNA and localizes to plastid and mitochondrial nucleoids, but fractionation and protein–protein interactions data indicate that MSH1 also associates with the thylakoid membrane. Plastid MSH1 depletion results in variegation, abiotic stress tolerance, variable growth rate, and delayed maturity. Depletion from mitochondria results in 7%–10% of plants altered in leaf morphology, heat tolerance, and mitochondrial genome stability. MSH1 does not localize within the nucleus directly, but plastid depletion produces non-genetic changes in flowering time, maturation, and growth rate that are heritable independent of MSH1. MSH1 depletion alters non-photoactive redox behavior in plastids and a sub-set of mitochondrially altered lines. Ectopic expression produces deleterious effects, underlining its strict expression control. Unraveling the complexity of the MSH1 effect offers insight into triggers of plant-specific, transgenerational adaptation behaviors.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Accumulation of lipids and oxidatively damaged DNA in hepatocytes exposed to particles

Exposure to particles has been suggested to generate hepatosteatosis by oxidative stress mechanisms. We investigated lipid accumulation in cultured human hepatocytes (HepG2) and rat liver after exposure to four different carbon-based particles. HepG2 cells were exposed to particles for 3 h and subsequently incubated for another 18 h to manifest lipid accumulation. In an animal model of metabolic syndrome we investigated the association between intake of carbon black (CB, 14 nm) particles and hepatic lipid accumulation, inflammation and gene expression of Srebp-1, Fasn and Scd-1 involved in lipid synthesis. There was a concentration-dependent increase in intracellular lipid content after exposure to CB in HepG2 cells, which was only observed after co-exposure to oleic/palmitic acid. Similar results were observed in HepG2 cells after exposure to diesel exhaust particles, fullerenes C{sub 60} or pristine single-walled carbon nanotubes. All four types of particles also generated oxidatively damaged DNA, assessed as formamidopyrimidine DNA glycosylase (FPG) sensitive sites, in HepG2 cells after 3 h exposure. The animal model of metabolic syndrome showed increased lipid load in the liver after one oral exposure to 6.4 mg/kg of CB in lean Zucker rats. This was not associated with increased iNOS staining in the liver, indicating that the oral CB exposure was associated with hepatic steatosis rather than steatohepatitis. The lipid accumulation did not seem to be related to increased lipogenesis because there were unaltered gene expression levels in both the HepG2 cells and rat livers. Collectively, exposure to particles is associated with oxidative stress and steatosis in hepatocytes. - Highlights: • Oral exposure to nanosized carbon black was associated with hepatosteatosis in rats. • In vitro studies included carbon black, C{sub 60}, diesel exhaust particles and SWCNTs. • Exposure to particles and free fatty acids increased lipid load in HepG2 cells. • Unaltered expression of lipogenesis genes despite oxidative stress in hepatocytes • Particles evoke hepatosteatosis by increased uptake rather than synthesis of lipids.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Metallothionein blocks oxidative DNA damage induced by acute inorganic arsenic exposure

We studied how protein metallothionein (MT) impacts arsenic-induced oxidative DNA damage (ODD) using cells that poorly express MT (MT-I/II double knockout embryonic cells; called MT-null cells) and wild-type (WT) MT competent cells. Arsenic (as NaAsO{sub 2}) was less cytolethal over 24 h in WT cells (LC{sub 50} = 11.0 ± 1.3 μM; mean ± SEM) than in MT-null cells (LC{sub 50} = 5.6 ± 1.2 μM). ODD was measured by the immuno-spin trapping method. Arsenic (1 or 5 μM; 24 h) induced much less ODD in WT cells (121% and 141% of control, respectively) than in MT-null cells (202% and 260%). In WT cells arsenic caused concentration-dependent increases in MT expression (transcript and protein), and in the metal-responsive transcription factor-1 (MTF-1), which is required to induce the MT gene. In contrast, basal MT levels were not detectable in MT-null cells and unaltered by arsenic exposure. Transfection of MT-I gene into the MT-null cells markedly reduced arsenic-induced ODD levels. The transport genes, Abcc1 and Abcc2 were increased by arsenic in WT cells but either showed no or very limited increases in MT-null cells. Arsenic caused increases in oxidant stress defense genes HO-1 and GSTα2 in both WT and MT-null cells, but to much higher levels in WT cells. WT cells appear more adept at activating metal transport systems and oxidant response genes, although the role of MT in these responses is unclear. Overall, MT protects against arsenic-induced ODD in MT competent cells by potential sequestration of scavenging oxidant radicals and/or arsenic. - Highlights: • Metallothionein blocks arsenic toxicity. • Metallothionein reduces arsenic-induced DNA damage. • Metallothionein may bind arsenic or radicals produced by arsenic.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Downregulation of UBE2E2 in rat liver cells after hepatocarcinogen treatment facilitates cell proliferation and slowing down of DNA damage response in GST-P-expressing preneoplastic lesions

We previously found downregulation of ubiquitin-conjugating enzyme E2E 2 (UBE2E2) in GST-P-positive {sup (+)} proliferative lesions produced by tumor promotion from early hepatocarcinogenesis stages in rats. Here we investigated the role of UBE2E2 downregulation in preneoplastic lesions of the liver and other target organs produced by tumor promotion in rats. Increased number of UBE2E2-related ubiquitination target proteins, phosphorylated c-MYC, KDM4A and KMT5A, was found in the UBE2E2-downregulated GST-P{sup +} foci, compared with GST-P{sup +} foci expressing UBE2E2. However, p21{sup WAF1/CIP1}, another UBE2E2 target protein, did not increase in the positive cells. Furthermore, the numbers of PCNA{sup +} cells and γH2AX{sup +} cells were increased in UBE2E2-downregulated foci. These results suggest sustained activation of c-MYC and stabilization of KMT5A to result in c-MYC-mediated transcript upregulation and following KMT5A-mediated protein stabilization of PCNA in GST-P{sup +} foci, as well as KDM4A stabilization resulting in slowing down of DNA damage response in these lesions. Similar results were also observed in GST-P{sup +} foci produced by repeated treatment of rats with a hepatocarcinogen, thioacetamide, for 90 days. Hepatocarcinogen treatment for 28 or 90 days also increased the numbers of liver cells expressing UBE2E2-related ubiquitination target proteins, as well as PCNA{sup +} or γH2AX{sup +} cells. Conversely, UBE2E2 downregulation was lacking in PPARα agonist-induced hepatocarcinogenesis, as well as in carcinogenic processes targeting other organs, suggestive of the loss of UBE2E2-related ubiquitination limited to hepatocarcinogenesis producing GST-P{sup +} proliferative lesions. Our results suggest that repeated hepatocarcinogen treatment of rats causes stabilization of UBE2E2-related ubiquitination target proteins in liver cells to promote carcinogenesis. - Highlights: • UBE2E2 downregulation was limited in GST-P-linked hepatocarcinogenesis. • UBE2E2 downregulation may stabilize c-MYC, KDM4A and KMT5A in GST-P{sup +} foci. • c-MYC and KMT5A may facilitate PCNA-mediated cell proliferation in GST-P{sup +} foci. • KDM4A may be responsible for slowing down of DNA damage response in GST-P{sup +} foci. • UBE2E2 downregulation triggers and further promotes carcinogenic responses.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2026

Construct design for precise DNA insertion in plants

Precise insertion of DNA sequences at targeted locations in plant genomes is pivotal for synthetic biology, genetics, and crop improvement. Construct design plays a critical role in achieving precise insertions, yet practical guidance remains limited. This review provides an in-depth overview of construct design principles and targeted DNA insertion (knock-in) strategies in plants. We assess the strengths, limitations, and construct requirements of current knock-in methods for specific applications, including short, large, and multifragment insertions. Additionally, we explore the potential of adopting advanced nonplant technologies to enhance knock-in efficiency and precision in plants. This review provides a valuable resource for facilitating the effective application of knock-in technologies to genetically improve crops with minimal off-target effects.

DNA construct↗
DOE OSTI2014

Plasmid DNA initiates replication of yellow fever vaccine in vitro and elicits virus-specific immune response in mice

Yellow fever (YF) causes an acute hemorrhagic fever disease in tropical Africa and Latin America. To develop a novel experimental YF vaccine, we applied iDNA infectious clone technology. The iDNA represents plasmid that encodes the full-length RNA genome of 17D vaccine downstream from a cytomegalovirus (CMV) promoter. The vaccine was designed to transcribe the full-length viral RNA and to launch 17D vaccine virus in vitro and in vivo. Transfection with 10 ng of iDNA plasmid was sufficient to start replication of vaccine virus in vitro. Safety of the parental 17D and iDNA-derived 17D viruses was confirmed in AG129 mice deficient in receptors for IFN-α/β/γ. Finally, direct vaccination of BALB/c mice with a single 20 μg dose of iDNA plasmid resulted in seroconversion and elicitation of virus-specific neutralizing antibodies in animals. We conclude that iDNA immunization approach combines characteristics of DNA and attenuated vaccines and represents a promising vaccination strategy for YF. - Highlights: • The iDNA{sup ®} platform combines advantages of DNA and live attenuated vaccines. • Yellow fever (YF) 17D vaccine was launched from iDNA plasmid in vitro and in vivo. • Safety of iDNA-generated 17D virus was confirmed in AG129 mice. • BALB/c mice seroconverted after a single-dose vaccination with iDNA. • YF virus-neutralizing response was elicited in iDNA-vaccinated mice.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Mitochondrial DNA disease—molecular insights and potential routes to a cure

Mitochondrial DNA diseases are common neurological conditions caused by mutations in the mitochondrial genome or nuclear genes responsible for its maintenance. Current treatments for these disorders are focussed on the management of the symptoms, rather than the correction of biochemical defects caused by the mutation. This review focuses on the molecular effects of mutations, the symptoms they cause and current work focusing on the development of targeted treatments for mitochondrial DNA disease. - Highlights: • We discuss several common disease causing mtDNA mutations. • We highlight recent work linking pathogenicity to deletion size and heteroplasmy. • We discuss recent advances in the development of targeted mtDNA disease treatments.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Identification and interplay of sequence specific DNA binding proteins involved in regulation of human Pregnane and Xenobiotic Receptor gene

Pregnane and Xenobiotic Receptor (PXR), a member of nuclear receptor superfamily, acts as a ‘xenosensor’ in our body and modulates a network of genes involved in xenobiotic metabolism and elimination. Expression levels of PXR in certain metabolic disorders including cancer are reported to be altered and its induced expression is associated with the development of resistance towards chemotherapy and adverse drug–drug interactions. Though the transcriptional regulation of PXR target genes have been elucidated in significant details, the structure and functional control of PXR promoter itself remains inadequately explored. In this work, we identify a Composite Element (CE) located within the proximal PXR promoter region that consists of multiple overlapping cis-elements and demonstrated that CE interacts specifically with some critical nuclear proteins. Subsequent DNA-protein interaction studies revealed mutually exclusive interactions on CE occurring between Sp1 and two unidentified DNA binding proteins with molecular masses of 50 and 54 kDa. Here, we report the identification of 54 kDa CE binding protein as a heterogeneous nuclear ribonucleoprotein K (hnRNPK) and demonstrate the effect of hnRNP K and Sp1 on PXR promoter transcriptional activity. Overall, the study indicates that PXR gene is tightly regulated to maintain a low receptor level. - Highlights: • Cis-regulatory elements present in proximal promoter of PXR are identified. • A composite element (CE) region is involved in PXR gene regulation. • Trans-acting factors binding to CE are identified as Sp1 and hnRNPK. • Mutually exclusive interactions between hnRNPK and other CEBFs at CE are observed.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

SPARC is down-regulated by DNA methylation and functions as a tumor suppressor in T-cell lymphoma

Highlights: • SPARC is significantly down-regulated in T-NHL tissues and cell lines. • High levels of SPARC demonstrated a favorable clinical outcome. • SPARC inhibits proliferation, invasion and migration, but promotes apoptosis of T-NHL cells. • 5-Aza-Cdr inhibits proliferation, invasion and migration, but promotes apoptosis of T-NHL cells. The aim of this study was to assess the functional role of SPARC in T-cell non-Hodgkin's lymphoma (T-NHL), as well as the underlying molecular mechanisms. Here, we first identified SPARC expression in T-NHL tissues and cell lines through western blot and real-time PCR (RT-PCR). Overall survival of T-NHL patients with different levels of SPARC was assessed by Kaplan-Meier survival curves. Then cell proliferation, apoptosis, migration and invasion of T-NHL cells with either knockdown or overexpression of SPARC were determined by MTT, flow cytometry, transwell migration and invasion assay, respectively. Finally, the molecular mechanism by which SPARC modulated T-NHL cell progression was assessed. We confirmed that SPARC was significantly down-regulated in T-NHL tissues and cell lines. T-NHL patients with high levels of SPARC demonstrated a favorable clinical outcome. SPARC significantly suppressed cell proliferation, migration and invasion, and EMT process, but facilitated cell apoptosis in T-NHL cells. Further, we found that loss of SPARC expression in T-NHL tissues and cell lines, both in mRNA and protein levels, was associated with the aberrant DNA methylation in SPRAC gene, and the disrupted SPARC expression could be rescued after treatment with the demethylating agent 5-Aza-2′-deoxycitydine (5-Aza-Cdr). Additionally, 5-Aza-Cdr reversed SPARC hypermethylation to restore its biological role as a tumor suppressor in T-NHL cells, including inhibiting cell proliferation, invasion and migration, while promoting cell apoptosis. Our data provided evidence that DNA methylation in SPARC gene may play a role in the progression of T-NHL.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

RPA1 downregulation enhances nasopharyngeal cancer radiosensitivity via blocking RAD51 to the DNA damage site

Highlights: • RPA1 is highly expressed in NPC patients with radioresistance. • RPA1 can be used as a predictor of radiosensitivity in NPC. • RPA1 silencing reduces the recruitment of RAD51 at the site of the DNA lesion and inhibits the repair of DSBs. Nasopharyngeal cancer (NPC) has a high local recurrence rate due to its resistance to ionizing radiation (IR). Replication protein A1 (RPA1) is one of the main elements in the homologous repair (HR) pathway, which is closely associated with the repair of DNA double strand breaks (DDBs). Studies on the relationship between RPA1 and the radiosensitivity of NPC are substantially limited. It was hypothesized that RPA1 plays a crucial role in predicting the radiosensitivity of NPC.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

A Small Covalent Allosteric Inhibitor of Human Cytomegalovirus DNA Polymerase Subunit Interactions

Human cytomegalovirus DNA polymerase comprises a catalytic subunit, UL54, and an accessory subunit, UL44, the interaction of which may serve as a target for the development of new antiviral drugs. Using a high-throughput screen, we identified a small molecule, (5-((dimethylamino)methylene-3-(methylthio)-6,7-dihydrobenzo[c]thiophen-4(5H)-one), that selectively inhibits the interaction of UL44 with a UL54-derived peptide in a time-dependent manner, full-length UL54, and UL44-dependent long-chain DNA synthesis. A crystal structure of the compound bound to UL44 revealed a covalent reaction with lysine residue 60 and additional noncovalent interactions that cause steric conflicts that would prevent the UL44 connector loop from interacting with UL54. Analyses of the reaction of the compound with model substrates supported a resonance-stabilized conjugation mechanism, and substitution of the lysine reduced the ability of the compound to inhibit UL44-UL54 peptide interactions. Furthermore, this novel covalent inhibitor of polymerase subunit interactions may serve as a starting point for new, needed drugs to treat human cytomegalovirus infections.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Non-equilibrium anisotropic colloidal single crystal growth with DNA

Abstract Anisotropic colloidal crystals are materials with novel optical and electronic properties. However, experimental observations of colloidal single crystals have been limited to relatively isotropic habits. Here, we show DNA-mediated crystallization of two types of nanoparticles with different hydrodynamic radii that form highly anisotropic, hexagonal prism microcrystals with AB 2 crystallographic symmetry. The DNA directs the nanoparticles to assemble into a non-equilibrium crystal shape that is enclosed by the highest surface energy facets (AB 2 (10 $$\overline 1$$ 1 ¯ 0) and AB 2 (0001)). Simulations and theoretical arguments show that this observation is a consequence of large energy barriers between different terminations of the AB 2 (10 $$\overline 1$$ 1 ¯ 0) facet, which results in a significant deceleration of the (10 $$\overline 1$$ 1 ¯ 0) facet growth rate. In addition to reporting a hexagonal colloidal crystal habit, this work introduces a potentially general plane multiplicity mechanism for growing non-equilibrium crystal shapes, an advance that will be useful for designing colloidal crystal habits with important applications in both optics and photocatalysis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

RAD51C-XRCC3 structure and cancer patient mutations define DNA replication roles

$RAD51C$ is an enigmatic predisposition gene for breast, ovarian, and prostate cancer. Currently, missing structural and related functional understanding limits patient mutation interpretation to homology-directed repair (HDR) function analysis. Here we report the RAD51C-XRCC3 (CX3) X-ray co-crystal structure with bound ATP analog and define separable RAD51C replication stability roles informed by its three-dimensional structure, assembly, and unappreciated polymerization motif. Mapping of cancer patient mutations as a functional guide confirms ATP-binding matching RAD51 recombinase yet highlights distinct CX3 interfaces. Analyses of CRISPR/Cas9-edited human cells with $RAD51C$ mutations combined with single-molecule, single-cell and biophysics measurements uncover discrete CX3 regions for DNA replication fork protection, restart and reversal, accomplished by separable functions in DNA binding and implied 5’ RAD51 filament capping. Collective findings establish CX3 as a cancer-relevant replication stress response complex, show how HDR-proficient variants could contribute to tumor development, and identify regions to aid functional testing and classification of cancer mutations.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Zinc-finger BED domains drive the formation of the active Hermes transpososome by asymmetric DNA binding

The Hermes DNA transposon is a member of the eukaryotic hAT superfamily, and its transposase forms a ring-shaped tetramer of dimers. Our investigation, combining biochemical, crystallography and cryo-electron microscopy, and in-cell assays, shows that the full-length Hermes octamer extensively interacts with its transposon left-end through multiple BED domains of three Hermes protomers contributed by three dimers explaining the role of the unusual higher-order assembly. By contrast, the right-end is bound to no BED domains at all. Thus, this work supports a model in which Hermes multimerizes to gather enough BED domains to find its left-end among the abundant genomic DNA, facilitating the subsequent interaction with the right-end.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Angle between DNA linker and nucleosome core particle regulates array compaction revealed by individual-particle cryo-electron tomography

Abstract The conformational dynamics of nucleosome arrays generate a diverse spectrum of microscopic states, posing challenges to their structural determination. Leveraging cryogenic electron tomography (cryo-ET), we determine the three-dimensional (3D) structures of individual mononucleosomes and arrays comprising di-, tri-, and tetranucleosomes. By slowing the rate of condensation through a reduction in ionic strength, we probe the intra-array structural transitions that precede inter-array interactions and liquid droplet formation. Under these conditions, the arrays exhibite irregular zig-zag conformations with loose packing. Increasing the ionic strength promoted intra-array compaction, yet we do not observe the previously reported regular 30-nanometer fibers. Interestingly, the presence of H1 do not induce array compaction; instead, one-third of the arrays display nucleosomes invaded by foreign DNA, suggesting an alternative role for H1 in chromatin network construction. We also find that the crucial parameter determining the structure adopted by chromatin arrays is the angle between the entry and exit of the DNA and the corresponding tangents to the nucleosomal disc. Our results provide insights into the initial stages of intra-array compaction, a critical precursor to condensation in the regulation of chromatin organization.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Control of transcription elongation and DNA repair by alarmone ppGpp

Second messenger (p)ppGpp (collectively guanosine tetraphosphate and guanosine pentaphosphate) mediates bacterial adaptation to nutritional stress by modulating transcription initiation. More recently, ppGpp has been implicated in coupling transcription and DNA repair; however, the mechanism of ppGpp engagement remained elusive. Here we present structural, biochemical and genetic evidence that ppGpp controls Escherichia coli RNA polymerase (RNAP) during elongation via a specific site that is nonfunctional during initiation. Structure-guided mutagenesis renders the elongation (but not initiation) complex unresponsive to ppGpp and increases bacterial sensitivity to genotoxic agents and ultraviolet radiation. Thus, ppGpp binds RNAP at sites with distinct functions in initiation and elongation, with the latter being important for promoting DNA repair. Our data provide insights on the molecular mechanism of ppGpp-mediated adaptation during stress, and further highlight the intricate relationships between genome stability, stress responses and transcription.

59 BASIC BIOLOGICAL SCIENCES↗