Nucleic Acid Processing Research Articles

Opioid-Induced Hyperalgesia Is Associated with Dysregulation of Circadian Rhythm and Adaptive Immune Pathways in the Mouse Trigeminal Ganglia and Nucleus Accumbens.

The benefits of opioid-based treatments to mitigate chronic pain can be hindered by the side effects of opioid-induced hyperalgesia (OIH) that can lead to higher consumption and risk of addiction. The present study advances the understanding of the molecular mechanisms associated with OIH by comparing mice presenting OIH symptoms in response to chronic morphine exposure (OIH treatment) relative to control mice (CON treatment). Using RNA-Seq profiles, gene networks were inferred in the trigeminal ganglia (TG), a central nervous system region associated with pain signaling, and in the nucleus accumbens (NAc), a region associated with reward dependency. The biological process of nucleic acid processing was over-represented among the 122 genes that exhibited a region-dependent treatment effect. Within the 187 genes that exhibited a region-independent treatment effect, circadian rhythm processes were enriched among the genes over-expressed in OIH relative to CON mice. This enrichment was supported by the differential expression of the period circadian clock 2 and 3 genes (Per2 and Per3). Transcriptional regulators in the PAR bZip family that are influenced by the circadian clock and that modulate neurotransmission associated with pain and drug addiction were also over-expressed in OIH relative to CON mice. Also notable was the under-expression in OIH relative to CON mice of the Toll-like receptor, nuclear factor-kappa beta, and interferon gamma genes and enrichment of the adaptive immune processes. The results from the present study offer insights to advance the effective use of opioids for pain management while minimizing hyperalgesia.

Exog Displays 5′-Exonuclease Activity on Both ssDNA and ssRNA

Human mitochondrial nuclease, EXOG (hEXOG), is essential for repair of damaged mtDNA. It shares sequence homology with the apoptotic endonuclease EndoG. However, apart from being endonuclease, hEXOG also possesses 5′-exonuclease activity which is necessary for the 5′-end damage removal and thus robust DNA repair in human mitochondria. Recently the X-ray crystal structures of hEXOG in apo and dsDNA-bound forms were solved. It was shown that a Wing domain of hEXOG confers a specific 5′-dsDNA exonuclease activity which provides an optimal substrate for subsequent gap-filling and strand ligation steps, thus efficient base-excision repair (BER). Still, the molecular mechanism underlying mitochondrial BER has yet to be clearly defined. Here we report structural and biochemical evidence of 5′-exonuclease activity of hEXOG on ssDNA as well as ssRNA. Our results demonstrate that hEXOG acts as a multifunctional enzyme which may contribute not only to the 5′-end damage removal in mitochondrial BER but also to elimination of unnecessary single-stranded DNA and RNA molecules that may arise during nucleic acid processing in mitochondria.

SU53 - GENOME-WIDE DNA METHYLATION ANALYSIS IN THE SALIVA OF CRACK COCAINE DEPENDENTS

Background DNA methylation has been increasingly recognized in the etiology of drug dependence. Due to the difficulties in assessing brain tissue in living humans, peripheral cells such as blood and saliva have been explored as alternatives to explore the involvement of DNA methylation in drug dependency. Here, we characterized DNA methylation changes in the saliva from crack cocaine users compared to health controls. Methods DNA was extracted from saliva of 128 crack cocaine dependents and 109 health controls (males, age 34.8 +/- 9.9 years). Bisulfite converted DNA was hybridized in the Illumina Infinium MethylationEPIC BeadChip arrays. Cellular composition heterogeneity was corrected using RefFreeEwas followed identification of differentially CpG sites using limma (DMSs). Using DMSs with pvalue Results Comparison between crack cocaine dependents and controls revealed 3,857 DMRs which were involved with metabolic processes related to nucleic acid, ncRNA and rRNA processing, gene expression and protein-DNA complex assembly. The gene-gene interaction analysis revealed three networks: one containing 11 genes, which were enriched for biological processes related to mitochondrial function; a second network composed by 36 genes which was enriched mainly for glycosylphosphatidylinositol (GPI) activity and extracellular membrane interaction biological processes; and a third network with 22 genes that was enriched for synapse related processes (GABaergic, serotononergic and cholinergic synapses) and, interestingly, biological processes related to visual perception. Discussion These results suggest that the use of saliva may help to understand the mechanisms underlying the pathology of drug abuse. Changes in DNA methylation found in the saliva revealed perturbation in pathways related to the reward system, often found affected in drug dependents. Moreover, the enrichment analysis of the networks replicated our previous study performed in peripheral blood where molecular functions related to protein activity were associated with cocaine and crack dependency.

Microfluidics for minute DNA sample analysis: open challenges for genetic testing of cell-free circulating DNA in blood plasma

Genetic testing based on the analysis of circulating cell-free DNA (cfDNA) in body fluids, especially blood plasma, is raising interest for the management and follow-up of many diseases, including cancer. Because the concentration of cfDNA is low and its composition mostly degraded, this material can only be assayed with the most sensitive nucleic acid processing technologies. cfDNA analysis therefore constitutes a model target and a driving force for innovation in microfluidic biotechnologies. Here, we overview the main physico-chemical characteristics of cfDNA, and provide a critical review on the different methods for its processing out of blood samples. Then, we describe recent microfluidic developments for high sensitivity DNA analysis, evaluate their practical relevance for cfDNA analysis, and identify a few challenges for technologists in the near future.

Structural Biology of the HEAT-Like Repeat Family of DNA Glycosylases.

DNA glycosylases remove aberrant DNA nucleobases as the first enzymatic step of the base excision repair (BER) pathway. The alkyl-DNA glycosylases AlkC and AlkD adopt a unique structure based on α-helical HEAT repeats. Both enzymes identify and excise their substrates without a base-flipping mechanism used by other glycosylases and nucleic acid processing proteins to access nucleobases that are otherwise stacked inside the double-helix. Consequently, these glycosylases act on a variety of cationic nucleobase modifications, including bulky adducts, not previously associated with BER. The related non-enzymatic HEAT-like repeat (HLR) proteins, AlkD2, and AlkF, have unique nucleic acid binding properties that expand the functions of this relatively new protein superfamily beyond DNA repair. Here, we review the phylogeny, biochemistry, and structures of the HLR proteins, which have helped broaden our understanding of the mechanisms by which DNA glycosylases locate and excise chemically modified DNA nucleobases.

Matrin 3-dependent neurotoxicity is modified by nucleic acid binding and nucleocytoplasmic localization.

Abnormalities in nucleic acid processing are associated with the development of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Mutations in Matrin 3 (MATR3), a poorly understood DNA- and RNA-binding protein, cause familial ALS/FTD, and MATR3 pathology is a feature of sporadic disease, suggesting that MATR3 dysfunction is integrally linked to ALS pathogenesis. Using a rat primary neuron model to assess MATR3-mediated toxicity, we noted that neurons were bidirectionally vulnerable to MATR3 levels, with pathogenic MATR3 mutants displaying enhanced toxicity. MATR3's zinc finger domains partially modulated toxicity, but elimination of its RNA recognition motifs had no effect on survival, instead facilitating its self-assembly into liquid-like droplets. In contrast to other RNA-binding proteins associated with ALS, cytoplasmic MATR3 redistribution mitigated neurodegeneration, suggesting that nuclear MATR3 mediates toxicity. Our findings offer a foundation for understanding MATR3-related neurodegeneration and how nucleic acid binding functions, localization, and pathogenic mutations drive sporadic and familial disease.

Effects of ketamine on the fetal transcriptomic response to umbilical cord occlusion: comparison with hypoxic hypoxia in the cerebral cortex.

The cerebral response to fetal asphyxia is characterized by an upregulation of nucleic acid and chromatin modification processes, as well as a downregulation of metabolic processes at 1h post-umbilical cord occlusion (UCO). Twenty-four hours post UCO, there was an upregulation of metabolic processes and protein modifications. UCO did not alter bacterial gene expression levels, nor did it produce a robust inflammatory response compared to maternal hypoxia. The administration of ketamine produced minimal effects on the fetal response to UCO in the cerebral cortex. Umbilical cord occlusion (UCO) is known to cause neurological disorders in the neonate. Previously, we have reported that hypoxic hypoxia (HH) stimulates the appearance of bacteria in the fetal brain and upregulates the expression of inflammatory markers in fetal cerebral cortex (CTX) and also that ketamine attenuates these responses. In the present study, we aimed to test the hypothesis that UCO, similar to HH, produces an inflammatory response in the fetal CTX and also that treatment with ketamine reduces these effects. In chronically instrumented fetal sheep (∼125days), 30min of partial UCO decreased fetal levels by ∼50%. Half of the fetuses received ketamine (3mgkg-1 ) 10min prior to UCO (n=4 per group). Fetal brains were collected 1 and 24h after the experiment and mRNA was extracted and hybridized for microarray analyses. Differentially-expressed genes were analysed for significant association with gene ontologies and pathways. After 1h, UCO upregulated nucleic acid processing and chromatin modification and downregulated metabolic processes compared to control. After 24h, UCO upregulated metabolic and protein modification processes. Ketamine produced minimal effects. UCO did not alter the abundance of bacterial DNA in fetal brain, nor did it upregulate inflammation pathways compared to HH. We conclude that UCO produced time-dependent responses that did not include bacterial invasion or upregulation of inflammation pathways in fetal CTX. This contrasts with the response to HH, which resulted in the appearance of bacteria in the CTX and upregulated inflammation pathways. These responses in fetal CTX to oxygen deprivation are therefore modified by the maternal or placental response to the stimulus.

An Update on Autoinflammatory Diseases: Interferonopathies.

Type I interferons (IFNαβ) induce the expression of hundreds of genes; thus, it is unsurprising that the initiation, transmission, and resolution of the IFNαβ-mediated immune response is tightly controlled. Mutations that alter nucleic acid processing and recognition, ablate IFNαβ-specific negative feedback mechanisms, or result in dysfunction of the proteasome system can all induce pathogenic IFNαβ signalling and are the focus of this review. Recent advances have delineated the precise cytoplasmic mechanisms that facilitate self-DNA to be recognised by cGAS and self-RNA to be recognised by RIG-I or MDA-5. This helps clarify interferonopathies associated with mutations in genes which code for DNase-II and ADAR1, among others. Similarly, loss of function mutations in Pol α, which lowers the presence of antagonistic ligands in the cytosol, or gain of function mutations in RIG-I and MDA-5, result in increased propensity for receptor activation and therefore IFNαβ induction. As the aetiology of monogenic autoinflammatory diseases are uncovered, novel and sometimes unsuspected molecular interactions and signalling pathways are being defined. This review covers developments that have come to light over the past 3years, with reference to the study of interferonopathies.

N‐π* and Other Atomic Level Interactions of Formamides with Nucleobases and Base Analogs in Water

To understand the molecular origins of favorable amide‐nucleobase interactions and predict or interpret effects of amides on nucleic acid processes, we quantify the preferential interactions of formamide and N‐methylated formamides with nucleobases and base analogs differing in amount and composition of water‐accessible surface area (ASA) by solubility assays. These formamide series solutes have much larger amide sp2C ASA than the alkylurea solutes studied previously. Using an established additive, ASA‐based analysis, we interpret these thermodynamic results to determine intrinsic strengths of interaction of each amide with unit area of the five types of nucleobase unified atoms (sp2C, sp3C; sp2N, sp3N; sp2O). All formamides interact favorably with all nucleobases and base analogs studied. “One‐way” analysis of results to date indicate favorable interactions of these formamides with all five types of unified atoms of nucleobases, and set the stage for a “two‐way” atom‐atom analysis quantifying interactions of the four atoms of amides (aliphatic sp3C; amide sp2C, sp2N and sp2O) with five atoms on nucleobase (aromatic ring sp2C and sp2N, methyl sp3C, amino sp3N, carbonyl sp2O). In particular, this “two‐way” analysis will quantify the favorable n‐π* interactions of nucleobase carbonyl O with amide sp2C and of formamide sp2O with nucleobase heterocyclic ring sp2C to compare with the very favorable n‐π* interactions of amide sp2O with homocyclic aromatic ring sp2C reported previously. Progress toward these goals will be reported.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Noncanonical substrate preference of lambda exonuclease for 5'-nonphosphate-ended dsDNA and a mismatch-induced acceleration effect on the enzymatic reaction.

Lambda exonuclease (λ exo) plays an important role in the resection of DNA ends for DNA repair. Currently, it is also a widely used enzymatic tool in genetic engineering, DNA-binding protein mapping, nanopore sequencing and biosensing. Herein, we disclose two noncanonical properties of this enzyme and suggest a previously undescribed hydrophobic interaction model between λ exo and DNA substrates. We demonstrate that the length of the free portion of the substrate strand in the dsDNA plays an essential role in the initiation of digestion reactions by λ exo. A dsDNA with a 5′ non-phosphorylated, two-nucleotide-protruding end can be digested by λ exo with very high efficiency. Moreover, we show that when a conjugated structure is covalently attached to an internal base of the dsDNA, the presence of a single mismatched base pair at the 5′ side of the modified base may significantly accelerate the process of digestion by λ exo. A detailed comparison study revealed additional π–π stacking interactions between the attached label and the amino acid residues of the enzyme. These new findings not only broaden our knowledge of the enzyme but will also be very useful for research on DNA repair and in vitro processing of nucleic acids.

A Strategically Located Arg/Lys Residue Promotes Correct Base Paring During Nucleic Acid Biosynthesis in Polymerases.

Polymerases (Pols) synthesize the double-stranded nucleic acids in the Watson-Crick (W-C) conformation, which is critical for DNA and RNA functioning. Yet, the molecular basis to catalyze the W-C base pairing during Pol-mediated nucleic acids biosynthesis remains unclear. Here, through bioinformatics analyses on a large data set of Pol/DNA structures, we first describe the conserved presence of one positively charged residue (Lys or Arg), which is similarly located near the enzymatic two-metal active site, always interacting directly with the incoming substrate (d)NTP. Incidentally, we noted that some Pol/DNA structures showing the alternative Hoogsteen base pairing were often solved with this specific residue either mutated, displaced, or missing. We then used quantum and classical simulations coupled to free-energy calculations to illustrate how, in human DNA Pol-η, the conserved Arg61 favors W-C base pairing through defined interactions with the incoming nucleotide. Taken together, these structural observations and computational results suggest a structural framework in which this specific residue is critical for stabilizing the incoming (d)NTP nucleotide and base pairing during Pol-mediated nucleic acid biosynthesis. These results may benefit enzyme engineering for nucleic acid processing and encourage new drug discovery strategies to modulate Pols function.

Highlights from the Faraday discussion on photoinduced processes in nucleic acids and proteins.

The Faraday meeting on photoinduced processes in nucleic acids and proteins was held in Thiruvananthapuram, India in January 2018, continuing the meetings initiated by the Royal Society over 110 years ago, with the first meeting taking place in London in 1907.

Photoinduced processes in nucleic acids and proteins: concluding remarks.

Light induced charge and energy transport in nucleic acids and proteins is the basis of fundamental biological processes such as photosynthesis, vision, DNA-photostability, DNA-photodamage and photosensing. This article summarises the concluding remarks given at the Faraday Discussions meeting on this topic. The specific themes arising from the meeting that are discussed herein include charge transfer in nucleic acids and proteins, excited state dynamics and topology, vibrations and dynamic disorder, proteins and charge transfer states, nanobiophotonics coupled to biomedical applications and photosynthesis.

Spiers Memorial Lecture.Introductory lecture: the impact of structure on photoinduced processes in nucleic acids and proteins.

Light is an important environmental variable and most organisms have evolved means to sense, exploit or avoid it and to repair detrimental effects on their genome. In general, light absorption is the task of specific chromophores, however other biomolecules such as oligonucleotides also do so which can result in undesired outcomes such as mutations and cancer. Given the biological importance of light-induced processes and applications for imaging, optogenetics, photodynamic therapy or photovoltaics, there is a great interest in understanding the detailed molecular mechanisms of photoinduced processes in proteins and nucleic acids. The processes are typically characterized by time-resolved spectroscopic approaches or computation, inferring structural information on transient species from stable ground state structures. Recently, however, structure determination of excited states or other short-lived species has become possible with the advent of X-ray free-electron lasers. This review gives an overview of the impact of structure on the understanding of photoinduced processes in macromolecules, focusing on systems presented at this Faraday Discussion meeting.

The risk of transfusion transmissible infections. Where do we stand in 2018?

Blood transfusion is an important aspect of medicine and patient safety plays a very important role. A myriad of blood-borne infectious agents can be potentially transmitted through transfusion of blood and blood products donated by apparently healthy and asymptomatic donors. The diversity of infectious agents includes human immunodeficiency viruses 1 and 2, hepatitis B virus, hepatitis C virus, malaria, syphilis, human T-lymphotropic virus types 1 and 2 and in certain circumstances, hepatitis A virus, cytomegalovirus, parvovirus B19, and many more. Besides the established viral, bacterial, and parasitic diseases, novel agents have now appeared and are still emerging as potential threats in transfusion medicine. While transmission of prion protein causing variant Creutzfeldt–Jakob disease is of prime concern in countries like the United Kingdom (UK), bacterial infections and viruses such as Dengue and Chikungunya are of prime concern in the Asian region. Transfusion centers employ three levels of screening to ensure blood safety: donor screening interventions to exclude high-risk donors, testing for infectious markers by serology, nucleic acid testing methods and processing technologies like leukoreduction, use of diversion pouch in blood collection, and use of aseptic blood collection technique. Implementing new technologies like pathogen reduction to destroy/inactivate pathological substances in blood components using chemicals or UV lights coupled with expanding testing for emerging diseases will certainly increase the safety of blood. The ultimate aim is to minimize the risks of transfusion-transmitted infection during the window period of infectious agent in a cost-effective and efficient manner. Although considerable effort and resources have been invested, no single technique is yet effective and we are far from achieving zero risk. The main challenges are faced by developing countries due to limited resources.

Second-Shell Basic Residues Expand the Two-Metal-Ion Architecture of DNA and RNA Processing Enzymes

SummarySynthesis and scission of phosphodiester bonds in DNA and RNA regulate vital processes within the cell. Enzymes that catalyze these reactions operate mostly via the recognized two-metal-ion mechanism. Our analysis reveals that basic amino acids and monovalent cations occupy structurally conserved positions nearby the active site of many two-metal-ion enzymes for which high-resolution (<3 Å) structures are known, including DNA and RNA polymerases, nucleases such as Cas9, and splicing ribozymes. Integrating multiple-sequence and structural alignments with molecular dynamics simulations, electrostatic potential maps, and mutational data, we found that these elements always interact with the substrates, suggesting that they may play an active role for catalysis, in addition to their electrostatic contribution. We discuss possible mechanistic implications of this expanded two-metal-ion architecture, including inferences on medium-resolution cryoelectron microscopy structures. Ultimately, our analysis may inspire future experiments and strategies for enzyme engineering or drug design to modulate nucleic acid processing.

Crystal structure of an ASCH protein from Zymomonas mobilis and its ribonuclease activity specific for single-stranded RNA

Activating signal cointegrator-1 homology (ASCH) domains were initially reported in human as a part of the ASC-1 transcriptional regulator, a component of a putative RNA-interacting protein complex; their presence has now been confirmed in a wide range of organisms. Here, we have determined the trigonal and monoclinic crystal structures of an ASCH domain-containing protein from Zymomonas mobilis (ZmASCH), and analyzed the structural determinants of its nucleic acid processing activity. The protein has a central β-barrel structure with several nearby α-helices. Positively charged surface patches form a cleft that runs through the pocket formed between the β-barrel and the surrounding α-helices. We further demonstrate by means of in vitro assays that ZmASCH binds nucleic acids, and degrades single-stranded RNAs in a magnesium ion-dependent manner with a cleavage preference for the phosphodiester bond between the pyrimidine and adenine nucleotides. ZmASCH also removes a nucleotide at the 5′-end. Mutagenesis studies, guided by molecular dynamics simulations, confirmed that three residues (Tyr47, Lys53, and Ser128) situated in the cleft contribute to nucleic acid-binding and RNA cleavage activities. These structural and biochemical studies imply that prokaryotic ASCH may function to control the cellular RNA amount.

Comparative Proteomics of Streptococcus pneumoniae Response to Vancomycin Treatment.

Streptococcus pneumoniae is a gram-positive pathogen that causes otitis media, pneumonia, meningitis, and other serious diseases. Vancomycin is one of the most important drugs currently used for the treatment of gram-positive bacterial infections, representing, importantly, the last line of defense against bacteria that have developed resistance to other antibiotics. While primary efforts of most investigations focused on the antibacterial mechanism of vancomycin, few studies have been performed to assess the tolerance mechanism of bacteria to vancomycin. In this work, whole cellular proteins were extracted from S. pneumoniae D39 with or without vancomycin treatment. Subsequently, differentially expressed proteins (DEPs) were identified with two-dimensional gel electrophoresis coupled with matrix-assisted laser desorption/ionization mass spectrometry (MS)/MS. In total, 27 proteins were upregulated and four proteins were downregulated in vancomycin-treated S. pneumoniae. Gene ontology analysis indicated that these DEPs were mainly involved in the nucleic acid, protein, and carbohydrate biosynthetic processes. Verification experiments with real-time quantitative polymerase chain reaction showed that the gene expression profiles were consistent with proteomic data. These new observations may serve as a valuable resource for future investigations of vancomycin tolerance mechanisms of S. pneumoniae.

DNA substrate recognition and processing by the full-length human UPF1 helicase.

UPF1 is a conserved helicase required for nonsense-mediated decay (NMD) regulating mRNA stability in the cytoplasm. Human UPF1 (hUPF1) is also needed for nuclear DNA replication. While loss of NMD is tolerated, loss of hUPF1 induces a DNA damage response and cell cycle arrest. We have analysed nucleic acid (NA) binding and processing by full-length hUPF1. hUPF1 unwinds non-B and B-form DNA and RNA substrates in vitro. Unlike many helicases involved in genome stability no hUPF1 binding to DNA structures stabilized by inter-base-pair hydrogen bonding was observed. Alternatively, hUPF1 binds to single-stranded NAs (ssNA) with apparent affinity increasing with substrate length and with no preference for binding RNA or DNA or purine compared to pyrimidine polynucleotides. However, the data show a pronounced nucleobase bias with a preference for binding poly (U) or d(T) while d(A) polymers bind with low affinity. Although the data indicate that hUPF1 must bind a ssNA segments to initiate unwinding they also raise the possibility that hUPF1 has significantly reduced affinity for ssNA structures with stacked bases. Overall, the NA processing activities of hUPF1 are consistent with its function in mRNA regulation and suggest that roles in DNA replication could also be influenced by base sequence.

Mechanisms of quinolone action and resistance: where do we stand?

Quinolone antibiotics represent one of the most important classes of anti-infective agents and, although still clinically valuable, their use has been compromised by the increasing emergence of resistant strains, which has become a prevalent clinical problem. Quinolones act by inhibiting the activity of DNA gyrase and topoisomerase IV - two essential bacterial enzymes that modulate the chromosomal supercoiling required for critical nucleic acid processes. The acquisition of quinolone resistance is recognized to be multifactorial and complex. The main resistance mechanism consists of one or a combination of target-site gene mutations that alter the drug-binding affinity of target enzymes. However, other mechanisms such as mutations that lead to reduced intracellular drug concentrations, by either decreased uptake or increased efflux, and plasmid-encoded resistance genes producing either target protection proteins, drug-modifying enzymes or multidrug efflux pumps are known to contribute additively to quinolone resistance. The understanding of these different resistance mechanisms has improved significantly in recent years; however, many details remain to be clarified and the contribution of less-studied mechanisms still needs to be better elucidated in order to fully understand this phenotype.