Base Excision DNA Repair in Plants: Arabidopsis and Beyond.

Base excision DNA repair (BER) is a key pathway safeguarding the genome of all living organisms from damage caused by both intrinsic and environmental factors. Most present knowledge about BER comes from studies of human cells, E. coli, and yeast. Plants may be under an even heavier DNA damage threat from abiotic stress, reactive oxygen species leaking from the photosynthetic system, and reactive secondary metabolites. In general, BER in plant species is similar to that in humans and model organisms, but several important details are specific to plants. Here, we review the current state of knowledge about BER in plants, with special attention paid to its unique features, such as the existence of active epigenetic demethylation based on the BER machinery, the unexplained diversity of alkylation damage repair enzymes, and the differences in the processing of abasic sites that appear either spontaneously or are generated as BER intermediates. Understanding the biochemistry of plant DNA repair, especially in species other than the Arabidopsis model, is important for future efforts to develop new crop varieties.

Genome-wide analysis of WD40 protein family and functional characterization of BvWD40-82 in sugar beet.

Sugar beet is one of the most important sugar crops in the world. It contributes greatly to the global sugar production, but salt stress negatively affects the crop yield. WD40 proteins play important roles in plant growth and response to abiotic stresses through their involvement in a variety of biological processes, such as signal transduction, histone modification, ubiquitination, and RNA processing. The WD40 protein family has been well-studied in Arabidopsis thaliana, rice and other plants, but the systematic analysis of the sugar beet WD40 proteins has not been reported. In this study, a total of 177 BvWD40 proteins were identified from the sugar beet genome, and their evolutionary characteristics, protein structure, gene structure, protein interaction network and gene ontology were systematically analyzed to understand their evolution and function. Meanwhile, the expression patterns of BvWD40s under salt stress were characterized, and a BvWD40-82 gene was hypothesized as a salt-tolerant candidate gene. Its function was further characterized using molecular and genetic methods. The result showed that BvWD40-82 enhanced salt stress tolerance in transgenic Arabidopsis seedlings by increasing the contents of osmolytes and antioxidant enzyme activities, maintaining intracellular ion homeostasis and increasing the expression of genes related to SOS and ABA pathways. The result has laid a foundation for further mechanistic study of the BvWD40 genes in sugar beet tolerance to salt stress, and it may inform biotechnological applications in improving crop stress resilience.

1 Cellular protein TTC4 and its cofactor HSP90 are pro-viral for bovine herpesvirus 1.

Bovine Herpesvirus Type 1 (BoHV-1) infection causes infectious bovine rhinotracheitis and genital disease in cattle, with significant economic and welfare impacts. However, the role of cellular host factors during viral replication remains poorly characterised. A previously performed genome-wide CRISPR knockout screen identified pro- and antiviral host factors acting during BoHV-1 replication. Herein we validate a pro-viral role for a candidate from this screen: the cellular protein tetracopeptide repeat protein 4 (TTC4). We show that TTC4 transcript production is upregulated during BoHV-1 infection. Depletion of TTC4 protein impairs BoHV-1 protein production but does not reduce production of infectious virions, whereas overexpression of exogenous TTC4 results in a significant increase in production of infectious BoHV-1 virions. TTC4 itself is poorly characterized (especially in the context of virus infection), but is a known co-chaperone of heat shock protein 90 (HSP90). HSP90 has a well-characterized pro-viral role during the replication of diverse herpesviruses, and we therefore hypothesized that HSP90 is also pro-viral for BoHV-1. Drug-mediated inhibition of HSP90 using geldanamycin at sub-cytotoxic concentrations inhibited both BoHV-1 protein production and viral genome replication, indicating a pro-viral role for HSP90 during BoHV-1 infection. Our data demonstrates pro-viral roles for both TTC4 and HSP90 during BoHV-1 replication; possibly, interactions between these two proteins are required for optimal BoHV-1 replication, or the two proteins may have independent pro-viral roles.

Noncatalytic Domains in DNA Glycosylases.

Many proteins consist of two or more structural domains: separate parts that have a defined structure and function. For example, in enzymes, the catalytic activity is often localized in a core fragment, while other domains or disordered parts of the same protein participate in a number of regulatory processes. This situation is often observed in many DNA glycosylases, the proteins that remove damaged nucleobases thus initiating base excision DNA repair. This review covers the present knowledge about the functions and evolution of such noncatalytic parts in DNA glycosylases, mostly concerned with the human enzymes but also considering some unique members of this group coming from plants and prokaryotes.

A Low-Activity Polymorphic Variant of Human NEIL2 DNA Glycosylase.

Human NEIL2 DNA glycosylase (hNEIL2) is a base excision repair protein that removes oxidative lesions from DNA. A distinctive feature of hNEIL2 is its preference for the lesions in bubbles and other non-canonical DNA structures. Although a number of associations of polymorphisms in the hNEIL2 gene were reported, there is little data on the functionality of the encoded protein variants, as follows: only hNEIL2 R103Q was described as unaffected, and R257L, as less proficient in supporting the repair in a reconstituted system. Here, we report the biochemical characterization of two hNEIL2 variants found as polymorphisms in the general population, R103W and P304T. Arg103 is located in a long disordered segment within the N-terminal domain of hNEIL2, while Pro304 occupies a position in the ß-turn of the DNA-binding zinc finger motif. Similar to the wild-type protein, both of the variants could catalyze base excision and nick DNA by ß-elimination but demonstrated a lower affinity for DNA. Steady-state kinetics indicates that the P304T variant has its catalytic efficiency (in terms of kcat/KM) reduced ~5-fold compared with the wild-type hNEIL2, whereas the R103W enzyme is much less affected. The P304T variant was also less proficient than the wild-type, or R103W hNEIL2, in the removal of damaged bases from single-stranded and bubble-containing DNA. Overall, hNEIL2 P304T could be worthy of a detailed epidemiological analysis as a possible cancer risk modifier.

A New Class of Uracil-DNA Glycosylase Inhibitors Active against Human and Vaccinia Virus Enzyme.

Uracil-DNA glycosylases are enzymes that excise uracil bases appearing in DNA as a result of cytosine deamination or accidental dUMP incorporation from the dUTP pool. The activity of Family 1 uracil-DNA glycosylase (UNG) activity limits the efficiency of antimetabolite drugs and is essential for virulence in some bacterial and viral infections. Thus, UNG is regarded as a promising target for antitumor, antiviral, antibacterial, and antiprotozoal drugs. Most UNG inhibitors presently developed are based on the uracil base linked to various substituents, yet new pharmacophores are wanted to target a wide range of UNGs. We have conducted virtual screening of a 1,027,767-ligand library and biochemically screened the best hits for the inhibitory activity against human and vaccinia virus UNG enzymes. Although even the best inhibitors had IC50 ≥ 100 µM, they were highly enriched in a common fragment, tetrahydro-2,4,6-trioxopyrimidinylidene (PyO3). In silico, PyO3 preferably docked into the enzyme's active site, and in kinetic experiments, the inhibition was better consistent with the competitive mechanism. The toxicity of two best inhibitors for human cells was independent of the presence of methotrexate, which is consistent with the hypothesis that dUMP in genomic DNA is less toxic for the cell than strand breaks arising from the massive removal of uracil. We conclude that PyO3 may be a novel pharmacophore with the potential for development into UNG-targeting agents.

Evolutionary Origins of DNA Repair Pathways: Role of Oxygen Catastrophe in the Emergence of DNA Glycosylases.

It was proposed that the last universal common ancestor (LUCA) evolved under high temperatures in an oxygen-free environment, similar to those found in deep-sea vents and on volcanic slopes. Therefore, spontaneous DNA decay, such as base loss and cytosine deamination, was the major factor affecting LUCA's genome integrity. Cosmic radiation due to Earth's weak magnetic field and alkylating metabolic radicals added to these threats. Here, we propose that ancient forms of life had only two distinct repair mechanisms: versatile apurinic/apyrimidinic (AP) endonucleases to cope with both AP sites and deaminated residues, and enzymes catalyzing the direct reversal of UV and alkylation damage. The absence of uracil-DNA N-glycosylases in some Archaea, together with the presence of an AP endonuclease, which can cleave uracil-containing DNA, suggests that the AP endonuclease-initiated nucleotide incision repair (NIR) pathway evolved independently from DNA glycosylase-mediated base excision repair. NIR may be a relic that appeared in an early thermophilic ancestor to counteract spontaneous DNA damage. We hypothesize that a rise in the oxygen level in the Earth's atmosphere ~2 Ga triggered the narrow specialization of AP endonucleases and DNA glycosylases to cope efficiently with a widened array of oxidative base damage and complex DNA lesions.

Displacement of Slow-Turnover DNA Glycosylases by Molecular Traffic on DNA.

In the base excision repair pathway, the initiating enzymes, DNA glycosylases, remove damaged bases and form long-living complexes with the abasic DNA product, but can be displaced by AP endonucleases. However, many nuclear proteins can move along DNA, either actively (such as DNA or RNA polymerases) or by passive one-dimensional diffusion. In most cases, it is not clear whether this movement is disturbed by other bound proteins or how collisions with moving proteins affect the bound proteins, including DNA glycosylases. We have used a two-substrate system to study the displacement of human OGG1 and NEIL1 DNA glycosylases by DNA polymerases in both elongation and diffusion mode and by D4, a passively diffusing subunit of a viral DNA polymerase. The OGG1-DNA product complex was disrupted by DNA polymerase ß (POLß) in both elongation and diffusion mode, Klenow fragment (KF) in the elongation mode and by D4. NEIL1, which has a shorter half-life on DNA, was displaced more efficiently. Hence, both possibly specific interactions with POLß and nonspecific collisions (KF, D4) can displace DNA glycosylases from DNA. The protein movement along DNA was blocked by very tightly bound Cas9 RNA-targeted nuclease, providing an upper limit on the efficiency of obstacle clearance.

Reading Targeted DNA Damage in the Active Demethylation Pathway: Role of Accessory Domains of Eukaryotic AP Endonucleases and Thymine-DNA Glycosylases.

Base excision DNA repair (BER) is an important process used by all living organisms to remove nonbulky lesions from DNA. BER is usually initiated by DNA glycosylases that excise a damaged base leaving an apurinic/apyrimidinic (AP) site, and an AP endonuclease then cuts DNA at the AP site, and the repair is completed by correct nucleotide insertion, end processing, and nick ligation. It has emerged recently that the BER machinery, in addition to genome protection, is crucial for active epigenetic demethylation in the vertebrates. This pathway is initiated by TET dioxygenases that oxidize the regulatory 5-methylcytosine, and the oxidation products are treated as substrates for BER. T:G mismatch-specific thymine-DNA glycosylase (TDG) and AP endonuclease 1 (APE1) catalyze the first two steps in BER-dependent active demethylation. In addition to the well-structured catalytic domains, these enzymes possess long tails that are structurally uncharacterized but involved in multiple interactions and regulatory functions. In this review, we describe the known roles of the tails in TDG and APE1, discuss the importance of order and disorder in their structure, and consider the evolutionary aspects of these accessory protein regions. We also propose that the tails may be important for the enzymes' oligomerization on DNA, an aspect of their function that only recently gained attention.

Photoinduced inhibition of DNA repair enzymes and the possible mechanism of photochemical transformations of the ruthenium nitrosyl complex [RuNO(ß-Pic)2(NO2)2OH].

In this work we have demonstrated that the ruthenium nitrosyl complex [RuNO(ß-Pic)2(NO2)2OH] is suitable for investigation of the inactivation of DNA repair enzymes in vitro. Photoinduced inhibition of DNA glycosylases such as E. coli Endo III, plant NtROS1, mammalian mNEIL1 and hNEIL2 occurs to an extent of ≥90% after irradiation with the ruthenium complex. The photophysical and photochemical processes of [RuNO(ß-Pic)2(NO2)2OH] were investigated using stationary and time-resolved spectroscopy, and mass spectrometry. A possible mechanism of the photo-processes was proposed from the combined spectroscopic study and DTF calculations, which reveal that the photolysis is multistage. The primary and secondary photolysis stages are the photo-induced cleavage of the Ru-NO bond with the formation of a free nitric oxide and RuIII complex followed by ligand exchange with solvent. For E. coli Endo III, covalent interaction with the photolysis product was confirmed by UV-vis and mass spectrometric methods.

Conformational Dynamics of Damage Processing by Human DNA Glycosylase NEIL1.

Endonuclease VIII-like protein 1 (NEIL1) is a DNA repair enzyme found in higher eukaryotes, including humans. It belongs to the helix-two turn-helix (H2TH) structural superfamily together with Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease VIII (Nei), and removes a variety of oxidized purine and pyrimidine bases from DNA. Structural, modeling and kinetic studies have established that the bacterial H2TH superfamily enzymes proceed through several conformational intermediates while recognizing and removing their cognate lesions. Here we apply stopped-flow kinetics with detection of intrinsic Trp fluorescence and Förster resonance energy transfer fluorescence to follow the conformational dynamics of human NEIL1 and DNA when the enzyme interacts with undamaged DNA, or DNA containing cleavable or non-cleavable abasic sites, or dihydrouracil lesions. NEIL1 processed a natural abasic site and a damaged base in DNA equally well but showed an additional fluorescently discernible step when DHU was present, likely reflecting additional rearrangements during base eversion into the enzyme's active site. With undamaged DNA and DNA containing a non-cleavable abasic site analog, (3-hydroxytetrahydrofuran-2-yl)methyl phosphate, NEIL1 was diverted to a non-productive DNA conformation early in the reaction. Our results support the view of NEIL1 as an enzyme that actively destabilizes damaged DNA and uses multiple checkpoints along the reaction coordinate to drive substrate lesions into the active site while rejecting normal bases and non-substrate lesions.

Cognitive disability in estrogenectomited and old rats with development of diabetes mellitus

Introduction: In recent years, there have been many works devoted to the study of the effects of sex hormones on cognitive function. Clinical studies have found that inmenopausal women, the tendency to develop type 2 diabetes will increase, the spatial and short-term working memory worsens, and there is a tendency to develop depression. The findings suggest that estrogens are involved in the mechanisms of insulin resistance in tissues, in the synthesis of mediators in the catecholaminergic systems of the brain, but many questions remain unresolved. The aim: Therefore, the aim of our study was to establish the effect of estrogens on the indices of spatial memory in ovariectomized and old rats against the background of the development of experimental diabetes. Material and methods: The study was conducted on 30 adult rats females 4-5 months and 20 months. The study groups were ovariectomized and reproduced experimental type 2 diabetes mellitus with protamine sulfate. The study of spatial memory was carried out in an eight-sleeved radial labyrinth. Results and conclusions: Results and conclusions: Ovariectomy caused the deterioration of spatial memory relative to the living control group, and diabetes mellitus aggravated pathological changes. The changes that occur after ovariectomy suggest estrogen involvement in the regulation of cognitive functions.

Data set on the synthesis and properties of 2',3'-dideoxyuridine triphosphate conjugated to SiO2 nanoparticles.

SiO2 nanoparticles were used as a transport system for cellular delivery of phosphorylated 2',3'-dideoxyuridine to increase its anticancer potency. This data set is related to the research article entitled "2',3'-Dideoxyuridine triphosphate conjugated to SiO2 nanoparticles: synthesis and evaluation of antiproliferative activity" (Vasilyeva et al., 2018) [1]. It includes a protocol for the synthesis of 2',3'-dideoxyuridine-5'-{N-[4-(prop-2-yn-1-yloxy)butyl]-γ-amino}-triphosphate, its identification by NMR, IR and ESI-MS, experimental procedure of covalent attachment to SiO2 nanoparticles with via Cu-catalyzed click-chemistry, experimental data on chemical stability of the conjugate at different pH values and cytotoxicity assessment of antiproliferative effect of the conjugate.

Oxidative damage to epigenetically methylated sites affects DNA stability, dynamics and enzymatic demethylation.

DNA damage can affect various regulatory elements of the genome, with the consequences for DNA structure, dynamics, and interaction with proteins remaining largely unexplored. We used solution NMR spectroscopy, restrained and free molecular dynamics to obtain the structures and investigate dominant motions for a set of DNA duplexes containing CpG sites permuted with combinations of 5-methylcytosine (mC), the primary epigenetic base, and 8-oxoguanine (oxoG), an abundant DNA lesion. Guanine oxidation significantly changed the motion in both hemimethylated and fully methylated DNA, increased base pair breathing, induced BI→BII transition in the backbone 3' to the oxoG and reduced the variability of shift and tilt helical parameters. UV melting experiments corroborated the NMR and molecular dynamics results, showing significant destabilization of all methylated contexts by oxoG. Notably, some dynamic and thermodynamic effects were not additive in the fully methylated oxidized CpG, indicating that the introduced modifications interact with each other. Finally, we show that the presence of oxoG biases the recognition of methylated CpG dinucleotides by ROS1, a plant enzyme involved in epigenetic DNA demethylation, in favor of the oxidized DNA strand. Thus, the conformational and dynamic effects of spurious DNA oxidation in the regulatory CpG dinucleotide can have far-reaching biological consequences.

2',3'-Dideoxyuridine triphosphate conjugated to SiO2 nanoparticles: Synthesis and evaluation of antiproliferative activity.

A conjugate of triphosphorylated 2',3'-dideoxyuridine (ddU) with SiO2 nanoparticles was obtained via the CuAAC click chemistry between a γ-alkynyl ddU triphosphate and azido-modified SiO2 nanoparticles. Assessment of cytotoxicity in human breast adenocarcinoma MCF7 cells demonstrated that ddU triphosphate conjugated to SiO2 nanoparticles exhibited a 50% decrease in cancer cell growth at a concentration of 183 ±â€¯57 µg/mL, which corresponds to 22 ±â€¯7 µM of the parent nucleotide, whereas the parent nucleoside, nucleotide and alkynyl triphosphate precursor do not show any cytotoxicity. The data provide an example of remarkable potential of novel conjugates of SiO2 nanoparticles with phosphorylated nucleoside analogues, even those, which have not been used previously as therapeutics, for application as new anticancer agents.

Improvement of myocardial infarction risk prediction via inflammation-associated metabolite biomarkers.

OBJECTIVE: The comprehensive assaying of low-molecular-weight compounds, for example, metabolomics, provides a unique tool to uncover novel biomarkers and understand pathways underlying myocardial infarction (MI). We used a targeted metabolomics approach to identify biomarkers for MI and evaluate their involvement in the pathogenesis of MI. METHODS AND RESULTS: Using three independent, prospective cohorts (KORA S4, KORA S2 and AGES-REFINE), totalling 2257 participants without a history of MI at baseline, we identified metabolites associated with incident MI (266 cases). We also investigated the association between the metabolites and high-sensitivity C reactive protein (hsCRP) to understand the relation between these metabolites and systemic inflammation. Out of 140 metabolites, 16 were nominally associated (p<0.05) with incident MI in KORA S4. Three metabolites, arginine and two lysophosphatidylcholines (LPC 17:0 and LPC 18:2), were selected as biomarkers via a backward stepwise selection procedure in the KORA S4 and were significant (p<0.0003) in a meta-analysis comprising all three studies including KORA S2 and AGES-REFINE. Furthermore, these three metabolites increased the predictive value of the Framingham risk score, increasing the area under the receiver operating characteristic score in KORA S4 (from 0.70 to 0.78, p=0.001) and AGES-REFINE study (from 0.70 to 0.76, p=0.02), but was not observed in KORA S2. The metabolite biomarkers attenuated the association between hsCRP and MI, indicating a potential link to systemic inflammatory processes. CONCLUSIONS: We identified three metabolite biomarkers, which in combination increase the predictive value of the Framingham risk score. The attenuation of the hsCRP-MI association by these three metabolites indicates a potential link to systemic inflammation.

Identification of immuno-reactive capsid proteins of malignant catarrhal fever viruses.

Malignant catarrhal fever (MCF) is a fatal disease of cattle and other ungulates caused by certain gamma-herpesviruses including alcelaphine herpesvirus-1 (AlHV-1) and ovine herpesvirus-2 (OvHV-2). An attenuated virus vaccine based on AlHV-1 has been shown to induce virus-neutralising antibodies in plasma and nasal secretions of protected cattle but the targets of virus-specific antibodies are unknown. Proteomic analysis and western blotting of virus extracts allowed the identification of eight candidate AlHV-1 virion antigens. Recombinant expression of selected candidates and their OvHV-2 orthologues confirmed that two polypeptides, the products of the ORF17.5 and ORF65 genes, were antigens recognised by antibodies from natural MCF cases or from AlHV-1 vaccinated cattle. These proteins have potential as diagnostic and/or vaccine antigens.

Cloning and characterization of a wheat homologue of apurinic/apyrimidinic endonuclease Ape1L.

BACKGROUND: Apurinic/apyrimidinic (AP) endonucleases are key DNA repair enzymes involved in the base excision repair (BER) pathway. In BER, an AP endonuclease cleaves DNA at AP sites and 3'-blocking moieties generated by DNA glycosylases and/or oxidative damage. A Triticum aestivum cDNA encoding for a putative homologue of ExoIII family AP endonucleases which includes E. coli Xth, human APE1 and Arabidopsis thaliana AtApe1L has been isolated and its protein product purified and characterized. METHODOLOGY/PRINCIPAL FINDINGS: We report that the putative wheat AP endonuclease, referred here as TaApe1L, contains AP endonuclease, 3'-repair phosphodiesterase, 3'-phosphatase and 3' → 5' exonuclease activities. Surprisingly, in contrast to bacterial and human AP endonucleases, addition of Mg(2+) and Ca(2+) (5-10 mM) to the reaction mixture inhibited TaApe1L whereas the presence of Mn(2+), Co(2+) and Fe(2+) cations (0.1-1.0 mM) strongly stimulated all its DNA repair activities. Optimization of the reaction conditions revealed that the wheat enzyme requires low divalent cation concentration (0.1 mM), mildly acidic pH (6-7), low ionic strength (20 mM KCl) and has a temperature optimum at around 20 °C. The steady-state kinetic parameters of enzymatic reactions indicate that TaApe1L removes 3'-blocking sugar-phosphate and 3'-phosphate groups with good efficiency (kcat/KM = 630 and 485 µM(-1) · min(-1), respectively) but possesses a very weak AP endonuclease activity as compared to the human homologue, APE1. CONCLUSIONS/SIGNIFICANCE: Taken together, these data establish the DNA substrate specificity of the wheat AP endonuclease and suggest its possible role in the repair of DNA damage generated by endogenous and environmental factors.

RNA-seq analysis of host and viral gene expression highlights interaction between varicella zoster virus and keratinocyte differentiation.

Varicella zoster virus (VZV) is the etiological agent of chickenpox and shingles, diseases characterized by epidermal skin blistering. Using a calcium-induced keratinocyte differentiation model we investigated the interaction between epidermal differentiation and VZV infection. RNA-seq analysis showed that VZV infection has a profound effect on differentiating keratinocytes, altering the normal process of epidermal gene expression to generate a signature that resembles patterns of gene expression seen in both heritable and acquired skin-blistering disorders. Further investigation by real-time PCR, protein analysis and electron microscopy revealed that VZV specifically reduced expression of specific suprabasal cytokeratins and desmosomal proteins, leading to disruption of epidermal structure and function. These changes were accompanied by an upregulation of kallikreins and serine proteases. Taken together VZV infection promotes blistering and desquamation of the epidermis, both of which are necessary to the viral spread and pathogenesis. At the same time, analysis of the viral transcriptome provided evidence that VZV gene expression was significantly increased following calcium treatment of keratinocytes. Using reporter viruses and immunohistochemistry we confirmed that VZV gene and protein expression in skin is linked with cellular differentiation. These studies highlight the intimate host-pathogen interaction following VZV infection of skin and provide insight into the mechanisms by which VZV remodels the epidermal environment to promote its own replication and spread.

Excision of 8-oxoguanine from methylated CpG dinucleotides by human 8-oxoguanine DNA glycosylase.

CpG dinucleotides are targets for epigenetic methylation, many of them bearing 5-methylcytosine (mCyt) in the human genome. Guanine in this context can be easily oxidized to 8-oxoguanine (oxoGua), which is repaired by 8-oxoguanine-DNA glycosylase (OGG1). We have studied how methylation affects the efficiency of oxoGua excision from damaged CpG dinucleotides. Methylation of the adjacent cytosine moderately decreased the oxoGua excision rate while methylation opposite oxoGua lowered the rate of product release. Cytosine methylation abolished stimulation of OGG1 by repair endonuclease APEX1. The OGG1 S326C polymorphic variant associated with lung cancer showed poorer base excision and lost sensitivity to the opposite-base methylation. The overall repair in the system reconstituted from purified proteins decreased for CpG with mCyt in the damaged strand.