AT-rich DNA Research Articles

An integrated computational approach to identify GC minor groove binders using various molecular docking scoring functions, dynamics simulations and binding free energy calculations

Understanding the DNA-ligand interaction mechanism is of utmost importance to design selective inhibitors targeting the GC- and AT-rich DNA. This forms a primary strategy to block the association of transcription factors to promoters and subsequently, reduce the expression of genes. We present here an integrated approach combining various docking scoring functions, selective ligand-based pharmacophore models, molecular dynamics simulations and binding free energy calculations to prioritize natural compounds specific to GC minor groove binding. The approach initially applies a selective ligand-based pharmacophore model built upon known GC minor groove binders to identify potential GC minor groove binders from natural compound repositories. These GC minor groove binders were then cross-examined with selective pharmacophore models (controls) based on AT-rich binders and GC intercalators to assess its unfitness. This approach involves the calculation of binding energies of known GC- and AT minor groove binders using three scoring functions without any constraint on groove specificity of GC- and AT-rich DNA. The evaluation of empirical scoring functions led to enumeration of a new parameter, the energy difference computed using Glide (sensitivity = 80%) to recognize GC-rich binders effectively. Molecular dynamics simulations and binding free energy calculations (MM/GBSA) constituted the final phase of this approach to analyze the interactions of natural molecules (hits) with GC-rich DNA comprehensively. Seven natural molecules were selected which exhibited fewer fluctuations in RMSD and RMSF profiles and better GC-rich DNA binding with low free energies of binding. These natural hits prioritized by this integrated approach can be tested in DNA binding assay.Communicated by Ramaswamy H. Sarma

Cellular Dynamics and Genomic Identity of Centromeres in Cereal Blast Fungus.

Precise kinetochore-microtubule interactions ensure faithful chromosome segregation in eukaryotes. Centromeres, identified as scaffolding sites for kinetochore assembly, are among the most rapidly evolving chromosomal loci in terms of the DNA sequence and length and organization of intrinsic elements. Neither the centromere structure nor the kinetochore dynamics is well studied in plant-pathogenic fungi. Here, we sought to understand the process of chromosome segregation in the rice blast fungus Magnaporthe oryzae High-resolution imaging of green fluorescent protein (GFP)-tagged inner kinetochore proteins CenpA and CenpC revealed unusual albeit transient declustering of centromeres just before anaphase separation of chromosomes in M. oryzae Strikingly, the declustered centromeres positioned randomly at the spindle midzone without an apparent metaphase plate per se Using CenpA chromatin immunoprecipitation followed by deep sequencing, all seven centromeres in M. oryzae were found to be regional, spanning 57-kb to 109-kb transcriptionally poor regions. Highly AT-rich and heavily methylated DNA sequences were the only common defining features of all the centromeres in rice blast. Lack of centromere-specific DNA sequence motifs or repetitive elements suggests an epigenetic specification of centromere function in M. oryzae PacBio genome assemblies and synteny analyses facilitated comparison of the centromeric/pericentromeric regions in distinct isolates of rice blast and wheat blast and in Magnaporthiopsis poae Overall, this study revealed unusual centromere dynamics and precisely identified the centromere loci in the top model fungal pathogens that belong to Magnaporthales and cause severe losses in the global production of food crops and turf grasses.IMPORTANCEMagnaporthe oryzae is an important fungal pathogen that causes a loss of 10% to 30% of the annual rice crop due to the devastating blast disease. In most organisms, kinetochores are clustered together or arranged at the metaphase plate to facilitate synchronized anaphase separation of sister chromatids in mitosis. In this study, we showed that the initially clustered kinetochores separate and position randomly prior to anaphase in M. oryzae Centromeres in M. oryzae occupy large genomic regions and form on AT-rich DNA without any common sequence motifs. Overall, this study identified atypical kinetochore dynamics and mapped functional centromeres in M. oryzae to define the roles of centromeric and pericentric boundaries in kinetochore assembly on epigenetically specified centromere loci. This study should pave the way for further understanding of the contribution of heterochromatin in genome stability and virulence of the blast fungus and its related species of high economic importance.

Abstract 4403: Regulation of SATB2 via miR-31 in Ni-induced malignant cell transformation

Abstract The special AT-rich DNA binding protein (SATB2) is a nuclear matrix associated protein and an important transcription factor for biological development, gene regulation, and chromatin remodeling. Under normal circumstances, SATB2 is not expressed in most cells, including epithelial cells of the lung. However, aberrant regulation of SATB2 has been found in various types of cancers including lung, colon, prostate, breast, gastric, and liver. Emerging studies have linked the possible role of SATB2 in carcinogenic metal-induced cell transformation. In fact, our previous microarray analysis showed that SATB2 is strongly induced in Ni-transformed cells. As an IARC Class I Human Carcinogen, there is well-established link between chronic nickel exposure and lung cancer; however, despite of considerable research efforts in regards to the epigenetic mechanisms, little is known about the role of microRNAs in Ni-induced carcinogenesis. As post-transcriptional regulators, miRNAs have great importance in maintaining normal cellular development. In particular, miR-31 is one of the most abundant types of miRNAs and commonly studied in disease development, including cancers of the lung, breast, and liver. Previous studies have shown the miR-31 can directly target the homeobox gene SATB, and here we report that miR-31 is capable of regulating SATB2 in Ni-induced tumorigenesis. First, our results confirmed that knocking down SATB2 in Ni-transformed BEAS-2B cells via shRNA significantly reduced the rate of migration, anchorage-independent growth. Notably, compared to scramble control shRNA transfected cells, SATB2 knockdown cells did not generate tumor growth in mice xenograft model. These results highlight the oncogenic role of SATB2 in cell transformation. In addition, our data indicate that both acute Ni-treated and Ni-transformed cells demonstrated significant reduction in miR-31 expression. Furthermore, we show that overexpressing miR-31 in Ni-transformed cells reduced rates of migration, anchorage-independent growth, and cellular invasion, indicating the importance of miR-31 in Ni-induced BEAS-2B cell transformation. Together, our results provide a novel mechanistic pathway for Ni-induced carcinogenesis, and add to the pool of existing research on the implications of SATB2 and miR-31 in tumorigenesis. Citation Format: Qiao Yi Chen, Yusha Zhu, Ashley Jordan, Jinquan Li, Hong Sun, Thomas Kluz, Max Costa. Regulation of SATB2 via miR-31 in Ni-induced malignant cell transformation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4403.

ERCC1/XPF Is Important for Repair of DNA Double-Strand Breaks Containing Secondary Structures

SummaryThe structure-specific endonuclease ERCC1/XPF plays an important role in nucleotide excision repair and interstrand cross-link repair. In this study, we identified new functions of ERCC1/XPF in DNA double-strand break (DSB) repair. We found that the conserved function of ERCC1/XPF to remove non-homologous sequences at DSBs is a rate-limiting step for homologous recombination in mammalian cells, and more importantly, we uncovered an indispensable role of ERCC1/XPF in repair of DSBs containing DNA secondary structures, including the structure-prone AT-rich DNA sequences derived from common fragile sites and G-quadruplexes (G4s). We also demonstrated a synthetic lethal interaction of XPF with DNA translocase FANCM that is involved in removing DNA secondary structures. Furthermore, inactivation of XPF sensitizes FANCM-deficient cells to G4-interacting compounds. These results suggest an important function of ERCC1/XPF in protecting DNA secondary structures and provide a rationale for targeted treatment of FANCM-deficient tumors through inhibition of XPF.

Synthesis, characterization, DNA binding and cleaving properties of photochemically activated phenanthrene dihydrodioxin

Dihydrodioxin compounds are formed upon visible light irradiation of a mixture of an ortho-quinone and a substituted alkene. The reverse photochemical reaction can result in the release of a highly reactive ortho-quinone that can induce oxidative DNA damage. A novel DNA binding and cleaving phenanthrene dihydrodioxin compound (1) was synthesized. The photochemical release of 9,10-phenanthrenequinone was studied under different light irradiation wavelengths. Interaction of 1 with DNA caused an increase in the viscosity of DNA as well as hypochromic effect and a bathochromic shift in the absorption of 1. These results indicated the intercalative mode of binding of 1 to DNA. Binding affinities of 1 to several DNA sequences were evaluated, and slightly preferential binding to AT-rich DNA oligomers was observed. Intercalation of 1 between CT-DNA base pairs induced a partial transition from B-form to Z-form of DNA. Significant stabilization of the DNA duplex was revealed by DNA optical melting experiments with 33 % GC 12-mer (∆Tm = 9 oC). ΦX174 photocleavage assay showed that 1 is capable of > 90 % single-strand DNA cleavage and ∼ 5 % of double-strand DNA breakage. Therefore, 1 further demonstrates the potential of masked orhto-quinones as efficient photoactivated DNA cleaving agents.

Retraction: The structure of SV40 large T hexameric helicase in complex with AT-rich origin DNA.

When a cell divides to form a new cell, it must also copy its DNA. An important step for starting DNA replication is to break or “melt” the bonds between the two strands of DNA that make up the double helix. This happens at a specific site on the DNA called the replication origin, and allows double-stranded DNA to partially unwind into two single strands. Each strand acts as a template to form a new copy of its partner strand. In many organisms, ring-shaped proteins called helicases attach directly to double-stranded DNA to melt the bonds at the replication origin. Viruses also have helicases that they can use to hijack the cell’s replication machinery and get it to copy the viral DNA. One such helicase called Large T antigen is an important part of a tumor virus called simian virus 40. Large T has a ‘hexameric’ structure, similar to the cell’s own helicases, and researchers often use it as a model for studying the replication process. However, key questions remain. How do hexameric helicases interact with the double-stranded DNA replication origin? And what are the molecular mechanisms by which these helicases melt bonds in double-stranded DNA? Gai et al. used a technique called X-ray crystallography to examine the molecular structure of the Large T helicase when it is attached to DNA. The resulting structures show that six subunits of Large T helicase assemble a ring around the replication origin, each making unique bonds with the DNA. The helicase ring squeezes the DNA, partially breaking the bonds between the DNA strands and causing local melting of the DNA. With the understanding of how the Large T hexamer helicase interacts with its DNA replication origin and how its assembly initiates DNA melting, the future challenge is to explain the process by which the partially melted origin opens up fully for replication to begin.

Deregulation of SATB2 in carcinogenesis with emphasis on miRNA-mediated control.

The special AT-rich DNA binding protein (SATB2) is a nuclear matrix-associated protein and an important transcription factor for biological development, gene regulation and chromatin remodeling. Aberrant regulation of SATB2 has been found to highly correlate with various types of cancers including lung, colon, prostate, breast, gastric and liver. Recent studies have revealed that a subset of small non-coding RNAs, termed microRNAs (miRNAs), are important regulators of SATB2 function. As post-transcriptional regulators, miRNAs have been found to have fundament importance maintaining normal cellular development. Evidence suggests that multiple miRNAs, including miR-31, miR-34, miR-182, miR-211, miR-599, are capable of regulating SATB2 in cancers of the lung, liver, colon and breast. This review examines the molecular functions of SATB2 and miRNAs in the text of cancer development and potential strategies for cancer therapy with a focus on systemic miRNA delivery.

Fluorescence enhancement strategy for evaluation of the minor groove binder DAPI to complementary ssDNA sequence including telomere mimics in (ssDNA@DAPI/LDH)n ultrathin films

Nucleic acids, the hereditary material of living beings, perform a vital role in major life activities including growth, heredity, and variation. The genetic information is coded in specific nitrogenous bases, which are the key players in genetic transformation and identification of hereditary diseases. Telomeres are specialized DNA structures present at the end of eukaryotic chromosomes, consisting of repetitive non-transcribed sequences (TTAGGG) and some binding proteins. They protect the chromosomal ends from sticking to each other, and play a key role in the maintenance of genomic integrity. Currently, a number of methods are reported for nucleic acids and telomeres detection but they are not easy going. Therefore, development of sensitive and fast DNA-biosensors to measure the nucleic acids and telomeres sequences is of great importance. This paper describes a systematic study on the preparation of simple ultrathin films (UTFs) composite of fluorescent dye 4′,6-diamidino-2-phenylindole (DAPI), having binding properties in the minor groove of AT-rich DNA segments, blending with single-stranded DNA (ssDNA) and incorporation into layered double hydroxides (LDH) nanosheets using layer-by-layer self-assembly method. The resulting UTFs exhibits excellent sensitivity, selectivity and reversibility for long complementary ssDNA sequence on the basis of DNA hybridization and fluorescence enhancement strategy. The dynamic range was 3–20 μg/mL with a detection limit of 20 μg/mL. This new (ssDNA @DAPI/LDHs)n UTFs, could be an efficient hybrid composite with potential applications in the field of bioluminescent sensoring materials.

Predicting the mechanism and rate of H-NS binding to AT-rich DNA.

Bacteria contain several nucleoid-associated proteins that organize their genomic DNA into the nucleoid by bending, wrapping or bridging DNA. The Histone-like Nucleoid Structuring protein H-NS found in many Gram-negative bacteria is a DNA bridging protein and can structure DNA by binding to two separate DNA duplexes or to adjacent sites on the same duplex, depending on external conditions. Several nucleotide sequences have been identified to which H-NS binds with high affinity, indicating H-NS prefers AT-rich DNA. To date, highly detailed structural information of the H-NS DNA complex remains elusive. Molecular simulation can complement experiments by modelling structures and their time evolution in atomistic detail. In this paper we report an exploration of the different binding modes of H-NS to a high affinity nucleotide sequence and an estimate of the associated rate constant. By means of molecular dynamics simulations, we identified three types of binding for H-NS to AT-rich DNA. To further sample the transitions between these binding modes, we performed Replica Exchange Transition Interface Sampling, providing predictions of the mechanism and rate constant of H-NS binding to DNA. H-NS interacts with the DNA through a conserved QGR motif, aided by a conserved arginine at position 93. The QGR motif interacts first with phosphate groups, followed by the formation of hydrogen bonds between acceptors in the DNA minor groove and the sidechains of either Q112 or R114. After R114 inserts into the minor groove, the rest of the QGR motif follows. Full insertion of the QGR motif in the minor groove is stable over several tens of nanoseconds, and involves hydrogen bonds between the bases and both backbone and sidechains of the QGR motif. The rate constant for the process of H-NS binding to AT-rich DNA resulting in full insertion of the QGR motif is in the order of 106 M−1s−1, which is rate limiting compared to the non-specific association of H-NS to the DNA backbone at a rate of 108 M−1s−1.

Properties and directionality of intragenic promoters in E. coli

The histone-like nucleoid structuring (H-NS) protein targets and binds AT-rich DNA. Oligomerisation of H-NS along AT-rich genes represses transcription from spurious intragenic promoters within the coding sequences of AT-rich genes. In this work, we sought to better understand why promoters occur so frequently within AT-rich DNA. To do this, we compared the properties of (i) canonical promoters (ii) promoters within H-NS bound genes and (iii) promoters generated by random combinations of nucleotides. We have identified and analysed many active intragenic promoters distributed within AT-rich genes in the E. coli genome. We show that these promoters, and promoters from randomly generated AT-rich DNA, differ from canonical promoters in several ways. In particular, spurious promoters are often dependent on AT-tracts upstream of the promoter −10 element. These AT-tracts play a key role by altering DNA curvature and facilitating interactions between the promoter and RNA polymerase sigma factor. Promoter regions inside genes are also often bidirectional.

Escherichia coli CrfC Protein, a Nucleoid Partition Factor, Localizes to Nucleoid Poles via the Activities of Specific Nucleoid-Associated Proteins.

The Escherichia coli CrfC protein is an important regulator of nucleoid positioning and equipartition. Previously we revealed that CrfC homo-oligomers bind the clamp, a DNA-binding subunit of the DNA polymerase III holoenzyme, promoting colocalization of the sister replication forks, which ensures the nucleoid equipartition. In addition, CrfC localizes at the cell pole-proximal loci via an unknown mechanism. Here, we demonstrate that CrfC localizes to the distinct subnucleoid structures termed nucleoid poles (the cell pole-proximal nucleoid-edges) even in elongated cells as well as in wild-type cells. Systematic analysis of the nucleoid-associated proteins (NAPs) and related proteins revealed that HU, the most abundant NAP, and SlmA, the nucleoid occlusion factor regulating the localization of cell division apparatus, promote the specific localization of CrfC foci. When the replication initiator DnaA was inactivated, SlmA and HU were required for formation of CrfC foci. In contrast, when the replication initiation was inhibited with a specific mutant of the helicase-loader DnaC, CrfC foci were sustained independently of SlmA and HU. H-NS, which forms clusters on AT-rich DNA regions, promotes formation of CrfC foci as well as transcriptional regulation of crfC. In addition, MukB, the chromosomal structure mainetanice protein, and SeqA, a hemimethylated nascent DNA region-binding protein, moderately stimulated formation of CrfC foci. However, IHF, a structural homolog of HU, MatP, the replication terminus-binding protein, Dps, a stress-response factor, and FtsZ, an SlmA-interacting factor in cell division apparatus, little or only slightly affected CrfC foci formation and localization. Taken together, these findings suggest a novel and unique mechanism that CrfC localizes to the nucleoid poles in two steps, assembly and recruitment, dependent upon HU, MukB, SeqA, and SlmA, which is stimulated directly or indirectly by H-NS and DnaA. These factors might concordantly affect specific nucleoid substructures. Also, these nucleoid dynamics might be significant in the role for CrfC in chromosome partition.

Tight Binding of Natural Polyphenols to the Intrinsically Disordered Mammalian High Mobility Group Protein At-Hook 2

The mammalian high mobility group protein AT-hook 2 (HMGA2) is an intrinsically disordered protein (IDP) that specifically recognizes the minor-groove of AT-rich DNA sequence. It works as a multifunctional DNA-binding protein and plays important roles in tumorigenesis and adipogenesis. Previous studies showed that HMGA2 is a potential therapeutic target of anticancer and anti-obesity drugs by inhibiting its DNA-binding activities. In this study, we found that several natural polyphenols, such as aurintricarboxylic acid, gallic acid, ellagic acid, and (−)-epigallocatechin gallate (EGCG) tightly bind to HMGA2 and, as a result, strongly inhibit HMGA2 binding to AT-rich DNA sequences. Since these polyphenols can inhibit adipogenesis in vitro and in animal models, our results suggest that their anti-obesity activities may stem from their inhibition of HMGA2-DNA interactions. Because polyphenols are naturally occurring chemical compounds enriched in vegetables and fruits, our discovery strongly support that vegetables and fruits are natural remedies to treat obesity.

A label-free and enzyme-free fluorescent aptasensor for sensitive detection of acetamiprid based on AT-rich dsDNA-templated copper nanoparticles

A label-free and enzyme-free aptasensor for sensitive assay of acetamiprid has been established using AT-rich double-stranded (ds) DNA-templated copper nanoparticles (CuNPs) as fluorescent probe. In this work, two hairpin DNA, HP1 and HP2, were elaborately designed with AT-rich DNA sequences in their loops. The aptamer of acetamiprid was located at the 3′-terminal of HP1, which was caged in the stem of HP1. Upon the addition of acetamiprid, the aptamer could combine with acetamiprid to form a target/aptamer complex, and thus its free 5′-terminal was released. Subsequently, the protruded 3′-terminal of HP2 could hybridize with the free 5′-terminal of HP1 to form a stable AT-rich dsDNA. When it interacted with Cu2+ and ascorbic acid (AA), the AT-rich dsDNA/CuNPs were generated with strong fluorescence, offering a “switch-on” detection of acetamiprid. The developed strategy could high selectively detect acetamiprid at the concentration as low as 2.37 nM. Moreover, the possibility of this strategy for the food sample analysis was also investigated. The obtained results demonstrate that the developed strategy has a promising application potential for acetamiprid assay in food safety fields.

Novel genetic code and record-setting AT-richness in the highly reduced plastid genome of the holoparasitic plant Balanophora

Plastid genomes (plastomes) vary enormously in size and gene content among the many lineages of nonphotosynthetic plants, but key lineages remain unexplored. We therefore investigated plastome sequence and expression in the holoparasitic and morphologically bizarre Balanophoraceae. The two Balanophora plastomes examined are remarkable, exhibiting features rarely if ever seen before in plastomes or in any other genomes. At 15.5 kb in size and with only 19 genes, they are among the most reduced plastomes known. They have no tRNA genes for protein synthesis, a trait found in only three other plastid lineages, and thus Balanophora plastids must import all tRNAs needed for translation. Balanophora plastomes are exceptionally compact, with numerous overlapping genes, highly reduced spacers, loss of all cis-spliced introns, and shrunken protein genes. With A+T contents of 87.8% and 88.4%, the Balanophora genomes are the most AT-rich genomes known save for a single mitochondrial genome that is merely bloated with AT-rich spacer DNA. Most plastid protein genes in Balanophora consist of ≥90% AT, with several between 95% and 98% AT, resulting in the most biased codon usage in any genome described to date. A potential consequence of its radical compositional evolution is the novel genetic code used by Balanophora plastids, in which TAG has been reassigned from stop to tryptophan. Despite its many exceptional properties, the Balanophora plastome must be functional because all examined genes are transcribed, its only intron is correctly trans-spliced, and its protein genes, although highly divergent, are evolving under various degrees of selective constraint.

Sequential backbone resonance assignment of AT-rich interaction domain of human BAF200.

BAF200 is a subunit of PBAF chromatin remodeling complex that contains an N-terminal AT-rich interaction domain (ARID). ARID domain in general has been shown to bind to the AT-rich DNA sequences. The human BAF200 ARID (~ 110 residues) has the potential to bind the DNA sequences with high affinity, however, the structure and the exact contribution of hBAF200 ARID in PBAF functions as well its DNA binding specificities have not been established. In this study, we have expressed and purified the hBAF200 ARID for NMR studies. We report the complete backbone 1H, 13C, and 15N chemical shift assignment and secondary structure of hBAF200 ARID domain.

Substituent specific bisbenzimidazole binding towards AT-rich DNA

Substituent specific bisbenzimidazole binding towards AT-rich DNA

Solid-phase enzyme catalysis of DNA end repair and 3\u2032 A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA

The use of next-generation sequencing (NGS) has been instrumental in advancing biological research and clinical diagnostics. To fully utilize the power of NGS, complete, uniform coverage of the entire genome is required. In this study, we identified the primary sources of bias observed in sequence coverage across AT-rich regions of the human genome with existing amplification-free DNA library preparation methods. We have found evidence that a major source of bias is the inefficient processing of AT-rich DNA in end repair and 3′ A-tailing, causing under-representation of extremely AT-rich regions. We have employed immobilized DNA modifying enzymes to catalyze end repair and 3′ A-tailing reactions, to notably reduce the GC bias observed with existing library construction methods.

Thermodynamic Study of Interactions of Distamycin A with Chromatin in Rat Liver Nuclei in the Presence of Polyamines.

We studied the thermodynamics of melting of isolated rat liver nuclei with different degrees of chromatin condensation determined by the concentration of polyamines (PA) and the solution ionic strength, as well as the effect of the antibiotic distamycin A (DM) on melting. Differential scanning calorimetry (DSC) profiles of nuclear preparations contained three peaks that reflected melting of three main chromatin domains. The number of peaks did not depend on the degree of condensation; however, nuclei with more condensed chromatin had a higher total enthalpy. DM stabilized peaks II and III corresponding to the melting of relaxed and topologically strained DNA, respectively, but destabilized peak I corresponding to the melting of nucleosome core histones. At the saturating concentration (DM/DNA molar ratio = 0.1), DM increased Tm of peaks II and III by ~5°C and decreased Tm of peak I by ~2.5°C. Based on the dependence of ΔH on DM concentration, we established that at low DM/DNA ratio (≤0.03), when DM interacted predominantly with AT-rich DNA regions, the enthalpy of peak II decreased in parallel with the increase in the enthalpy of peak III, which indicated that DM induces structural transitions in the nuclear chromatin associated with the increase in torsional stress in DNA. An increase in free energy under saturation conditions was equal to the change in the free energy of DM interaction with DNA. However, the increase in the enthalpy of melting of the nuclei in the presence of DM was much greater than the enthalpy of titration of nuclei with DM. This indicates a significant increase in the strength of interaction between the two DNA strands apparently due, among other things, to changes in the torsional stress of DNA in the nuclei. Titration of the nuclei with increasing PA concentrations resulted in the decrease in the number of DM-binding sites and the non-monotonous dependence of the enthalpy and entropy contribution to the binding free energy on the PA content. We suggested that the observed differences in the thermodynamic parameters were due to the different width of the minor groove in the nuclear chromatin DNA, which depends on PA concentration.

Varicella-zoster virus CNS vasculitis and RNA polymerase III gene mutation in identical twins.

ObjectiveDeficiency in the cytosolic DNA sensor RNA Polymerase III (POL III) was recently described in children with severe varicella-zoster virus (VZV) infection in the CNS or lungs. Here, we describe a pair of monozygotic female twins, who both experienced severe recurrent CNS vasculitis caused by VZV reactivation. The clinical presentation and findings included recurrent episodes of headache, dizziness, and neurologic deficits, CSF with pleocytosis and intrathecal VZV antibody production, and MRI of the brain showing ischemic lesions.MethodsWe performed whole-exome sequencing and identified a rare mutation in the POL III subunit POLR3F. Subsequently, antiviral responses in patient peripheral blood mononuclear cells (PBMCs) were examined and compared with healthy controls.ResultsThe identified R50W POLR3F mutation is predicted by bioinformatics to be damaging, and when tested in functional assays, patient PBMCs exhibited impaired antiviral and inflammatory responses to the POL III agonist poly(dA:dT) and increased viral replication compared with controls.ConclusionsAltogether, these cases add genetic and immunologic evidence to the novel association between defects in sensing of AT-rich DNA present in the VZV genome and increased susceptibility to severe manifestations of VZV infection in the CNS in humans.

Peptide Sequence Influence on the Conformational Dynamics and DNA binding of the Intrinsically Disordered AT-Hook 3 Peptide

The intrinsically disordered ATHP3 was studied at native conditions and in complex with DNA using single amino acid substitutions and high-resolution ion mobility spectrometry coupled to mass spectrometry (trapped IMS-MS). Results showed that ATHP3 can exist in multiple conformations at native conditions (at least 10 conformers were separated), with a variety of proline cis/trans orientations, side chain orientations and protonation sites. When in complex with AT rich DNA hairpins, the -RGRP- core is essential for stabilizing the ATHP3: DNA complex. In particular, the arginine in the sixth position plays an important role during binding to AT-rich regions of hairpin DNA, in good agreement with previous NMR and X-ray data. Mobility based correlation matrices are proposed as a way to reveal differences in structural motifs across the peptide mutants based on the conformational space and relative conformer abundance.