Putative DNA Research Articles

Computational genome-wide survey of odorant receptors from two solitary bees Dufourea novaeangliae (Hymenoptera: Halictidae) and Habropoda laboriosa (Hymenoptera: Apidae)

Olfactory/odorant receptors (ORs) probably govern eusocial behaviour in honey bees through detection of cuticular hydrocarbons (CHCs) and queen mandibular gland pheromones (QMP). CHCs are involved in nest-mate recognition whereas QMP acts as sex pheromone for drones and as retinue pheromone for female workers. Further studies on the effect of eusociality on the evolution of ORs are hindered by the non-availability of comprehensive OR sets of solitary species. We report complete OR repertoires from two solitary bees Dufourea novaeangliae (112 ORs) and Habropoda laboriosa (151 ORs). We classify these ORs into 34 phylogenetic clades/subfamilies. Differences in the OR sets of solitary and eusocial bees are observed in individual subfamilies like subfamily 9-exon (putative CHC receptors) and L (contains putative QMP receptor group). A subfamily (H) including putative floral scent receptors is expanded in the generalist honey bees only, but not in the specialists. On the contrary, subfamily J is expanded in all bees irrespective of their degree of social complexity or food preferences. Finally, we show species-lineage specific and OR-subfamily specific differences in the putative cis-regulatory DNA motifs of the ORs from six hymenopteran species. Out of these, [A/G]CGCAAGCG[C/T] is a candidate master transcription factor binding site for multiple olfactory genes.

Copper homeostasis networks in the bacterium Pseudomonas aeruginosa

Bacterial copper (Cu+) homeostasis enables both precise metallation of diverse cuproproteins and control of variable metal levels. To this end, protein networks mobilize Cu+ to cellular targets with remarkable specificity. However, the understanding of these processes is rather fragmented. Here, we use genome-wide transcriptomic analysis by RNA-Seq to characterize the response of Pseudomonas aeruginosa to external 0.5 mm CuSO4, a condition that did not generate pleiotropic effects. Pre-steady-state (5-min) and steady-state (2-h) Cu+ fluxes resulted in distinct transcriptome landscapes. Cells quickly responded to Cu2+ stress by slowing down metabolism. This was restored once steady state was reached. Specific Cu+ homeostasis genes were strongly regulated in both conditions. Our system-wide analysis revealed induction of three Cu+ efflux systems (a P1B-ATPase, a porin, and a resistance-nodulation-division (RND) system) and of a putative Cu+-binding periplasmic chaperone and the unusual presence of two cytoplasmic CopZ proteins. Both CopZ chaperones could bind Cu+ with high affinity. Importantly, novel transmembrane transporters probably mediating Cu+ influx were among those largely repressed upon Cu+ stress. Compartmental Cu+ levels appear independently controlled; the cytoplasmic Cu+ sensor CueR controls cytoplasmic chaperones and plasma membrane transporters, whereas CopR/S responds to periplasmic Cu+ Analysis of ΔcopR and ΔcueR mutant strains revealed a CopR regulon composed of genes involved in periplasmic Cu+ homeostasis and its putative DNA recognition sequence. In conclusion, our study establishes a system-wide model of a network of sensors/regulators, soluble chaperones, and influx/efflux transporters that control the Cu+ levels in P. aeruginosa compartments.

Characterization of a cold-adapted DNA photolyase from C. psychrerythraea 34H.

The phrB gene encoding a putative cold-adapted DNA photolyase was cloned from the bacterial genomic DNA of Colwellia psychrerythraea 34H, a psychrophilic bacterium. Recombinant DNA photolyase, rCpPL, was overexpressed and purified from three different vectors. rCpPL binds its DNA substrate by flipping a cyclobutane pyrimidine dimer (CPD) into its active site and repairs CPD-containing DNA in vitro. rCpPL contains one catalytic flavin adenine dinucleotide (FAD) cofactor, but displays promiscuity in cofactor binding, in which either a flavin mononucleotide (FMN) or a methenyltetrahydrofolate (MTHF) molecule is bound as an antenna molecule and found in sub-stoichiometric amounts. The UV/Vis spectrum of oxidized rCpPL shows that the FADOX absorption maximum is the most red-shifted reported for a PL, suggesting a unique cavity electrostatic environment. Modest FAD vibronic structure suggests that the binding pocket is more flexible than warmer PLs, corroborating the hypothesis that psychrophilic proteins must be highly flexible to function at low temperatures. Fluorescence excitation data show that the freshly purified flavin cofactor is in its fully reduced state (FADH¯). A homology analysis of PL protein structures spanning 70°C in growth temperature supports the data that the structure of CpPL is quite different from its warmer cousins.

Identification of a prototypical single-stranded uracil DNA glycosylase from Listeria innocua

A recent phylogenetic study on UDG superfamily estimated a new clade of family 3 enzymes (SMUG1-like), which shares a lower homology with canonic SMUG1 enzymes. The enzymatic properties of the newly found putative DNA glycosylase are unknown. To test the potential UDG activity and evaluate phylogenetic classification, we isolated one SMUG1-like glycosylase representative from Listeria innocua (Lin). A biochemical screening of DNA glycosylase activity in vitro indicates that Lin SMUG1-like glycosylase is a single-strand selective uracil DNA glycosylase. The UDG activity on DNA bubble structures provides clue to its physiological significance in vivo. Mutagenesis and molecular modeling analyses reveal that Lin SMUG1-like glycosylase has similar functional motifs with SMUG1 enzymes; however, it contains a distinct catalytic doublet S67-S68 in motif 1 that is not found in any families in the UDG superfamily. Experimental investigation shows that the S67M-S68N double mutant is catalytically more active than either S67M or S68N single mutant. Coupled with mutual information analysis, the results indicate a high degree of correlation in the evolution of SMUG1-like enzymes. This study underscores the functional and catalytic diversity in the evolution of enzymes in UDG superfamily.

Capturing Environmental Plant Memories in DNA, with a Little Help from Chromatin.

Plants are eukaryotes living mostly immotile in harsh environments. On occasion, it is beneficial for their survival to maintain a transcriptional response to an environmental stress longer than the stress lasts (transcriptional memory) and even to reiterate such a response more quickly or more strongly when the same stress is re-encountered (priming memory). In eukaryotes, transcription takes place in the context of chromatin, the packaging material of DNA. Chromatin regulation is often invoked when it comes to environmental transcriptional and priming memory in plants, but rarely chromatin-based regulation can be accurately assigned to a given aspect of transcription in vivo. The conserved eukaryotic chromatin-modifying system Polycomb/Trithorax can support both long-term stability and flexibility of gene expression in Drosophila. The main principles of Polycomb/Trithorax regulation will be outlined and illustrated with the best-studied case of environmental memory from Arabidopsis. Despite being complex, the Polycomb/Trithorax system relies on experimentally tractable elements in the form of DNA, termed Polycomb/Trithorax Responsive Elements. PREs/TREs are essentially memory DNA elements. Here, relevant information to identify PRE/TRE-like elements in plants is highlighted. Examples of priming memory in plants are discussed in relation to the first two reported putative memory DNA elements. Arguably, similar cases from plants can be conducive in dissecting the contribution of DNA-based from chromatin-based regulation of transcription, when two types of DNA elements are defined: those representing binding sites for the transcription factors determining the environmental response and those controlling memory by regulating chromatin modification dynamics, ultimately maintaining the corresponding transcriptional state.

Whole Genome Sequencing of Mycobacterium tuberculosis Isolates From Extrapulmonary Sites.

Tuberculosis (TB) remains one of the leading causes of morbidity and mortality worldwide. Extrapulmonary tuberculosis (EPTB) constitutes around 15-20% of TB cases in immunocompetent individuals. Extrapulmonary sites that are affected by TB include bones, lymph nodes, meningitis, pleura, and genitourinary tract. Whole genome sequencing has emerged as a powerful tool to map genetic diversity among Mycobacterium tuberculosis (MTB) isolates and identify the genomic signatures associated with drug resistance, pathogenesis, and disease transmission. Several pulmonary isolates of MTB have been sequenced over the years. However, availability of whole genome sequences of MTB isolates from extrapulmonary sites is limited. Some studies suggest that genetic variations in MTB might contribute to disease presentation in extrapulmonary sites. This can be addressed if whole genome sequence data from large number of extrapulmonary isolates becomes available. In this study, we have performed whole genome sequencing of five MTB clinical isolates derived from EPTB sites using next-generation sequencing platform. We identified 1434 nonsynonymous single nucleotide variations (SNVs), 143 insertions and 105 deletions. This includes 279 SNVs that were not reported before in publicly available datasets. We found several mutations that are known to confer resistance to drugs. All the five isolates belonged to East-African-Indian lineage (lineage 3). We identified 9 putative prophage DNA integrations and 14 predicted clustered regularly interspaced short palindromic repeats (CRISPR) in MTB genome. Our analysis indicates that more work is needed to map the genetic diversity of MTB. Whole genome sequencing in conjunction with comprehensive drug susceptibility testing can reveal clinically relevant mutations associated with drug resistance.

The LacI family protein GlyR3 co-regulates the celC operon and manB in Clostridium thermocellum

BackgroundClostridium thermocellum utilizes a wide variety of free and cellulosomal cellulases and accessory enzymes to hydrolyze polysaccharides present in complex substrates. To date only a few studies have unveiled the details by which the expression of these cellulases are regulated. Recent studies have described the auto regulation of the celC operon and determined that the celC–glyR3–licA gene cluster and nearby manB–celT gene cluster are co-transcribed as polycistronic mRNA.ResultsIn this paper, we demonstrate that the GlyR3 protein mediates the regulation of manB. We first identify putative GlyR3 binding sites within or just upstream of the coding regions of manB and celT. Using an electrophoretic mobility shift assay (EMSA), we determined that a higher concentration of GlyR3 is required to effectively bind to the putative manB site in comparison to the celC site. Neither the putative celT site nor random DNA significantly binds GlyR3. While laminaribiose interfered with GlyR3 binding to the celC binding site, binding to the manB site was unaffected. In the presence of laminaribiose, in vivo transcription of the celC–glyR3–licA gene cluster increases, while manB expression is repressed, compared to in the absence of laminaribiose, consistent with the results from the EMSA. An in vitro transcription assay demonstrated that GlyR3 and laminaribiose interactions were responsible for the observed patters of in vivo transcription.ConclusionsTogether these results reveal a mechanism by which manB is expressed at low concentrations of GlyR3 but repressed at high concentrations. In this way, C. thermocellum is able to co-regulate both the celC and manB gene clusters in response to the availability of β-1,3-polysaccharides in its environment.

Crystal structure of the crenarchaeal ExoIII AP endonuclease SisExoIII reveals a conserved disulfide bond endowing the protein with thermostability

AP endonuclease recognizes and cleaves apurinic/apyrimidinic (AP) sites and plays a critical role in base excision repair. Many ExoIII and EndoIV family AP endonucleases have been characterized both biochemically and structurally in Eukaryote and Bacteria. However, relatively fewer have been studied in Euryarchaeota and there is no such report on an AP endonuclease from Crenarchaeota. Here we report, for the first time, the crystal structure of a crenarchaeal ExoIII AP endonuclease, SisExoIII, from Sulfolobus islandicus REY15A. SisExoIII comprises a two-layer core formed by 10 β-sheets and a shell formed by 9 surrounding α-helices. A disulfide bond connecting β8 and β9 is formed by Cys142 and Cys215. This intra-molecular linkage is conserved among crenarchaeal ExoIII homologs and site-directed mutagenesis revealed that it endows the protein with thermostability, however, disruption of the disulfide bond only has a slight effect on the AP endonuclease activity. We also observed that several key residues within the catalytic center including conserved Glu35 and Asn9 show different conformation compared with known ExoIII proteins and form various intra-molecular salt bridges. The protein possesses three putative DNA binding loops with higher flexibility and hydrophobicity than those of ExoIIIs from other organisms. These features may result in low AP endonuclease activity and defect of exonuclease activity of SisExoIII. The study has deepened our understanding in the structural basis of crenarchaeal ExoIII catalysis and clarified a role of the disulfide bond in maintaining protein thermostability.

Characterization and Temperature Dependence of Arctic Micromonas polaris Viruses.

Global climate change-induced warming of the Artic seas is predicted to shift the phytoplankton community towards dominance of smaller-sized species due to global warming. Yet, little is known about their viral mortality agents despite the ecological importance of viruses regulating phytoplankton host dynamics and diversity. Here we report the isolation and basic characterization of four prasinoviruses infectious to the common Arctic picophytoplankter Micromonas. We furthermore assessed how temperature influenced viral infectivity and production. Phylogenetic analysis indicated that the putative double-stranded DNA (dsDNA) Micromonas polaris viruses (MpoVs) are prasinoviruses (Phycodnaviridae) of approximately 120 nm in particle size. One MpoV showed intrinsic differences to the other three viruses, i.e., larger genome size (205 ± 2 vs. 191 ± 3 Kb), broader host range, and longer latent period (39 vs. 18 h). Temperature increase shortened the latent periods (up to 50%), increased the burst size (up to 40%), and affected viral infectivity. However, the variability in response to temperature was high for the different viruses and host strains assessed, likely affecting the Arctic picoeukaryote community structure both in the short term (seasonal cycles) and long term (global warming).

Abstract MIP-062: THE EMSY THREONINE 207 PHOSPHO–SITE IS REQUIRED FOR EMSY–DRIVEN SUPPRESSION OF DNA DAMAGE REPAIR

Abstract PURPOSE OF STUDY: EMSY, a putative DNA damage repair gene, is amplified in over 10% of high-grade serous ovarian carcinoma (HGSOC) cases. EMSY overexpression has been hypothesized to antagonize BRCA2 via direct interaction and compromise the homology-directed repair (HDR) of DNA double strand breaks. EMSY's role as a transcription factor has been described in a protein kinase AKT1 phosphorylation-dependent manner. The purpose of this study was to decipher EMSY's role in HDR and to assess the importance of its phosphorylation in this context. EXPERIMENTAL PROCEDURES: We measured HDR activity in several cell lines (U2OS osteosarcoma, H1299 non-small cell lung carcinoma and OVCAR8 HGSOC) using the DR-GFP reporter assay and RAD51 foci assessment. Endogenous immunoprecipitations (IPs) were performed with low stringency lysis buffer and protein A/G-plus agarose. V5-tagged EMSY constructs were made using the Invitrogen's Gateway TOPO cloning system. These constructs were further used to create EMSY phospho-mutants. Cells were transfected by either electroporation or FuGene reagent. For the in vitro kinase assays, EMSY constructs were sub-cloned and expressed in BL21 STAR bacteria and purified using Invitrogen's Champion pET102 Expression kit. Recombinant protein kinases were obtained from Active Motif and CellSignaling. Forskolin and H-89 were obtained from Santa Cruz Biotechnology. SUMMARY OF THE DATA: EMSY overexpression resulted in decreased HDR activity in all three DR-GFP cell lines, thus supporting the hypothesis that EMSY overexpression impairs HDR. V5-tagged EMSY overexpressing and endogenous immunoprecipitation experiments demonstrated no interaction between EMSY and BRCA2, suggesting EMSY's role in HDR to be BRCA2-independent. We confirmed that EMSY is phosphorylated by AKT1 at serine 209 phospho-site and identified a previously unknown phospho-site at threonine 207. We identified protein kinase A (PKA) as a kinase targeting EMSY T207. Furthermore, by performing both DR-GFP assay and RAD51 foci assessment in OVCAR8 cells that overexpress WT EMSY or either phospho-mutant, we demonstrated that mutant EMSY-S209A affects HDR activity similar to the WT EMSY while EMSY-T207A does not. This suggests the importance of PKA and the T207 phospho site for the EMSY-driven HDR suppression. CONCLUSIONS: EMSY-overexpressing cells show decreased HDR activity, demonstrating EMSY's relevance to the HDR pathway. Our data support the notion that EMSY-driven HDR impairment is BRCA2-interaction-independent and challenges the currently held impression that EMSY overexpression mimics the BRCA2-depleted phenotype via direct interaction. We found a new phospho-site at EMSY T207 and identified PKA as a targeting kinase. Phosphorylation of EMSY at T207, but not S209 phospho-site is necessary for EMSY-driven suppression of HDR. We suggest that an increase in EMSY's T207 phosphorylation in patients bearing EMSY-amplified tumors could enhance BRCAness and render these patients more sensitive to drugs effective in HDR-impaired setting, such as PARP inhibitors. Citation Format: Petar Jelinic, Laura A. Eccles, Jill Tseng, Paulina Cybulska, Simon N. Powell, Douglas A. Levine. THE EMSY THREONINE 207 PHOSPHO–SITE IS REQUIRED FOR EMSY–DRIVEN SUPPRESSION OF DNA DAMAGE REPAIR [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr MIP-062.

Abstract A03: The EMSY threonine 207 phospho-site is required for EMSY-driven suppression of DNA damage repair

Abstract Introduction: EMSY, a putative DNA damage repair gene, is amplified in over 10% of high-grade serous ovarian carcinoma (HGSOC) cases. EMSY overexpression has been hypothesized to antagonize BRCA2 via direct interaction and compromise the homology-directed repair (HDR) of DNA double strand breaks. Its role as a transcription factor has been described in a protein kinase AKT1 phosphorylation-dependent manner. The purpose of this study was to decipher EMSY's role in HDR and to assess the importance of its phosphorylation in this context. Experimental procedures: We measured HDR activity in several cell lines (U2OS osteosarcoma, H1299 non-small cell lung carcinoma and OVCAR8 HGSOC) using the DR-GFP reporter assay and RAD51 foci assessment. Endogenous immunoprecipitations (IPs) were performed with low stringency lysis buffer and protein A/G-plus agarose. V5-tagged EMSY constructs were made using the Invitrogen's Gateway TOPO cloning system. These constructs were further used to create EMSY phospho-mutants. Cells were transfected by either electroporation or FuGene reagent. For the in vitro kinase assays, EMSY constructs were sub-cloned and expressed in BL21 STAR bacteria and purified using Invitrogen's Champion pET102 Expression kit. Recombinant protein kinases were obtained from Active Motif and CellSignaling. Forskolin and H-89 were obtained from Santa Cruz Biotechnology. Summary of the data: EMSY overexpression resulted in decreased HDR activity in all three DR-GFP cell lines, thus supporting the hypothesis that EMSY overexpression impairs HDR. V5-tagged EMSY overexpressing and endogenous immunoprecipitation experiments demonstrated no interaction between EMSY and BRCA2, suggesting EMSY's role in HDR to be BRCA2-independent. We confirmed that EMSY is phosphorylated by AKT1 at serine 209 phospho-site and identified a previously unknown phospho-site at threonine 207. We identified protein kinase A (PKA) as a kinase targeting EMSY T207. Furthermore, by performing both DR-GFP assay and RAD51 foci assessment in OVCAR8 cells that overexpress WT EMSY or either phospho-mutant, we demonstrated that mutant EMSY-S209A affects HDR activity similar to the WT EMSY while EMSY-T207A does not. This suggests the importance of PKA and the T207 phospho site for the EMSY-driven HDR suppression. Conclusions: EMSY-overexpressing cells show decreased HDR activity, demonstrating EMSY's relevance to the HDR pathway. Our data support the notion that EMSY-driven HDR impairment is BRCA2-interaction-independent and challenges the currently held impression that EMSY overexpression mimics the BRCA2-depleted phenotype via direct interaction. We found a new phospho-site at EMSY T207 and identified PKA as a targeting kinase. Phosphorylation of EMSY at T207, but not S209 phospho-site is necessary for EMSY-driven suppression of HDR. We suggest that an increase in EMSY's T207 phosphorylation in patients bearing EMSY¬-amplified tumors could enhance BRCAness and render these patients more sensitive to drugs effective in HDR-impaired setting, such as PARP inhibitors. Citation Format: Petar Jelinic, Laura Eccles, Jill Tseng, Paulina Cybulska, Simon N. Powell, Douglas Levine. The EMSY threonine 207 phospho-site is required for EMSY-driven suppression of DNA damage repair [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr A03.

NFAT5 and SLC4A10 Loci Associate with Plasma Osmolality.

Disorders of water balance, an excess or deficit of total body water relative to body electrolyte content, are common and ascertained by plasma hypo- or hypernatremia, respectively. We performed a two-stage genome-wide association study meta-analysis on plasma sodium concentration in 45,889 individuals of European descent (stage 1 discovery) and 17,637 additional individuals of European descent (stage 2 replication), and a transethnic meta-analysis of replicated single-nucleotide polymorphisms in 79,506 individuals (63,526 individuals of European descent, 8765 individuals of Asian Indian descent, and 7215 individuals of African descent). In stage 1, we identified eight loci associated with plasma sodium concentration at P<5.0 × 10-6 Of these, rs9980 at NFAT5 replicated in stage 2 meta-analysis (P=3.1 × 10-5), with combined stages 1 and 2 genome-wide significance of P=5.6 × 10-10 Transethnic meta-analysis further supported the association at rs9980 (P=5.9 × 10-12). Additionally, rs16846053 at SLC4A10 showed nominally, but not genome-wide, significant association in combined stages 1 and 2 meta-analysis (P=6.7 × 10-8). NFAT5 encodes a ubiquitously expressed transcription factor that coordinates the intracellular response to hypertonic stress but was not previously implicated in the regulation of systemic water balance. SLC4A10 encodes a sodium bicarbonate transporter with a brain-restricted expression pattern, and variant rs16846053 affects a putative intronic NFAT5 DNA binding motif. The lead variants for NFAT5 and SLC4A10 are cis expression quantitative trait loci in tissues of the central nervous system and relevant to transcriptional regulation. Thus, genetic variation in NFAT5 and SLC4A10 expression and function in the central nervous system may affect the regulation of systemic water balance.

Kinetic analysis, size profiling, and bioenergetic association of DNA released by selected cell lines in vitro.

Although circulating DNA (cirDNA) analysis shows great promise as a screening tool for a wide range of pathologies, numerous stumbling blocks hinder the rapid translation of research to clinical practice. This is related directly to the inherent complexity of the in vivo setting, wherein the influence of complex systems of interconnected cellular responses and putative DNA sources creates a seemingly arbitrary representation of the quantitative and qualitative properties of the cirDNA in the blood of any individual. Therefore, to evaluate the potential of in vitro cell cultures to circumvent the difficulties encountered in in vivo investigations, the purpose of this work was to elucidate the characteristics of the DNA released [cell-free DNA (cfDNA)] by eight different cell lines. This revealed three different forms of cfDNA release patterns and the presence of nucleosomal fragments as well as actively released forms of DNA, which are not only consistently observed in every tested cell line, but also in plasma samples. Correlations between cfDNA release and cellular origin, growth rate, and cancer status were also investigated by screening and comparing bioenergetics flux parameters. These results show statistically significant correlations between cfDNA levels and glycolysis, while no correlations between cfDNA levels and oxidative phosphorylation were observed. Furthermore, several correlations between growth rate, cancer status, and dependency on aerobic glycolysis were observed. Cell cultures can, therefore, successfully serve as closed-circuit models to either replace or be used in conjunction with biofluid samples, which will enable sharper focus on specific cell types or DNA origins.

Bug mapping and fitness testing of chemically synthesized chromosome X.

Debugging a genome sequence is imperative for successfully building a synthetic genome. As part of the effort to build a designer eukaryotic genome, yeast synthetic chromosome X (synX), designed as 707,459 base pairs, was synthesized chemically. SynX exhibited good fitness under a wide variety of conditions. A highly efficient mapping strategy called pooled PCRTag mapping (PoPM), which can be generalized to any watermarked synthetic chromosome, was developed to identify genetic alterations that affect cell fitness ("bugs"). A series of bugs were corrected that included a large region bearing complex amplifications, a growth defect mapping to a recoded sequence in FIP1, and a loxPsym site affecting promoter function of ATP2 PoPM is a powerful tool for synthetic yeast genome debugging and an efficient strategy for phenotype-genotype mapping.

Permanganate/S1 Nuclease Footprinting Reveals Non-B DNA Structures with Regulatory Potential across a Mammalian Genome

DNA in cells is predominantly B-form double helix. Though certain DNA sequences invitro may fold into other structures, such as triplex, left-handed Zform, or quadruplex DNA, the stability and prevalence of these structures invivo are not known. Here, using computational analysis of sequence motifs, RNA polymerase II binding data, and genome-wide potassium permanganate-dependent nuclease footprinting data, we map thousands of putative non-B DNA sites at high resolution in mouse B cells. Computational analysis associates these non-B DNAs with particular structures and indicates that they form at locations compatible with an involvement in gene regulation. Further analyses support the notion that non-B DNA structure formation influences the occupancy and positioning of nucleosomes in chromatin. These results suggest that non-B DNAs contribute to the control of a variety of critical cellular and organismal processes.

IFI16 and cGAS cooperate in the activation of STING during DNA sensing in human keratinocytes

Many human cells can sense the presence of exogenous DNA during infection though the cytosolic DNA receptor cyclic GMP-AMP synthase (cGAS), which produces the second messenger cyclic GMP-AMP (cGAMP). Other putative DNA receptors have been described, but whether their functions are redundant, tissue-specific or integrated in the cGAS-cGAMP pathway is unclear. Here we show that interferon-γ inducible protein 16 (IFI16) cooperates with cGAS during DNA sensing in human keratinocytes, as both cGAS and IFI16 are required for the full activation of an innate immune response to exogenous DNA and DNA viruses. IFI16 is also required for the cGAMP-induced activation of STING, and interacts with STING to promote STING phosphorylation and translocation. We propose that the two DNA sensors IFI16 and cGAS cooperate to prevent the spurious activation of the type I interferon response.

Identification of Symmetrical RNA Editing Events in the Mitochondria of Salvia miltiorrhiza by Strand-specific RNA Sequencing

Salvia miltiorrhiza is one of the most widely-used medicinal plants. Here, we systematically analyzed the RNA editing events in its mitochondria. We developed a pipeline using REDItools to predict RNA editing events from stand-specific RNA-Seq data. The predictions were validated using reverse transcription, RT-PCR amplification and Sanger sequencing experiments. Putative sequences motifs were characterized. Comparative analyses were carried out between S. miltiorrhiza, Arabidopsis thaliana and Oryza sativa. We discovered 1123 editing sites, including 225 “C to U” sites in the protein-coding regions. Fourteen of sixteen (87.5%) sites were validated. Three putative DNA motifs were identified around the predicted sites. The nucleotides on both strands at 115 of the 225 sites had undergone RNA editing, which we called symmetrical RNA editing (SRE). Four of six these SRE sites (66.7%) were experimentally confirmed. Re-examination of strand-specific RNA-Seq data from A. thaliana and O. sativa identified 327 and 369 SRE sites respectively. 78, 20 and 13 SRE sites were found to be conserved among A. thaliana, O. sativa and S. miltiorrhiza respectively. This study provides a comprehensive picture of RNA editing events in the mitochondrial genome of S. miltiorrhiza. We identified SREs for the first time, which may represent a universal phenomenon.

Investigation of a Putative Novel Papillomavirus that causes InvertedPapillomas in Horses

The Papillomaviridae family comprises a large number of viruses that can infect a broad range of hosts giving rise to benign lesions of the skin or mucosal membranes. Until today, seven viruses termed EcPV1-7 were identified in equine papillomas. Two trotter stallion siblings at the age of two were referred to the Equine Clinic of Free University of Berlin, because of erythema, alopecia and crusting. After a diagnosis of immune-mediated skin disease, the horses underwent long-term treatment with glucocorticoids and azathioprine. Two months later, both horses developed hundreds of small wart-like proliferations on the head, neck, chest and trunk. Six healthy horses, housed in the same stable, were used to assess the occurrence of the virus in healthy skin. Biopsies were obtained for histopathology and for amplification and cloning of the papillomavirus genome as well as for sequence analysis. Histopathology revealed well demarcated cup-shaped epidermal proliferations. Foci were covered by parakeratotic, or abnormally formed, keratin. In deeper layers, numerous enlarged cells with cytoplasmic eosinophilic inclusion-like structures and occasional intranuclear basophilic inclusion bodies were striking, existing beside cells with a peripheral cytoplasmic clearing (koilocytes). Histopathological findings were consistent with endophytic papilloma similar to the Le Net-type described in immunosuppressed dogs caused by a distinct canine papillomavirus. Furthermore, papillomavirus DNA was detected in tissue samples using a broad range PCR analysis. Preliminary phylogenetic analysis of a partial sequence from the L1 gene indicated a putative new papillomavirus that has not been described in horses so far. Biopsies of the control horses showed no abnormalities in histopathology and were tested negative for the putative novel papilloma virus DNA.

Putative DNA modification methylase DR_C0020 of Deinococcus radiodurans is an atypical SAM dependent C-5 cytosine DNA methylase

BackgroundControl of cellular processes by epigenetic modification of cytosine in DNA is widespread among living organisms, but, is hitherto unknown in the extremely radioresistant microbe D. radiodurans. MethodsC-5 methyl cytosines (m5C) were detected by immuno-blotting with m5C-specific antibody. Site of cytosine methylation by DR_C0020 encoded protein was investigated by bisulfite sequencing. The DR_C0020 knockout mutant (Δdcm), constructed by site directed mutagenesis, was assessed for effect on growth, radiation resistance and proteome. Proteins were identified by mass spectrometry. ResultsMethylated cytosines were detected in the D. radiodurans genome. The DR_C0020 encoded protein (Dcm, NCBI accession: WP_034351354.1), whose amino acid sequence resembles m4C methylases, was shown to be the lone SAM-dependent C-5 cytosine methyltransferase. Purified Dcm protein was found to methylate CpN sequence with a preference for methylation of two consecutive cytosines. The Δdcm strain completely lost m5C modification from its genome, had no effect on growth but became radiation sensitive. The Δdcm cells exhibited minor alterations in the abundance of several proteins involved primarily in protein homeostasis, oxidative stress defense, metabolism, etc. ConclusionDR_C0020 encoded SAM-dependent methyltransferase Dcm is solely responsible for C-5cytosine methylation at CpN sites in the genome of D. radiodurans and regulates protein homeostasis under normal growth conditions. The protein is an unusual case of an amino methyltransferase that has evolved to producing m5C. General significanceAlthough, dispensable under optimal growth conditions, the presence of m5C may be important for recognition of parent strand and, thus, could contribute to the extraordinary DNA repair in D. radiodurans.

Structural and functional analysis of BF2549, a PadR-like transcription factor from Bacteroides fragilis

A phenolic acid decarboxylase (padC) regulator, PadR and its homologs proteins belong to the PadR family. Despite the growing numbers of the PadR family members and their various roles in bacteria, such as detoxifications, drug transports and circadian rhythms, biochemical and biophysical studies of the PadR family are very limited. Thus, a ligand-induced regulatory mechanism of the PadR family transcription factors remains to be elucidated. Here, we report a crystal structure of a Bacteroides fragilis PadR-like protein, BF2549 and revealed its interaction with putative operator DNA and ligand molecules. Comparative structural and primary sequence analyses provide a PadR-specific motif that is conserved in the PadR family but deviated from the MarR family. Furthermore, putative ligand binding sites are observed in the BF2549 structure. Finally, a homology-based structure model of BF2549 and 29-mer dsDNA propose regulatory mechanisms of the PadR family in transcriptional derepression.