N-terminal Basic Domain Research Articles

Autoregulation of TRF2 through G-Quadruplex-Specific Interaction between the Gene and N-Terminal Domain of the Protein.

Telomere repeat-binding factor 2 (TRF2) is a key component of the shelterin complex which guards the integrity of the telomere. Most of the TRF2 discussed previously was focused on the telomere, and relatively less is discussed on aspects other than that. It is proved that TRF2 also localizes to other potential G-quadruplex-forming sequences among the whole genome besides the telomere. Therefore, it may participate in regulating genes generally except for the well-known function of protecting telomeres. Here, we demonstrate that the N-terminal basic domain of TRF2 (TRF2B) can interact with the G-quadruplex formed by the 5'-UTR sequence of its gene. Subsequently, this interaction was identified as G-quadruplex-specific. Using a reporter gene system, we proved that the translation of the reporter gene was dramatically reduced, triggered by the interaction between TRF2B and the G-quadruplex. Altogether, we propose that TRF2 can be "auto-regulated" through the G-quadruplex formed by its own gene sequence. This finding indicates a potential feedback mechanism in the regulation of the TRF2 gene. Additionally, it suggests a common mode in gene regulation involving the cooperation of TRF2 and the G-quadruplex.

The impact of mutations affecting highly conserved amino acids in the simian immunodeficiency virus nucleocapsid protein on virion assembly, genomic RNA packaging and viral infectivity

The nucleocapsid (NC) domain of the retroviral Gag polyproteins mediates the incorporation of the viral genomic RNA into virions. Although SIV is widely used as a model for human immunodeficiency virus type 1 (HIV-1) infections, the SIV NC has been the subject of few studies which have provided discrepant data on the relative contribution of the two NC zinc finger motifs to genomic RNA encapsidation. Here, we demonstrate that mutations affecting the first cysteine in the distal zinc finger motif (C33S) or the N-terminal NC basic domain (R7A/K8A) drastically impair virion assembly and viral RNA binding. By contrast, amino acid substitutions targeting the first cysteine of the proximal zinc finger (C12S) or the basic region connecting both zinc fingers (R29A/R30A) allow substantial particle production and genomic RNA encapsidation. Our results help define the relative contribution of the SIV NC zinc finger motifs and basic regions to the NC biological properties.

Activation of the Ca2+/NFAT Pathway by Assembly of Hepatitis C Virus Core Protein into Nucleocapsid-like Particles

Hepatitis C virus (HCV) is the primary pathogen responsible for liver cirrhosis and hepatocellular carcinoma. The main virion component, the core (C) protein, has been linked to several aspects of HCV pathology, including oncogenesis, immune evasion and stress responses. We and others have previously shown that C expression in various cell lines activates Ca2+ signaling and alters Ca2+ homeostasis. In this study, we identified two distinct C protein regions that are required for the activation of Ca2+/NFAT signaling. In the basic N-terminal domain, which has been implicated in self-association of C, amino acids 1–68 were critical for NFAT activation. Sedimentation analysis of four mutants in this domain revealed that association of the C protein into nucleocapsid-like particles correlated with NFAT-activated transcription. The internal, lipid droplet-targeting domain was not required for NFAT-activated transcription. Finally, the C-terminal ER-targeting domain was required in extenso for the C protein to function. Our results indicate that targeting of HCV C to the ER is necessary but not sufficient for inducing Ca2+/NFAT signaling. Taken together, our data are consistent with a model whereby proteolytic intermediates of C with an intact transmembrane ER-anchor assemble into pore-like structures in the ER membrane.

Mitotic phosphorylation by NEK6 and NEK7 reduces the microtubule affinity of EML4 to promote chromosome congression.

EML4 is a microtubule-associated protein that promotes microtubule stability. We investigated its regulation across the cell cycle and found that EML4 was distributed as punctate foci along the microtubule lattice in interphase but exhibited reduced association with spindle microtubules in mitosis. Microtubule sedimentation and cryo-electron microscopy with 3D reconstruction revealed that the basic N-terminal domain of EML4 mediated its binding to the acidic C-terminal tails of α- and β-tubulin on the microtubule surface. The mitotic kinases NEK6 and NEK7 phosphorylated the EML4 N-terminal domain at Ser144 and Ser146 in vitro, and depletion of these kinases in cells led to increased EML4 binding to microtubules in mitosis. An S144A-S146A double mutant not only bound inappropriately to mitotic microtubules but also increased their stability and interfered with chromosome congression. In addition, constitutive activation of NEK6 or NEK7 reduced the association of EML4 with interphase microtubules. Together, these data support a model in which NEK6- and NEK7-dependent phosphorylation promotes the dissociation of EML4 from microtubules in mitosis in a manner that is required for efficient chromosome congression.

Shelterin Components Modulate the Phase-Separation Propensity of Telomeres

Telomeres are nucleoprotein complexes that provide a mechanism to shelter the ends of chromosomes and are essential to maintain chromosome stability in Eukaryotes. In cells, individual telomeres are bound by the six-protein shelterin complex (TRF2-hRap1-Tin2-TRF1-TPP1-Pot1) and form distinct condensed globules of finite size. Interestingly, incells that maintain their telomeres via an alternative lengthening mechanism, multiple telomeric DNAs and shelterin proteins coalesce in membrane-less promyelocytic leukemia (PML) nuclear bodies. The mechanism controlling this dual nature of telomere organization is currently not understood. Here, we propose that shelterin proteins modulate the phase-separation propensity of telomeres, leading to either the condensation of single DNA chains or the coalescence of multiple ones. In particular, we use a combination of optical and fluorescence microscopy and single-molecule experiments to quantify the contribution of shelterin components to phase-separation and condensation of single DNA chains. Our experiments reveal that TRF2 promotes phase-separation of TRF2-DNA mixtures. Even though the N-terminal basic domain and the dimerization domain of TRF2 are sufficient to promote phase-separation, the C-terminal DNA-binding domain imparts specificity for telomeric DNA repeats. Moreover, addition of shelterin components, such as hRap1, provides a fine-tuning of the properties of the resulting dense and de-mixed phase.

Biphasic recruitment of TRF2 to DNA damage sites promotes non-sister chromatid homologous recombination repair.

TRF2 (TERF2) binds to telomeric repeats and is critical for telomere integrity. Evidence suggests that it also localizes to non-telomeric DNA damage sites. However, this recruitment appears to be precarious and functionally controversial. We find that TRF2 recruitment to damage sites occurs by a two-step mechanism: the initial rapid recruitment (phase I), and stable and prolonged association with damage sites (phase II). Phase I is poly(ADP-ribose) polymerase (PARP)-dependent and requires the N-terminal basic domain. The phase II recruitment requires the C-terminal MYB/SANT domain and the iDDR region in the hinge domain, which is mediated by the MRE11 complex and is stimulated by TERT. PARP-dependent recruitment of intrinsically disordered proteins contributes to transient displacement of TRF2 that separates two phases. TRF2 binds to I-PpoI-induced DNA double-strand break sites, which is enhanced by the presence of complex damage and is dependent on PARP and the MRE11 complex. TRF2 depletion affects non-sister chromatid homologous recombination repair, but not homologous recombination between sister chromatids or non-homologous end-joining pathways. Our results demonstrate a unique recruitment mechanism and function of TRF2 at non-telomeric DNA damage sites.

Genome-wide Control of Heterochromatin Replication by the Telomere Capping Protein TRF2

Hard-to-replicate regions of chromosomes (e.g., pericentromeres, centromeres, and telomeres) impede replication fork progression, eventually leading, in the event of replication stress, to chromosome fragility, aging, and cancer. Our knowledge of the mechanisms controlling the stability of these regions is essentially limited to telomeres, where fragility iscounteracted by the shelterin proteins. Here we show that the shelterin subunit TRF2 ensures progression of the replication fork through pericentromeric heterochromatin, but not centromeric chromatin. In a process involving its N-terminal basic domain, TRF2 binds to pericentromeric Satellite III sequences during S phase, allowing the recruitment of the G-quadruplex-resolving helicase RTEL1 to facilitate fork progression. We also show that TRF2 is required for the stability of other heterochromatic regions localized throughout the genome, paving the way for future research on heterochromatic replication and its relationship with aging and cancer.

TRF1 participates in chromosome end protection by averting TRF2-dependent telomeric R loops.

The shelterin protein TRF2 assembles protective T-loops at chromosome ends by stimulating intramolecular invasion of the telomeric G-rich ssDNA overhang into the duplex telomeric array. The other shelterin factor TRF1 is thought to mainly facilitate telomeric dsDNA replication without directly participating in end protection. Here we show that in vitro human TRF2 stimulates invasion of G-rich TERRA-like RNA into telomeric dsDNA leading to formation of telomeric RNA:DNA hybrids (telR-loops). The N-terminal basic domain of TRF2 binds to TERRA-like RNA and enables TRF2 to promote efficient RNA invasion. TRF1, through its N-terminal acidic domain, counteracts TRF2-mediated RNA but not ssDNA invasion. In vivo, when TRF1 is depleted or replaced with a variant lacking the acidic domain, TRF2 induces formation of telR-loops, which in turn cause telomere loss. Hence, uncontrolled TRF2 threatens telomere integrity and TRF1 directly supports end protection by suppressing harmful telR-loops.

Basic domain of telomere guardian TRF2 reduces D-loop unwinding whereas Rap1 restores it.

Telomeric repeat binding factor 2 (TRF2) folds human telomeres into loops to prevent unwanted DNA repair and chromosome end-joining. The N-terminal basic domain of TRF2 (B-domain) protects the telomeric displacement loop (D-loop) from cleavage by endonucleases. Repressor activator protein 1 (Rap1) binds TRF2 and improves telomeric DNA recognition. We found that the B-domain of TRF2 stabilized the D-loop and thus reduced unwinding by BLM and RPA, whereas the formation of the Rap1–TRF2 complex restored DNA unwinding. To understand how the B-domain of TRF2 affects DNA binding and D-loop processing, we analyzed DNA binding of full-length TRF2 and a truncated TRF2 construct lacking the B-domain. We quantified how the B-domain improves TRF2’s interaction with DNA via enhanced long-range electrostatic interactions. We developed a structural envelope model of the B-domain bound on DNA. The model revealed that the B-domain is flexible in solution but becomes rigid upon binding to telomeric DNA. We proposed a mechanism for how the B-domain stabilizes the D-loop.

Basic domain of telomere guardian TRF2 reduces D-loop unwinding whereas Rap1 restores it.

Telomeric repeat binding factor 2 (TRF2) folds human telomeres into loops to prevent unwanted DNA repair and chromosome end-joining. The N-terminal basic domain of TRF2 (B-domain) protects the telomeric displacement loop (D-loop) from cleavage by endonucleases. Repressor activator protein 1 (Rap1) binds TRF2 and improves telomeric DNA recognition. We found that the B-domain of TRF2 stabilized the D-loop and thus reduced unwinding by BLM and RPA, whereas the formation of the Rap1–TRF2 complex restored DNA unwinding. To understand how the B-domain of TRF2 affects DNA binding and D-loop processing, we analyzed DNA binding of full-length TRF2 and a truncated TRF2 construct lacking the B-domain. We quantified how the B-domain improves TRF2’s interaction with DNA via enhanced long-range electrostatic interactions. We developed a structural envelope model of the B-domain bound on DNA. The model revealed that the B-domain is flexible in solution but becomes rigid upon binding to telomeric DNA. We proposed a mechanism for how the B-domain stabilizes the D-loop.

Major satellite repeat RNA stabilize heterochromatin retention of Suv39h enzymes by RNA-nucleosome association and RNA:DNA hybrid formation.

The Suv39h1 and Suv39h2 histone lysine methyltransferases are hallmark enzymes at mammalian heterochromatin. We show here that the mouse Suv39h2 enzyme differs from Suv39h1 by containing an N-terminal basic domain that facilitates retention at mitotic chromatin and provides an additional affinity for major satellite repeat RNA. To analyze an RNA-dependent interaction with chromatin, we purified native nucleosomes from mouse ES cells and detect that Suv39h1 and Suv39h2 exclusively associate with poly-nucleosomes. This association was attenuated upon RNaseH incubation and entirely lost upon RNaseA digestion of native chromatin. Major satellite repeat transcripts remain chromatin-associated and have a secondary structure that favors RNA:DNA hybrid formation. Together, these data reveal an RNA-mediated mechanism for the stable chromatin interaction of the Suv39h KMT and suggest a function for major satellite non-coding RNA in the organization of an RNA-nucleosome scaffold as the underlying structure of mouse heterochromatin.

MS2 Lysis of Escherichia coli Depends on Host Chaperone DnaJ.

The L protein of the single-stranded RNA phage MS2 causes lysis of Escherichia coli without inducing bacteriolytic activity or inhibiting net peptidoglycan (PG) synthesis. To find host genes required for L-mediated lysis, spontaneous Ill (insensitivity to Llysis) mutants were selected as survivors of L expression and shown to have a missense change of the highly conserved proline (P330Q) in the C-terminal domain of DnaJ. In the dnaJP330Q mutant host, L-mediated lysis is completely blocked at 30°C without affecting the intracellular levels of L. At higher temperatures (37°C and 42°C), both lysis and L accumulation are delayed. The lysis block at 30°C in the dnaJP330Q mutant was recessive and could be suppressed by Lovercomes dnaJ (Lodj ) alleles selected for restoration of lysis. All three Lodj alleles lack the highly basic N-terminal half of the lysis protein and cause lysis ∼20 min earlier than full-length L. DnaJ was found to form a complex with full-length L. This complex was abrogated by the P330Q mutation and was absent with the Lodj truncations. These results suggest that, in the absence of interaction with DnaJ, the N-terminal domain of L interferes with its ability to bind to its unknown target. The lysis retardation and DnaJ chaperone dependency conferred by the nonessential, highly basic N-terminal domain of L resembles the SlyD chaperone dependency conferred by the highly basic C-terminal domain of the E lysis protein of ϕX174, suggesting a common theme where single-gene lysis can be modulated by host factors influenced by physiological conditions.IMPORTANCE Small single-stranded nucleic acid lytic phages (Microviridae and Leviviridae) lyse their host by expressing a single "protein antibiotic." The protein antibiotics from two out of three prototypic small lytic viruses have been shown to inhibit two different steps in the conserved PG biosynthesis pathway. However, the molecular basis of lysis caused by L, the lysis protein of the third prototypic virus, MS2, is unknown. The significance of our research lies in the identification of DnaJ as a chaperone in the MS2 L lysis pathway and the identification of the minimal lytic domain of MS2 L. Additionally, our research highlights the importance of the highly conserved P330 residue in the C-terminal domain of DnaJ for specific protein interactions.

Heterochiral Jun and Fos bZIP peptides form a coiled-coil heterodimer that is competent for DNA binding.

Coiled coils, consisting of at least two α-helices, have important roles in the regulation of transcription, cell differentiation, and cell growth. Peptides composed of d-amino acids (d-peptides) have received great attention for their potential in biomedical applications, because they give large diversity for the design of peptidyl drug and are more resistant to proteolytic digestion than l-peptides. However, the interactions between l-peptides/l-protein and d-peptides in the formation of complex are poorly understood. In this study, stereoisomer-specific peptides were constructed corresponding to regions of the basic-leucine-zipper domains of Jun and Fos proteins. basic-leucine-zipper domains consist of an N-terminal basic domain, which is responsible for DNA binding, and a C-terminal domain that enables homodimerization or heterodimerization via formation of a coiled-coil. By combining peptides with different stereochemistries, the d-l heterochiral Jun-Fos heterodimer formation induced DNA binding by the basic domains of Jun-Fos. Our study provides new insight into the interaction between l-peptide and d-peptide enantiomers for developing d-peptide materials and drugs. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

FlhF Is Required for Swarming Motility and Full Pathogenicity of Bacillus cereus.

Besides sporulation, Bacillus cereus can undergo a differentiation process in which short swimmer cells become elongated and hyperflagellated swarmer cells that favor migration of the bacterial community on a surface. The functionally enigmatic flagellar protein FlhF, which is the third paralog of the signal recognition particle (SRP) GTPases Ffh and FtsY, is required for swarming in many bacteria. Previous data showed that FlhF is involved in the control of the number and positioning of flagella in B. cereus. In this study, in silico analysis of B. cereus FlhF revealed that this protein presents conserved domains that are typical of SRPs in many organisms and a peculiar N-terminal basic domain. By proteomic analysis, a significant effect of FlhF depletion on the amount of secreted proteins was found with some proteins increased (e.g., B component of the non-hemolytic enterotoxin, cereolysin O, enolase) and others reduced (e.g., flagellin, L2 component of hemolysin BL, bacillolysin, sphingomyelinase, PC-PLC, PI-PLC, cytotoxin K) in the extracellular proteome of a ΔflhF mutant. Deprivation of FlhF also resulted in significant attenuation in the pathogenicity of this strain in an experimental model of infection in Galleria mellonella larvae. Our work highlights the multifunctional role of FlhF in B. cereus, being this protein involved in bacterial flagellation, swarming, protein secretion, and pathogenicity.

Time-Resolved Detection of Light-Induced Dimerization of Monomeric Aureochrome-1 and Change in Affinity for DNA.

Aureochrome (Aureo) is a recently discovered blue light sensor protein initially from Vaucheria frigida, in which it controls blue light-dependent branch formation and/or development of a sex organ by a light-dependent change in the affinity for DNA. Although photochemical reactions of Aureo-LOV (LOV is a C-terminal light-oxygen-voltage domain) and the N-terminal truncated construct containing a bZIP (N-terminal basic leucine zipper domain) and a LOV domain have previously been reported, the reaction kinetics of the change in affinity for DNA have never been elucidated. The reactions of Aureo where the cysteines are replaced by serines (AureoCS) as well as the kinetics of the change in affinity for a target DNA are investigated in the time-domain. The dimerization rate constant is obtained as 2.8 × 10(4) M(-1) s(-1), which suggests that the photoinduced dimerization occurs in the LOV domain and the bZIP domain dimerizes using the interaction with DNA. Surprisingly, binding with the target DNA is completed very quickly, 7.7 × 10(4) M(-1) s(-1), which is faster than the protein dimerization rate. It is proposed that the nonspecific electrostatic interaction, which is observed as a weak binding with DNA, may play a role in the efficient searching for the target sequence within the DNA.

Overview of Lactoferrin as a Natural Immune Modulator

Lactoferrin is thought to be the most polyvalent protein present in host defense against tissue injuries and infections in vertebrates. Owing to the propensity of its basic N-terminal domain to interact with various microbial and host targets, lactoferrin not only has antimicrobial properties, but also modulates the innate and adaptive immune responses. Lactoferrin may indeed up- and downregulate immune cell activation, migration, and growth. Whereas the immunomodulatory properties of lactoferrin are evidenced from invivo studies using either lactoferrin-knockout, lactoferrin-overexpressing transgenic models, and dietary lactoferrin, few mechanisms from invitro studies have been proposed to explain these properties. The best characterized lactoferrin targets are negatively charged molecules. They encompass pro-inflammatory microbial molecules, such as pathogen-associated molecular patterns (eg, lipopolysaccharide), but also host components such as DNA, the glycosaminoglycan chains of proteoglycans, and surface cell receptors. Signaling through these receptors is thought to be the main lever used by lactoferrin to influence immune cells and cytokine-balance-controlling cell activity. This article aims to review our current understanding, though incomplete, of the many ways lactoferrin influences the complex immune machinery and the known and putative mechanisms that may explain its properties.

Stereospecific Inhibitory Effects of CCG-1423 on the Cellular Events Mediated by Myocardin-Related Transcription Factor A.

CCG-1423 suppresses several pathological processes including cancer cell migration, tissue fibrosis, and the development of atherosclerotic lesions. These suppressions are caused by inhibition of myocardin-related transcription factor A (MRTF-A), which is a critical factor for epithelial–mesenchymal transition (EMT). CCG-1423 can therefore be a potent inhibitor for EMT. CCG-1423 and related compounds, CCG-100602 and CCG-203971 possess similar biological activities. Although these compounds are comprised of two stereoisomers, the differences in their biological activities remain to be assessed. To address this issue, we stereoselectively synthesized optically pure isomers of these compounds and validated their biological activities. The S-isomer of CCG-1423 rather than the R-isomer exhibited modestly but significantly higher inhibitory effects on the cellular events triggered by MRTF-A activation including serum response factor-mediated gene expression and cell migration of fibroblasts and B16F10 melanoma cells. Accordingly, the S-isomer of CCG-1423 more potently blocked the serum-induced nuclear import of MRTF-A than the R-isomer. No such difference was observed in cells treated with each of two stereoisomers of CCG-100602 or CCG-203971. We previously reported that the N-terminal basic domain (NB), which functions as a nuclear localization signal of MRTF-A, is a binding site for CCG-1423. Consistent with the biological activities of two stereoisomers of CCG-1423, docking simulation demonstrated that the S-isomer of CCG-1423 was more likely to bind to NB than the R-isomer. This is a first report demonstrating the stereospecific biological activities of CCG-1423.

Mitotic lifecycle of chromosomal 3xHMG-box proteins and the role of their N-terminal domain in the association with rDNA loci and proteolysis.

The high mobility group (HMG)-box is a DNA-binding domain characteristic of various eukaryotic DNA-binding proteins. 3xHMG-box proteins (containing three copies of the HMG-box domain and a unique basic N-terminal domain) are specific for plants and the Arabidopsis genome encodes two versions termed 3xHMG-box1 and 3xHMG-box2, whose expression is cell cycle-dependent, peaking during mitosis. Here, we analysed in detail the spatiotemporal expression, subcellular localisation and chromosome association of the Arabidopsis thaliana 3xHMG-box proteins. Live cell imaging and structured illumination microscopy revealed that the expression of the 3xHMG-box proteins is induced in late G2 phase of the cell cycle and upon nuclear envelope breakdown in prophase they rapidly associate with the chromosomes. 3xHMG-box1 associates preferentially with 45S rDNA loci and the basic N-terminal domain is involved in the targeting of rDNA loci. Shortly after mitosis the 3xHMG-box proteins are degraded and an N-terminal destruction-box mediates the proteolysis. Ectopic expression/localisation of 3xHMG-box1 in interphase nuclei results in reduced plant growth and various developmental defects including early bolting and abnormal flower morphology. The remarkable conservation of 3xHMG-box proteins within the plant kingdom, their characteristic expression during mitosis, and their striking association with chromosomes, suggest that they play a role in the organisation of plant mitotic chromosomes.

Identification and characterization of the EIN3/EIL gene family in Hevea brasiliensis

Ethylene-insensitive3/ethylene-insensitive3-like (EIN3/EIL) transcription factor is a key transcription factor involved in the ethylene signal transduction pathway in plants. Although the genome-wide analysis of this family has been carried out in certain plant species, little is known about EIN3/EIL genes in rubber tree (Hevea brasiliensis). In this study, we identified four EIN3/EIL genes (designated as HbEIN3-1 to HbEIN3-4) in the rubber tree genome annotation. Tissue-specific expression profiles showed that four HbEIN3s were expressed in at least one of the tested tissues. The four HbEIN3s responded to ethylene (ET) and jasmonic acid (JA) treatment, which suggested that HbEIN3s were involved in response to JA and ET signaling. HbEIN3s showed transactivation activity in yeast. HbEIN3-1 bound to the promoter of the rubber transferase gene, suggesting that HbEIN3-1 may be involved in the regulation of natural rubber biosynthesis. The N-terminal basic domain II was required for the HbEIN3-1 binding with the promoter of the rubber transferase gene.

Nucleocapsid Annealing-Mediated Electrophoresis (NAME) Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral proteins involved in the regulation of the virus activity recognize several nucleic acids. The nucleocapsid protein NCp7 (NC) is a key protein regulating several processes during virus replication. NC is in fact a chaperone destabilizing the secondary structures of RNA and DNA and facilitating their annealing. The inactivation of NC is a new approach and an interesting target for anti-HIV therapy. The Nucleocapsid Annealing-Mediated Electrophoresis (NAME) assay was developed to identify molecules able to inhibit the melting and annealing of RNA and DNA folded in thermodynamically stable tridimensional conformations, such as hairpin structures of TAR and cTAR elements of HIV, by the nucleocapsid protein of HIV-1. The new assay employs either the recombinant or the synthetic protein, and oligonucleotides without the need of their previous labeling. The analysis of the results is achieved by standard polyacrylamide gel electrophoresis (PAGE) followed by conventional nucleic acid staining. The protocol reported in this work describes how to perform the NAME assay with the full-length protein or its truncated version lacking the basic N-terminal domain, both competent as nucleic acids chaperones, and how to assess the inhibition of NC chaperone activity by a threading intercalator. Moreover, NAME can be performed in two different modes, useful to obtain indications on the putative mechanism of action of the identified NC inhibitors.