Polymerase Binding Research Articles

Fidaxomicin resistance in Clostridioides difficile: a systematic review and predictive modeling with RNA polymerase binding sites.

Fidaxomicin (FDX), an RNA polymerase (RNAP) inhibitor antibiotic, is a guideline-recommended therapy for Clostridioides difficile infection. Mutations associated with reduced FDX minimum inhibitory concentrations (MICs) have been identified. However, the molecular characterization of these mutations on FDX binding and the development of FDX resistance have not been studied. The purpose of this systematic review was to identify FDX resistance in C. difficile isolates and determine whether single nucleotide polymorphisms associated with increased FDX MIC aligned with the RNAP binding pocket interacting residues. A systematic literature search was done in PubMed (1991-2023) with identified articles and their bibliographies searched for papers that included C. difficile genetic mutations and increased FDX MIC. Visualization of FDX-RNAP interactions was performed on Schrödinger Maestro using the publicly available C. difficile RNAP with fidaxomicin sequence (code 7L7B) on the Protein Data Bank. Seven articles were identified after applying inclusion and exclusion criteria. The most common mutation in clinical and laboratory isolates was at position V1143 of the β subunit, which accounted for approximately 50% of the identified mutations. Most other mutations occurred within the β' subunit of RNAP. Approximately one-third of the identified mutation aligned directly with FDX interacting residues with C. difficile RNAP (7/20) with most of the remainder occurring within 5 Å of the binding residues. C. difficile strains with elevated FDX MIC align closely with the known RNAP binding residues. These data demonstrate the potential to identify genomic methods to identify emerging FDX resistance.

Antioxidant: Antimycobacterial and Antibiofilm Activities of Acetone Extract and Subfraction Artemisia afra Jacq. ex Willd. Against Mycobacterium smegmatis

Tuberculosis is a worldwide prevalent and recurring disease that contributes significantly to high mortality rates. This study aimed to investigate the antioxidant, anti-mycobacterial, and antibiofilm activities of Artemisia afra acetone crude extract. Methodology: The crude acetone extract was fractionated using column chromatography and characterized by liquid chromatography–mass spectroscopy (LC-MS). A 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay was used to assess the antioxidant activity. The antimycobacterial activity against Mycobacterium smegmatis was screened using bioautography, broth microdilution, and growth curve assays. Molecular docking was used to predict the possible mechanisms of action of the LC-MS-identified ligands. Crystal violet was used to screen for anti-cell adherence and biofilm inhibition activities. Results: The crude extract scavenged 77% of the free radical at 16 μg/mL. The subfraction had a lower minimum inhibitory concentration (MIC) (0.078 mg/mL) compared to the crude extract (0.313–0.833 mg/mL). The subfraction had a concentration-dependent inhibition effect (>50%) on mycobacterial cell adherence and early biofilms. However, the mature biofilms were resistant. Two propanoate compounds, [(2S)-3-[6-acetyl-4,6-dihydroxy-3-[(1R)-1-hydroxyethyl]tetrahydropyran-2-yl]-2-hydroxy-propyl] (2R)-2-amino-3-(1H-imidazol-5-yl)propanoate and 3-(6-aminopurin-9-yl)propyl 3-(2,4-dioxo-1,3-diazaspiro[4.5]decan-3-yl) propanoate, had binding energies of −5.4 kcal/mol and −6.3 kcal/mol, respectively, against the RNA polymerase binding protein. Conclusions: The results show that A. afra acetone crude extract has antioxidant and antimycobacterial activities that can be improved by fractionation.

Unveiling the genetic basis and metabolic rewiring behind the galactose-positive phenotype in a Streptococcus thermophilus mutant

Streptococcus thermophilus (S. thermophilus) is a widely used starter culture in dairy fermentation, but most strains are galactose-negative and only metabolize glucose from lactose hydrolysis. In this study, we aimed to uncover the mechanisms underlying the acquisition of a stable galactose-positive (Gal+) phenotype in a mutant strain of S. thermophilus IMAU10636. By treating the wild-type strain with the mutagenic agent N-methyl-N-nitro-N-nitrosoguanidine, we successfully isolated a Gal+ mutant, S. thermophilus IMAU10636Y. Comparative enzyme activity assays revealed that the mutant exhibited higher β-galactosidase and galactokinase activities, but lower glucokinase and pyruvate kinase activities compared to the wild-type. High-performance liquid chromatography analysis confirmed the mutant’s enhanced ability to utilize lactose and galactose, leading to increased glucose secretion. Integrated genome and transcriptomics analyses provided deeper insights into the underlying genetic and metabolic mechanisms. We found that the metabolism regulatory network of the glycolysis / Leloir pathway was altered in the mutant, possibly due to the upregulation of the gene expression in the galR-galK intergenic region. This likely led to increased RNA polymerase binding and transcription of the gal operon, ultimately promoting the Gal+ phenotype. Additionally, we identified a mutation in the scrR gene, encoding a LacI family transcriptional repressor, which also contributed to the Gal+ phenotype. These findings offer new perspectives on the metabolic rewiring and regulatory mechanisms that enable S. thermophilus to acquire the ability to metabolize galactose. This knowledge can inform strategies for engineering and selecting Gal+ strains with desirable fermentation characteristics for dairy applications.

Visualizing Viral RNA Packaging Signals in Action

Here we confirm, using genome-scale RNA fragments in assembly competition assays, that multiple sub-sites (Packaging Signals, PSs) across the 5′ two-thirds of the gRNA of Satellite Tobacco Necrosis Virus-1 make sequence-specific contacts to the viral CPs helping to nucleate formation of its T = 1 virus-like particle (VLP). These contacts explain why natural virions only package their positive-sense genomes. Asymmetric cryo-EM reconstructions of these VLPs suggest that interactions occur between amino acid residues in the N-terminal ends of the CP subunits and the gRNA PS loop sequences. The base-paired stems of PSs also act non-sequence-specifically by electrostatically promoting the assembly of CP trimers. Importantly, alterations in PS-CP affinity result in an asymmetric distribution of bound PSs inside VLPs, with fuller occupation of the higher affinity 5′ PS RNAs around one vertex, decreasing to an RNA-free opposite vertex within the VLP shell. This distribution suggests that gRNA folding regulates cytoplasmic genome extrusion so that the weakly bound 3′ end of the gRNA, containing the RNA polymerase binding site, extrudes first. This probably occurs after cation-loss induced swelling of the CP-shell, weakening contacts between CP subunits. These data reveal for the first time in any virus how differential PS folding propensity and CP affinities support the multiple roles genomes play in virion assembly and infection. The high degree of conservation between the CP fold of STNV-1 and those of the CPs of many other viruses suggests that these aspects of genome function will be widely shared.

An intrinsically disordered region in MED13 turns Mediator on/off on cue

Complementary studies by Zhao etal.1 and Chen etal.2 reveal how an intrinsically disordered region in MED13 controls mutually exclusive binding of RNA Polymerase II and CDK8 kinase module to Mediator, switching Mediator and transcription activation on and off.

A Transient, Excited Species of the Autoinhibited α-State of the Bacterial Transcription Factor RfaH May Represent an Early Intermediate on the Fold-Switching Pathway.

RfaH is a two-domain transcription factor in which the C-terminal domain switches fold from an α-helical hairpin to a β-roll upon binding the ops-paused RNA polymerase. To ascertain the presence of a sparsely populated excited state that may prime the autoinhibited resting state of RfaH for binding ops-paused RNA polymerase, we carried out a series of NMR-based exchange experiments to probe for conformational exchange on the millisecond time scale. Quantitative analysis of these data reveals exchange between major ground (∼95%) and sparsely populated excited (∼5%) states with an exchange lifetime of ∼3 ms involving residues at the interface between the N-terminal and C-terminal domains formed by the β3/β4 hairpin and helix α3 of the N-terminal domain and helices α4 and α5 of the C-terminal domain. The largest 15N backbone chemical shift differences are associated with the β3/β4 hairpin, leading us to suggest that the excited state may involve a rigid body lateral displacement/rotation away from the C-terminal domain to adopt a position similar to that seen in the active RNA polymerase-bound state. Such a rigid body reorientation would result in a reduction in the interface between the N- and C-terminal domains with the possible introduction of a cavity or cavities. This hypothesis is supported by the observation that the population of the excited species and the exchange rate of interconversion between ground and excited states are reduced at a high (2.5 kbar) pressure. Mechanistic implications for fold switching of the C-terminal domain in the context of RNA polymerase binding are discussed.

Insights into the association of the Chlamydia trachomatis type III secretion chaperone complex, Scc4:Scc1, from sequential expression in Escherichia coli

Chlamydia trachomatis (CT) is the bacterial pathogen responsible for causing the most common sexually transmitted disease in the United States. This obligate, intracellular Gram-negative bacterium has a type III secretion system (T3SS) to invade host cells. CopN is an important effector, plug protein that mediates early interactions between the host and Chlamydia. CopN is chaperoned by a heterodimer, T3SS chaperone complex containing Scc4 and Scc1. Scc4 is a unique, bifunctional protein that, in addition to its T3SS chaperone activity, acts as an RNA polymerase (RNAP) binding protein. We hypothesized that the two functions occur at different points in CT's developmental cycle with Scc4 acting alone in the early-to-mid stages and the Scc4:Scc1 complex chaperoning CopN in the mid-to-late stages. To study the Scc4:Scc1 complex by NMR, we previously explored various methods of associating Scc4 and Scc1 in vitro to produce the complex with chain-selective isotopic labeling. Though co-expressed Scc4 and Scc1 form a stable complex, the in vitro association studies suggest that partial protein denaturation and/or components in E. coli lysate are necessary to form the stable complex. In this study Scc4 and Scc1 were sequentially expressed in E. coli under the control of different promoters, allowing separate isotopic labeling of each chain and complex formation in vivo. Sequential expression resulted in no or unstable complex formation depending on the culture medium used. These results, taken together with previous in vitro association studies, suggest that Scc4 and Scc1 assemble co-translationally to form the stable Scc4:Scc1 complex in E. coli.

Dissociation of transcription factor MYB94 and histone deacetylases HDA907/908 alleviates oxidative damage in poplar.

Drought is one of the major threats to forest productivity. Oxidation stress is common in drought-stressed plants, and plants need to maintain normal life activities through complex reactive oxygen scavenging mechanisms. However, the molecular links between epigenetics, oxidation stress, and drought in poplar (Populus) remain poorly understood. Here, we found that Populus plants overexpressing PtrMYB94, which encodes an R2R3-MYB transcription factor that regulates the abscisic acid signaling pathway, displayed increased tolerance to extreme drought stress via upregulation of embryogenic cell phosphoprotein 44 (PtrECPP44) expression. Further investigation revealed that PtrMYB94 could recruit the histone deacetylases PtrHDA907/908 to the promoter of PtrECPP44 and decrease acetylation at lysine residues 9, 14, and 27 of histone H3, leading to relatively low transcriptional expression levels under normal conditions. Drought induced the expression of PtrMYB94 while preventing interaction of PtrMYB94 with PtrHDA907/908, which relaxed the chromatin structure and facilitated the binding of RNA polymerase II to the PtrECPP44 promoter. The upregulation of PtrECPP44 helped poplar alleviate oxidative damage and maintain normal cell activities. This study establishes a PtrMYB94-PtrECPP44 transcriptional regulatory module modified by PtrHDA907/908 in modulating drought-induced oxidative stress recovery. Therefore, our study reveals an oxidative regulatory mechanism in response to drought stress and provides insights into molecular breeding for stress resistance in poplar.

First Report of Gray Mold Caused by Botrytis cinerea on Dragon Fruit (Hylocereus undatus) in Mexico.

The dragon fruit is native of Mexico, and Puebla is the third-largest producing state (SIAP 2023). In June 2023, field sampling was conducted in El Paraíso, Atlixco (18° 49' 5.275" N, 98° 26' 52.353" W), Puebla, Mexico. The mean temperature and relative humidity were 20 °C and 75% for seven consecutive days. Dragon fruits cv. 'Delight' close to harvest with gray mold symptoms were found in a commercial area of 2 ha, with an incidence of 35 to 40% and an estimated severity of 75% on infected fruit. The symptoms included necrosis at the apex, which later spread throughout the fruit, along with a soft, black rot covered in abundant mycelium and sporulation. The fungus was isolated from 40 symptomatic fruits by disinfesting pieces of necrotic tissue with 3% NaClO for one minute, rinsing with sterile distilled water (SDW), plating on Petri dishes with potato dextrose agar, and incubating at 25 °C in the dark. One isolate was obtained from each diseased fruit by the hyphal-tip method. The colonies were initially white with a growth rate of 1.15-1.32 cm per day and turned gray after 10 days; the mycelium was dense and aerial. Spherical and irregular sclerotia were formed, measuring 0.9-1.4 × 0.6-1.1 mm (n = 100). Each Petri dish produced 56-278 sclerotia (n = 40) after 11 days; these were initially white and gradually turned dark brown. Brown to olive conidiophores were straight, septate, and branched, measuring 1075-1520 × 10-21 μm, with elliptical hyaline to light brown conidia of 6.6-11.5 × 5-8.1 μm (n=100). The isolates were tentatively identified as Botrytis cinerea based on morphological characteristics (Ellis 1971). Two representative isolates were chosen for molecular identification and genomic DNA was extracted by the CTAB protocol. The ITS region and the heat shock protein (HSP60), RNA polymerase binding II (RPB2) and glyceraldehyde 3-phosphate dehydrogenase (G3PDH) genes were sequenced (White et al. 1990; Staats et al. 2005). The sequences of a representative isolate (BcPh5) were deposited in GenBank (ITS-OR582337; HSP60-OR636622; RPB2-OR636623; and G3PDH-OR636621). BLAST analysis of the partial sequences of ITS (479 bp), HSP60 (1006 bp), RPB2 (1126 bp), and G3PDH (907 bp) showed 100% similarity to B. cinerea isolates (GenBank: KM840848, MH796663, MK919495, MF480679). Phylogenetic analysis confirmed that BcPh5 clustered with B. cinerea strains. Pathogenicity was confirmed by inoculating the non-wounded surface of 20 detached dragon fruits cv. 'Delight' using the BcPh5 isolate by depositing 20 μl of a 105 conidia/ml suspension with a sterile syringe. The fruits were placed on the rim of a plastic container and inserted in a moisture box with 2 cm of water at the bottom. The box was covered with a plastic sheet to maintain humidity. Control fruits were inoculated with SDW. The inoculated fruits became covered with abundant white to gray mycelium, and soft rot developed within eight days, while no symptoms were observed on the controls. The fungus was re-isolated from the inoculated fruits as described above, fulfilling Koch's postulates. The pathogenicity tests were repeated three times. Gray mold caused by B. cinerea was also recently reported in Mexico on pomegranate (Hernández et al. 2023) and rose apple (Isodoro et al. 2023). As far as we know, this is the first report of B. cinerea causing gray mold on dragon fruit in Mexico. This research is essential for designing integrated management strategies against gray mold on dragon fruits.

Investigating G-quadruplex structures in RPGR gene: Implications for understanding X-linked retinal degeneration

AimsThis pilot study investigates the potential pathogenic role of G-quadruplex (G4) structures in RPGR-associated retinal degeneration, starting from a case of suspected X-linked form affected family. We hypothesize that the stabilization of these structures might alter DNA replication and transcription, inducing genetic instability and influencing gene expression. Main methodsWe conducted whole genome amplification experiments and next-generation sequencing to detect the blockade of polymerase activity by G4 structures. Our specific focus was the RPGR gene, which hosts a high concentration of predicted G4-forming motifs and is implicated in most X-linked retinal degeneration cases. To understand the potential interference of G4 structures, we applied computational and 3D molecular modeling to visualize interferences in DNA replication and transcription regulation. Key findingsOur data confirmed the obstruction of DNA polymerase enzymes by G4 structures, particularly when stabilized by the compound pyridostatin. This obstruction was evident in the reduced amplification of RPGR gene regions and a shift in the start/end sites of putative G4 motifs. Moreover, the modeling indicated a potential disruption of critical promoter elements and RNA polymerase binding, which could drastically alter gene expression. SignificanceOur findings suggest that G4 formation in the RPGR gene could lead to genetic instability and affect the expression of RPGR, contributing to retinal dystrophy. Moreover, this study underscores the broader implications of G4 structures in other genetic disorders. Improved understanding of G4 structures could reveal novel therapeutic targets to combat genetic disorders, promoting the advancement of personalized medicine and precision health.

SN-38, an active metabolite of irinotecan, inhibits transcription of nuclear factor erythroid 2-related factor 2 and enhances drug sensitivity of colorectal cancer cells.

Nuclear factor erythroid 2-related factor 2 (Nrf2) significantly contributes to drug resistance of cancer cells, and Nrf2 inhibitors have been vigorously pursued. Repurposing of existing drugs, especially anticancer drugs, is a straightforward and promising strategy to find clinically available Nrf2 inhibitors and effective drug combinations. Topoisomerase inhibitors SN-38 (an active metabolite of irinotecan), topotecan, mitoxantrone, and epirubicin were found to significantly suppress Nrf2 transcriptional activity in cancer cells. SN-38, the most potent one among them, significantly inhibited the transcription of Nrf2, as indicated by decreased mRNA level and binding of RNA polymerase II to NFE2L2 gene, while no impact on Nrf2 protein or mRNA degradation was observed. SN-38 synergized with Nrf2-sensitive anticancer drugs such as mitomycin C in killing colorectal cancer cells, and irinotecan and mitomycin C synergistically inhibited the growth of SW480 xenografts in nude mice. Our study identified SN-38 and three other topoisomerase inhibitors as Nrf2 inhibitors, revealed the Nrf2-inhibitory mechanism of SN-38, and indicate that clinically feasible drug combinations could be designed based on their interactions with Nrf2 signaling.

Alteration of the -35 and -10 sequences and deletion the upstream sequence of the -35 region of the promoter A1 of the phage T7 in dsDNA confirm the contribution of non-specific interactions with E. coli RNA polymerase to the transcription initiation process.

Transcription initiation is a multi-step process, in which the RNA polymerase holoenzyme binds to the specific promoter sequences to form a closed complex, which, through intermediate stages, isomerizes into an open complex capable of initiating the productive phase of transcription. The aim of this work was to determine the contribution of the -10 and -35 regions of the promoter, as well as the role of non-specific interactions, in the binding of RNA polymerase and the formation of an active initiation complex capable of transcription. Therefore, fragments of promoter DNA, derived from the strong promoter A1 of the phage T7, containing completely and partially altered elements -35 and -10, and devoid of an upstream region, were constructed using genetic engineering methods. Functional analyses of modified promoter fragments were carried out, checking their ability to form binary complexes with Escherichia coli RNA polymerase (RNAP) and the efficiency of converting binary complexes into triple complexes characteristic of the productive phase of transcription. The obtained results suggest that, in relation to the A1 promoter of the T7 phage, the most important role of the -35 region is carrying the open complex through the next phases of transcription initiation. The weakening of specific impacts within the region -35 is a reason for the defect associated with the transformation of the open complex, formed by a DNA fragment containing the completely altered -35 region, into elongation and the impairment of RNA synthesis. This leads to breaking contacts with the RNA polymerase holoenzyme, and destabilization and disintegration of the complex in the initial phase of productive transcription. This confirms the hypothesis of the so-called stressed intermediate state associated with the stage of transition from the open complex to the elongation complex. The experiments carried out in this work confirm also that the process of promoter localization and recognition, as well as the formation of binary complexes, is sequential in nature, and that the region located upstream of the -35 hexamer, and the hexamer itself, plays here an additive role.

Structural Analysis of the Hepatitis B Virus RNA Encapsidation Signal ε by Selective 2'-Hydroxyl Acylation Analyzed by Primer Extension (SHAPE).

RNA structure is crucial for RNA function, including in viral cis-elements such as the hepatitis B virus (HBV) RNA encapsidation signal ε. Interacting with the viral polymerase ε mediates packaging of the pregenomic (pg) RNA into capsids, initiation of reverse transcription, and it affects the mRNA functions of pgRNA. As free RNA, the 61-nucleotide (nt) ε sequence adopts a bipartite stem-loop structure with a central bulge and an apical loop. Due to stable Watson-Crick base pairing, this was already predicted by early RNA folding programs and confirmed by classical enzymatic and chemical structure probing. A newer, high-resolution probing technique exploits the selective acylation of solvent-accessible 2'-hydroxyls in the RNA backbone by electrophilic compounds such as 2-methylnicotinic acid imidazolide (NAI), followed by mapping of the modified sites by primer extension. This SHAPE principle has meanwhile been extended to numerous applications. Here we provide a basic protocol for NAI-based SHAPE of isolated HBV ε RNA which already provided insights into the impact of mutations, and preliminarily, of polymerase binding on the RNA structural dynamics. While the focus is on NAI modification, we also briefly cover target RNA preparation by in vitro transcription, primer extension using a radiolabeled primer, and analysis of the resulting cDNAs by denaturing polyacrylamide gelelectrophoresis (PAGE). Given the high tolerance of SHAPE chemistry to different conditions, including applicability in live cells, we expect this technique to greatly facilitate deciphering the conformational dynamics underlying the various functions of the ε element, especially in concert with the recently solved three-dimensional structure of the free RNA.

Joint Efforts of Replicative Helicase and SSB Ensure Inherent Replicative Tolerance of G-Quadruplex.

G-quadruplex (G4) is a four-stranded noncanonical DNA structure that has long been recognized as a potential hindrance to DNA replication. However, how replisomes effectively deal with G4s to avoid replication failure is still obscure. Here, using single-molecule and ensemble approaches, the consequence of the collision between bacteriophage T7 replisome and an intramolecular G4 located on either the leading or lagging strand is examined. It is found that the adjacent fork junctions induced by G4 formation incur the binding of T7 DNA polymerase (DNAP). In addition to G4, these inactive DNAPs present insuperable obstacles, impeding the progression of DNA synthesis. Nevertheless, T7 helicase can dismantle them and resolve lagging-strand G4s, paving the way for the advancement of the replication fork. Moreover, with the assistance of the single-stranded DNA binding protein (SSB) gp2.5, T7 helicase is also capable of maintaining a leading-strand G4 structure in an unfolded state, allowing for a fraction of T7 DNAPs to synthesize through without collapse. These findings broaden the functional repertoire of a replicative helicase and underscore the inherent G4 tolerance of a replisome.

Canonical binding of Chaetomium thermophilum DNA polymerase δ/ζ subunit PolD3 and flap endonuclease Fen1 to PCNA.

The sliding clamp PCNA is a key player in eukaryotic genome replication and stability, acting as a platform onto which components of the DNA replication and repair machinery are assembled. Interactions with PCNA are frequently mediated via a short protein sequence motif known as the PCNA-interacting protein (PIP) motif. Here we describe the binding mode of a PIP motif peptide derived from C-terminus of the PolD3 protein from the thermophilic ascomycete fungus C. thermophilum, a subunit of both DNA polymerase δ (Pol δ) and the translesion DNA synthesis polymerase Pol ζ, characterised by isothermal titration calorimetry (ITC) and protein X-ray crystallography. In sharp contrast to the previously determined structure of a Chaetomium thermophilum PolD4 peptide bound to PCNA, binding of the PolD3 peptide is strictly canonical, with the peptide adopting the anticipated 310 helix structure, conserved Gln441 inserting into the so-called Q-pocket on PCNA, and Ile444 and Phe448 forming a two-fork plug that inserts into the hydrophobic surface pocket on PCNA. The binding affinity for the canonical PolD3 PIP-PCNA interaction determined by ITC is broadly similar to that previously determined for the non-canonical PolD4 PIP-PCNA interaction. In addition, we report the structure of a PIP peptide derived from the C. thermophilum Fen1 nuclease bound to PCNA. Like PolD3, Fen1 PIP peptide binding to PCNA is achieved by strictly canonical means. Taken together, these results add to an increasing body of information on how different proteins bind to PCNA, both within and across species.

Unraveling the salt tolerance of Phi29 DNA polymerase using compartmentalized self-replication and microfluidics platform.

In Phi29-α-hemolysin (α-HL) nanopore sequencing systems, a strong electrochemical signal is dependent on a high concentration of salt. However, high salt concentrations adversely affect polymerase activity. Sequencing by synthesis (SBS) requires the use of phi29 polymerase without exonuclease activity to prevent the degradation of modified nucleotide tags; however, the lack of exonuclease activity also affects polymerase processivity. This study aimed to optimize phi29 polymerase for improved salt tolerance and processivity while maintaining its lack of exonuclease activity to meet the requirements of nanopore sequencing. Using salt tolerance compartmentalized self-replication (stCSR) and a microfluidic platform, we obtained 11 mutant sites with enhanced salt tolerance attributes. Sequencing and biochemical analyses revealed that the substitution of conserved amino acids such as G197D, Y369E, T372N, and I378R plays a critical role in maintaining the processivity of exonuclease-deficient phi29 polymerase under high salt conditions. Furthermore, Y369E and T372N have been identified as important determinants of DNA polymerase binding affinity. This study provides insights into optimizing polymerase processability under high-salt conditions for real-time polymerase nanopore sequencing, paving the way for improved performance and applications in nanopore sequencing technologies.

Implementation of computational strategies to combat drug resistance in Mycobacterium tuberculosis

Mycobacterium tuberculosis (MTB) is an infectious pathogen that causes tuberculosis (TB). Even though it is curable, the first-line anti-TB medication rifampicin has been a source of rising concern for human health across the globe. However, rifampicin resistance results from mutations in the RNA polymerase's binding site of the target protein. This infectious pathogen can survive in macrophages and can induce a long-lasting latent infection. The prolonged survival of mycobacterial species was achieved with the aid of serine/threonine protein kinase G (PknG) which performs a preventive role in this situation. Thus, PknG is believed to be an essential target to prevent the pathogen from entering the latency stage. The present investigation was oriented towards molecular-based virtual screening carried out using AutoDock for the FDA-approved and nutraceutical subsets of compounds from the DrugBank repository. Initially, a total of 3035 compounds were screened based on their absorption, distribution, metabolism, excretion and toxicity (ADMET) properties. The screened molecules were taken for docking purposes after which post-docking analysis was performed by calculating the random forest (RF) score. Further, the structural characterization of hit compounds was validated using interaction studies and in silico bioactivity prediction studies. Finally, the anti-myotubular activity prediction was carried out in the search for potential inhibitors. Importantly, four potential ligands, namely DB00080, DB00807, DB06249 and DB06274 were preferred after a thorough investigation. These ligands showed significant structural and protein-ligand interactions, suggesting that they might be potentially used as therapeutic candidates to combat MTB.

Identifying Innate Resistance Hotspots for SARS-CoV-2 Antivirals Using In Silico Protein Techniques.

The development and approval of antivirals against SARS-CoV-2 has further equipped clinicians with treatment strategies against the COVID-19 pandemic, reducing deaths post-infection. Extensive clinical use of antivirals, however, can impart additional selective pressure, leading to the emergence of antiviral resistance. While we have previously characterized possible effects of circulating SARS-CoV-2 missense mutations on proteome function and stability, their direct effects on the novel antivirals remains unexplored. To address this, we have computationally calculated the consequences of mutations in the antiviral targets: RNA-dependent RNA polymerase and main protease, on target stability and interactions with their antiviral, nucleic acids, and other proteins. By analyzing circulating variants prior to antiviral approval, this work highlighted the inherent resistance potential of different genome regions. Namely, within the main protease binding site, missense mutations imparted a lower fitness cost, while the opposite was noted for the RNA-dependent RNA polymerase binding site. This suggests that resistance to nirmatrelvir/ritonavir combination treatment is more likely to occur and proliferate than that to molnupiravir. These insights are crucial both clinically in drug stewardship, and preclinically in the identification of less mutable targets for novel therapeutic design.

Mutations in the DNA polymerase binding pathway affect the immune microenvironment of patients with small-cell lung cancer and enhance the efficacy of platinum-based chemotherapy.

Small-cell lung cancer (SCLC) is characterized by its high malignancy and is associated with a poor prognosis. In the early stages of the disease, platinum-based chemotherapy is the recommended first-line treatment and has demonstrated efficacy. However, SCLC is prone to recurrence and is generally resistant to chemotherapy in its later stages. Here, we collected samples from SCLC patients who received platinum-based chemotherapy, performed genomic and transcriptomic analyses, and validated our results with publicly available data. SCLC patients with DNA polymerase binding pathway mutations had an improved prognosis after platinum chemotherapy compared with patients without such mutations. Patients in the mutant (MT) group had higher infiltration of T cells, B cells, and M1 macrophages compared with patients without DNA polymerase binding pathway mutations. DNA polymerase binding pathway mutations can be used as prognostic markers for platinum-based chemotherapy in SCLC.

Novel pyrimidine Schiff bases and their selenium-containing nanoparticles as dual inhibitors of CDK1 and tubulin polymerase: design, synthesis, anti-proliferative evaluation, and molecular modelling

Nanotechnology-based strategies can overcome the limitations of conventional cancer therapies. Hence, novel series of pyrimidine Schiff bases (4–9) were employed in the synthesis of selenium nanoparticle forms (4NPs–9NPs). All selenium nano-sized forms exerted greater inhibitions than normal-sized compounds, far exceeding 5-fluorouracil activity. Compound 4 showed effective anti-proliferative activity against MCF-7(IC50 3.14 ± 0.04 µM), HepG-2(IC50 1.07 ± 0.03 µM), and A549(IC50 1.53 ± 0.01 µM) cell lines, while its selenium nanoform 4NPs showed excellent inhibitory effects, with efficacy increased by 96.52%, 96.45%, and 93.86%, respectively. Additionally, 4NPs outperformed 4 in selectivity against the Vero cell line by 4.5-fold. Furthermore, 4NPs exhibited strong inhibition of CDK1(IC50 0.47 ± 0.3 µM) and tubulin polymerase(IC50 0.61 ± 0.04 µM), outperforming 4 and being comparable to roscovitine (IC50 0.27 ± 0.03 µM) and combretastatin-A4(IC50 0.25 ± 0.01 µM), respectively. Moreover, both 4 and 4NPs arrested the cell cycle at G0/G1 phase and significantly forced the cells towards apoptosis. Molecular docking demonstrated that 4 and 4NPs were able to inhibit CDK1 and tubulin polymerase binding sites.