Search Results (456)

Bacillus subtilis DinG/XPD-like paralogues, DinG and YpvA, have been implicated in overcoming replication stress. DinG possesses a DEDD exonuclease and DNA helicase domains, whereas YpvA lacks the DEDD exonuclease domain. We report that DinG·Mg²⁺ (hereafter referred to as DinG) degrades linear single-stranded (lss) DNA with 3′→5′ polarity and binds lssDNA with higher affinity than its exonuclease-deficient mutant DinG D10A E12A. DinG’s ssDNA-dependent ATPase activity neither stimulates nor inhibits DNA degradation. When bound to the 3′-end of forked DNA, DinG destabilises and degrades the substrate; however, in the presence of ATP, DinG dissociates before reaching the duplex junction. DinG degrades the RNA strand within RNA–DNA hybrids but does not cleave lssRNA unless complexed with Mn²⁺. DinG removes genomic R-loops, as RnhC and PcrA do. DinG physically interacts with RecA and PolA and functions in the same pathway as translesion synthesis (TLS) DNA polymerases (DNAPs) to respond to both spontaneous and methyl methanesulphonate (MMS)-induced mutagenesis. DinG-mGold forms spontaneous foci at or near replication forks, which become enriched following MMS or rifampicin treatment. We propose that DinG contributes to mitigating replication stress by degrading R-loop barriers and facilitating TLS, potentially via RecA-linked mechanisms. Full article

RNA polymerases are essential enzymes that catalyze DNA transcription into RNA, vital for protein synthesis, gene expression regulation, and cellular responses. Non-template-dependent RNA polymerases, which synthesize RNA without a template, are valuable in biological research due to their flexibility in producing RNA without predefined sequences. However, their substrate polymerization mechanisms are not well understood. This study examines Poly (A) polymerase (PAP), a nucleotide transferase superfamily member, to explore its substrate selectivity using computational methods. Previous research shows PAP’s polymerization efficiency for nucleoside triphosphates (NTPs) ranks ATP > GTP > CTP > UTP, though the reasons remain unclear. Using 500 ns Gaussian accelerated molecular dynamics simulations, stability analysis, secondary structure analysis, MM-PBSA calculations, and Markov state modeling, we investigate PAP’s differential polymerization efficiencies. Results show that ATP binding enhances PAP’s structural flexibility and increases solvent-accessible surface area, likely strengthening protein–substrate or protein–solvent interactions and affinity. In contrast, polymerization of other NTPs leads to a more open conformation of PAP’s two domains, facilitating substrate dissociation from the active site. Additionally, ATP binding induces a conformational shift in residues 225–230 of the active site from a loop to an α-helix, enhancing regional rigidity and protein stability. Both ATP and GTP form additional π–π stacking interactions with PAP, further stabilizing the protein structure. This theoretical study of PAP polymerase’s substrate selectivity mechanisms aims to clarify the molecular basis of substrate recognition and selectivity in its catalytic reactions. These findings offer valuable insights for the targeted engineering and optimization of polymerases and provide robust theoretical support for developing novel polymerases for applications in drug discovery and related fields. Full article

In this study, 944 Subo Merino sheep, a high-quality fine wool breed, were selected as research subjects. The SNP typing of the FAT3 gene was performed using the Fluidigm BiomarkTM HD system, and 11 missense mutation sites were identified. The analysis of population polymorphism of single-nucleotide polymorphisms was conducted. It is noteworthy that a substantial strong linkage disequilibrium was identified between SNP 5 and SNP 6 (r² > 0.8). The association between SNPs of the FAT3 gene and wool traits showed that multiple SNPs were significantly correlated with several different wool traits (p < 0.05). Furthermore, the investigation delved into the impact of the FAT3 gene on wool fiber through the utilization of quantitative polymerase chain reaction (qPCR), which yielded findings that this gene was notably expressed in fine wool fiber (FW) (p < 0.001). To predict the subcellular localization and protein transmembrane structure of FAT3, we employed the PSORT II Prediction and TMHMM online software. It was determined that the protein contains a transmembrane domain. This study provides molecular markers for the improvement of the selection and breeding of ultrafine-wool sheep and offers experimental evidence for accelerating the genetic breeding of sheep. Full article

The research aimed to study the genome of Cryptococcus neoformans isolated from bird excreta. Thirteen isolates were cultured, colony stained, and underwent biochemical testing confirmation by nested polymerase chain reaction using ITS1-ITS4 and CN4-CN5 primers, respectively. Antifungal susceptibility testing and whole-genomic sequencing were analyzed. The results determined that all isolates were susceptible to amphotericin B (100%), fluconazole, and itraconazole (92.3%). One isolate (DOP3) showed resistance to fluconazole and itraconazole (MIC >64 and >8 µg/mL, respectively). A phylogenetic tree showed the identity of C. neoformans (serotype A). The genome of resistant (DOP3) and non-resistant isolates (DOP3.1) had 14 chromosomes. DOP3 consisted of 38 candidate antifungal resistance genes, which were the most active against azoles (14). The annotated genes in the azole group mostly were in the ATP-binding cassette transporter transmembrane superfamily. Resistance genes against FCZ were in the transcription factors (HAP2, HAP5), zinc finger (NRG1), cytochrome P450 (ERG11), and Myb-like DNA-binding domain (REB1). The most frequent resistance genes against ITZ were cytochrome P450 (ERG5 and ERG11) and a transcription factor (HAP5). DOP3.1 also consisted of 26 candidate resistance genes against azoles. Resistance genes against the azole group belong to the ABC transporter transmembrane superfamily. Resistance genes against FCZ belong to cytochrome P450 (ERG11), the zinc finger (NRG1), and the CCAAT binding transcription factor (HAP2). Resistance genes belonging to cytochrome P450 (ERG5) were found against ITZ. This research provides the first report of C. neoformans (serotype A) in zebra dove excreta, drug susceptibility to a resistant strain, and identification of resistance genes. Farm sanitation should be strictly applied, and immunocompetent people should avoid contact with zebra dove excreta. Full article

The hepatitis B virus (HBV) is a DNA virus of major public health concern whose error-prone polymerase has driven the emergence of ten distinct genotypes and a multitude of resistance-associated mutations (RAMs). Herein, we conducted a retrospective observational study analyzing 8152 hepatitis B virus (HBV) sequences from 27 regions across the Americas, retrieved from GenBank, to construct a database and examine associations among HBV genotypes/subtypes, geographic distribution, resistance-associated mutations (RAMs), and resistance to nucleos(t)ide analogs (NAs) used in the treatment of chronic infection. Following phylogenetic analysis, mutations at clinically relevant sites in the reverse transcriptase domain were identified and classified by resistance to NAs. Genotypes A (21.1% A2 and 14.7% A1) and D predominated across the retrieved database, whereas genotypes E, G, H, and I each accounted for fewer than 3% of the sequences. Among the sequences in the database, 10.6% harbored RAMs, with genotypes G, A, and H predominating in this category. The most frequently observed RAM was L180M + M204V/I, which is associated with resistance to LMV, ETV, and TBV, whereas resistance to ADV and TDF remained rare. Genotypes G and A2 were significantly associated with a higher likelihood of harboring multiple RAMs (as evaluated by logistic regression), along with an increased risk of resistance to LMV, ETV, and TBV; the opposite was true for subtype A1. Notably, genotypes H and B5 were associated with an elevated risk of TDF resistance. A comprehensive understanding of RAMs and circulating genotypes in the Americas is essential for identifying high-risk populations and establishing geographically targeted therapeutic strategies. Full article

DNA polymerase epsilon (POLe) is the leading strand replicative polymerase. POLe mutations located primarily in the proofreading domain cause replication errors and increase mutation burden in cancer cells. Consequently, POLe has been classified as a cancer driver gene. Certain POLe frameshift mutations that affect the proofreading domain are purified in cancer cells, but point mutations in other domains have also been reported. Here we use an artificial intelligence algorithm to determine what other mutations co-occur with POLe mutations in colorectal cancers. We partitioned POLe mutations into driver, passenger, and WT (no mutation), then assessed mutations in other genes in these three groups. We found that a driver POLe mutation is not likely to associate with driver mutations in other genes. Thus, driver mutations in colorectal cancers appear to purify in a manner that is independent of POLe. Mutations that affect POLe function do not necessarily increase the frequency of driver mutations in other genes. Structural analysis shows that many POLe driver mutations affect coordination of the Mg²⁺ ion in the active site. Our data show that the accumulation of colorectal cancer mutations is driven by complex factors. Full article

A novel barna-like virus was found to be associated with field-collected Afrina sporoboliae plant-parasitic nematodes. The positive-sense, single-stranded RNA genome of this virus, named Afrina barna-like virus (AfBLV), comprises 4020 nucleotides encoding four open reading frames (ORFs). ORF 1 encodes a protein product spanning a transmembrane, a peptidase, and VPg domains, whereas an overlapping ORF 2 encodes an RNA-dependent RNA polymerase (RdRP). ORF2 may be expressed via a −1 translational frameshift. In phylogenetic reconstructions, the RdRP of AfBLV was placed inside a separate clade of barna and barna-like viruses related to but distinct from the genera in the Solemoviridae and Alvernaviridae families, within the overall lineage of Sobelivirales. ORF 3 of AfBLV encodes a protein product of 206 amino acids (aa) long with homology to a putative protein encoded by a similarly positioned gene of an uncharacterized virus sequence identified previously as Barnaviridae sp. ORF 4 encodes a 161 aa protein with no significant similarities to sequences in the GenBank databases. AfBLV is the first barnavirus found in a nematode. Sequence comparisons of the AfBLV genome and genomes of other barna-like viruses suggested that a recombination event was involved in the evolution of AfBLV. Analyses of the phylogeny of RdRPs and genome organizations of barna-like and solemo-like viruses support the re-classification of Barnavirus and Dinornavirus genera as members of the Solemoviridae family. Full article

Poly(ADP-ribosyl)ation is a crucial posttranslational modification that governs gene expression, chromatin remodeling, and cellular homeostasis. This dynamic process is mediated by the opposing activities of poly(ADP-ribose) polymerases (PARPs), which synthesize poly(ADP-ribose) (pADPr), and poly(ADP-ribose) glycohydrolase (PARG), which degrades it. While PARP function has been extensively studied, the structural and mechanistic basis of PARG-mediated pADPr degradation remain incompletely understood. To investigate the role of PARG in pADPr metabolism, we employed CRISPR/Cas9-based genome editing to generate a novel Parg^29b mutant mouse embryonic stem cell (ESC) line carrying a precise deletion within the PARG catalytic domain. This deletion completely abolished pADPr hydrolytic activity, resulting in massive nuclear pADPr accumulation, yet ESC viability, proliferation, and cell cycle progression remained unaffected. Using Drosophila melanogaster as a model system, we demonstrated that this mutation completely disrupted the pADPr pathway and halted developmental progression, highlighting the essential role of PARG and pADPr turnover in organismal development. Our results define a critical structural determinant of PARG catalytic function, underscore the distinct requirements for pADPr metabolism in cellular versus developmental contexts, and provide a genetically tractable model for studying the regulation of poly(ADP-ribose) dynamics and therapeutic responses to PARP inhibition in vivo. Full article

Bougainvillea spp. possesses vibrantly pigmented bracts that exhibit high ornamental value. Zinc finger proteins (ZFPs), one of the most extensive transcription factor families in plants, are implicated in diverse biological functions, including plant morphogenesis, transcriptional regulation, and responses to abiotic stress. Nevertheless, their regulatory roles in bract pigmentation in Bougainvillea remain unexplored. In the present investigation, 105 BbZFP genes were identified from the Bougainvillea genome via bioinformatic analyses and subsequently categorized into five subgroups according to the quantity and arrangement of their structural domains. Analysis of physicochemical characteristics demonstrated that the BbZFP family encompasses both acidic and basic proteins, all of which are hydrophilic and predominantly classified as unstable proteins. Gene structure analysis revealed that the majority of BbZFP genes comprise between one and five– introns. Cis-regulatory element analysis suggested that BbZFP promoter regions harbor multiple elements associated with abiotic stress responses, hormonal regulation, and light responsiveness, implying their possible participation in these physiological processes. Transcriptomic data analysis revealed distinct expression patterns of BbZFP genes among bracts of different colors. A quantitative real-time polymerase chain reaction (RT-qPCR) further confirmed that Bou_68928, Bou_1096, Bou_4400, and Bou_17631 were markedly upregulated in yellow bracts relative to white bracts, suggesting their involvement in flavonoid biosynthesis regulation. Meanwhile, Bou_1096 and Bou_17631 exhibited markedly elevated expression in red-purple bracts compared to white bracts, potentially regulating betacyanin biosynthesis in Bougainvillea. These findings offer candidate genes for molecular breeding strategies aimed at enhancing floral coloration in Bougainvillea. The next step will involve elucidating the functions of these genes in bract coloration. Full article

Long interspersed element-1 (LINE-1) is the only active autonomous transposon comprising about 17% of human genomes. LINE-1 transposition can cause the mutation and rearrangement of the host’s genomic DNA. The host has, therefore, developed multiple mechanisms to restrict LINE-1 mobility. Here, we report that SLFN11, a member of the Schlafen family, can restrict LINE-1 retrotransposition, and the inhibitory activity requires its helicase domain. Mechanistically, SLFN11 specifically binds to the LINE-1 5′ untranslated region (5′UTR) and blocks RNA polymerase II recruitment, thereby suppressing its transcription. Furthermore, SLFN11 promotes heterochromatinization, suggesting an epigenetic inhibition pathway. Full article

Background/Objectives: This study aimed to analyze optic nerve parameters, retinal nerve fiber layer thickness (RNFLT), ganglion cell layer thickness (GCLT), and subfoveal choroidal thickness (ChT) in patients who have recovered from coronavirus disease 2019 (COVID-19). Methods: This comparative study included 78 recovered COVID-19 patients (16 men, 62 women) and 56 age- and sex-matched healthy controls (18 men, 38 women). COVID-19 was confirmed in all patients, either through the detection of viral RNA in nasopharyngeal swabs via reverse transcriptase polymerase chain reaction or by serological testing for SARS-CoV-2 antibodies. Spectral-domain optical coherence tomography (SD-OCT) was used to assess optic nerve parameters, RNFLT, GCLT, and ChT. Results: The mean age was 35.0 ± 8.3 years in the COVID-19 group and 31.5 ± 8.3 years in the control group, with no statistically significant differences in age or sex distribution between groups (p = 0.41 and p = 0.16, respectively). Optic nerve parameters and RNFLT (overall and across the four peripapillary quadrants) did not differ significantly between the COVID-19 and control groups. However, the mean ganglion cell–inner plexiform layer (GC-IPL) thickness was significantly reduced in all quadrants in the COVID-19 group compared to the controls. No significant difference was observed in mean subfoveal ChT between groups. Conclusions: A significant reduction in ganglion GCLT was observed in recovered COVID-19 patients compared to healthy controls, suggesting a potential neurodegenerative effect of the disease on the optic nerve. Full article

ABC transporters are a large family of proteins that mediate the export or import of a variety of molecules, including capsular polysaccharides. The capsules are an important virulence factor that protect bacteria from host immune system attacks, antibiotics, and physicochemical changes in their environment. In some Gram-negative pathogenic bacteria, ABC transporter-dependent systems facilitate the export of capsular polysaccharides. These transport systems are composed of three parts: the ABC transporter and the polysaccharide co-polymerase protein in the inner membrane and the outer membrane polysaccharide export protein in the outer membrane. The glycolipid anchor of the capsular polysaccharide binds to a pocket between the two subunits of the ABC transporter transmembrane domain. The three parts of the ABC transporter-dependent system form a tunnel, through which the capsular polysaccharide is exported using energy from ATP hydrolysis. Knowledge of the ABC transporter-dependent system and its function is incomplete, requiring further research to better understand the processes of capsular polysaccharide export. This may also allow, in the future, to develop new molecules that inhibit capsular polysaccharide export, which would help the host immune system fight Gram-negative pathogenic bacteria coated with capsular polysaccharides. This review presents the latest findings on ABC transporter-dependent systems that export capsular polysaccharides in Gram-negative pathogenic bacteria. Full article

Missense mutations in the BCR::ABL1 kinase domain are found in approximately 12–80% of patients with chronic myeloid leukemia (CML). Clinically significant mutations include T315I, M244V, Y253H/F, E255K/V, V299L, and F359V. The aim of this study was to create a diagnostic system for rapid and inexpensive detection of the above mutations. We used genomic DNA and RNA from peripheral blood and bone marrow cells of 57 patients with a Ph-positive CML diagnosis established in the chronic phase. We have developed a method to detect mutations in the BCR::ABL1 gene based on allele-specific real-time polymerase chain reaction (AS-PCR). In parallel, we analyzed the RNA sequence of the protein kinase domain of the same samples by next-generation sequencing (NGS) covering the points of putative mutations. In this work, we compared the results obtained by both methods for mutation detection and variant allele frequency (VAF) estimation of mutated vs. normal alleles. The sensitivity and specificity of our diagnostic system were also evaluated. It was found that AS-PCR gives reliable results at VAF up to 0.01%. AS-PCR has high sensitivity and may serve as an alternative for the more time-consuming NGS in some cases, as well as for monitoring CML treatment and for analyzing archival material. Full article

The regulation of downstream responsive genes by transcription factors (TFs) is a critical step in the stress response system of plants. While bZIP transcription factors are known to play important roles in stress reactions, their functional characterization in soybeans remains limited. Here, we identified a soybean bZIP gene, GmbZIP60, which encodes a protein containing a typical bZIP domain with a basic region and a leucine zipper region. Subcellular localization studies confirmed that GmbZIP60 is localized in the nucleus. Expression analysis demonstrated that GmbZIP60 is induced by salt stress, drought stress, and various plant hormone treatments, including abscisic acid (ABA), ethylene (ETH), and methyl jasmonate acid (MeJA). Overexpressing GmbZIP60 (OE-GmbZIP60) in transgenic soybean and rice enhanced tolerance to both salt and drought stresses. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that the expression levels of abiotic stress-responsive genes were significantly higher in transgenic plants than in wild-type (WT) plants under stress conditions. Chromatin immunoprecipitation-qPCR (ChIP-qPCR) analysis further confirmed that GmbZIP60 directly binds to the promoters of abiotic stress-related genes induced by ABA, ETH, JA, and salicylic acid (SA). Overall, these findings revealed GmbZIP60 as a positive regulator of salt and drought stress tolerance. Full article

Background: The location of BRCA mutations within functional domains may affect sensitivity to poly (ADP-ribose) polymerase (PARP) inhibitors and platinum-based chemotherapy. This study aimed to evaluate the progression-free survival (PFS) benefit from the PARP inhibitor in relation to the location of mutations in BRCA1/BRCA2 in newly diagnosed ovarian cancer. Materials and methods: Patients with advanced stage III-IV epithelial ovarian cancer who had deleterious BRCA1 or BRCA2 were analyzed. PFS and clinical and molecular data were compared between patients who received olaparib or niraparib as frontline maintenance therapy and those who did not. Subgroup analyses were conducted based on the location of BRCA mutations within the functional domain or the ovarian cancer cluster region (OCCR). Results: Of the 380 patients, 242 (63.7%) harbored BRCA1 mutation, 137 (36.1%) harbored BRCA2, and one (0.3%) harbored both BRCA1 and BRCA2. With a median follow-up of 35.8 months, the DNA binding domain in BRCA1 (HR, 0.34; 95% CI, 0.15–0.79; p = 0.01) and BRCA2 (HR, 0.25; 95% CI, 0.08–0.78; p = 0.01) demonstrated particularly significant benefit. In patients who harbored BRCA1 mutation in the C-terminal domain (BRCT), no statistically significant PFS benefit from PARP inhibitor was observed (HR, 0.76; 95% CI, 0.39–1.52; p = 0.44). PFS benefit from PARP inhibitor maintenance was observed in both OCCR (HR, 0.49; 95% CI, 0.32–0.74; p < 0.01) and non-OCCR (HR, 0.51; 95% CI, 0.27–0.63; p < 0.01). Conclusions: Frontline PARP inhibitor maintenance therapy demonstrated a significant PFS benefit in patients with BRCA1/2 mutations, with particularly pronounced benefits for those with mutations located in the DBD of BRCA1 and BRCA2. However, the benefit was less evident for patients with BRCA1 mutations located in the BRCT domain. Full article