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Microorganisms May 2024Replication of the mitochondrial (mt) genome in filamentous fungi is under-studied, and knowledge is based mainly on data from yeasts and higher eukaryotes. In this...
Replication of the mitochondrial (mt) genome in filamentous fungi is under-studied, and knowledge is based mainly on data from yeasts and higher eukaryotes. In this study, the mitochondrial DNA polymerase γ (Mip1) of the entomopathogenic fungus is characterized and analyzed with disruption experiments and its in silico interactions with key proteins implicated in mt gene transcription, i.e., mt RNA polymerase Rpo41 and mt transcription factor Mtf1. Disruption of 1 gene and its partial expression influences cell growth, morphology, germination and stress tolerance. A putative in silico model of Mip1-Rpo41-Mtf1, which is known to be needed for the initiation of replication, was proposed and helped to identify potential amino acid residues of Mip1 that interact with the Rpo41-Mtf1 complex. Moreover, the reduced expression of 1 indicates that Mip1 is not required for efficient transcription but only for replication. Functional differences between the Mip1 and its counterparts from and higher eukaryotes are discussed.
PubMed: 38930434
DOI: 10.3390/microorganisms12061052 -
Life (Basel, Switzerland) May 2024Theoretical and experimental approaches have been applied to study the polymer physics underlying the compaction of DNA in the bacterial nucleoid. Knowledge of the... (Review)
Review
Theoretical and experimental approaches have been applied to study the polymer physics underlying the compaction of DNA in the bacterial nucleoid. Knowledge of the compaction mechanism is necessary to obtain a mechanistic understanding of the segregation process of replicating chromosome arms (replichores) during the cell cycle. The first part of this review discusses light microscope observations demonstrating that the nucleoid has a lower refractive index and thus, a lower density than the cytoplasm. A polymer physics explanation for this phenomenon was given by a theory discussed at length in this review. By assuming a phase separation between the nucleoid and the cytoplasm and by imposing equal osmotic pressure and chemical potential between the two phases, a minimal energy situation is obtained, in which soluble proteins are depleted from the nucleoid, thus explaining its lower density. This theory is compared to recent views on DNA compaction that are based on the exclusion of polyribosomes from the nucleoid or on the transcriptional activity of the cell. These new views prompt the question of whether they can still explain the lower refractive index or density of the nucleoid. In the second part of this review, we discuss the question of how DNA segregation occurs in in the absence of the so-called active ParABS system, which is present in the majority of bacteria. How is the entanglement of nascent chromosome arms generated at the origin in the parental DNA network of the nucleoid prevented? Microscopic observations of the position of fluorescently-labeled genetic loci have indicated that the four nascent chromosome arms synthesized in the initial replication bubble segregate to opposite halves of the sister nucleoids. This implies that extensive intermingling of daughter strands does not occur. Based on the hypothesis that leading and lagging replichores synthesized in the replication bubble fold into microdomains that do not intermingle, a passive four-excluding-arms model for segregation is proposed. This model suggests that the key for segregation already exists in the structure of the replication bubble at the very start of DNA replication; it explains the different patterns of chromosome arms as well as the segregation distances between replicated loci, as experimentally observed.
PubMed: 38929644
DOI: 10.3390/life14060660 -
Children (Basel, Switzerland) Jun 2024A ~3-kb deletion-type DNA copy number variation (CNV, esv3587290) located at intron 7 of the gene (1p13.1, MIM*610132) has been proposed as a genetic factor in lupus...
Does the esv3587290 Copy Number Variation in the Gene Differ as a Genetic Factor for Developing Nephritis in Mexican Childhood-Onset Systemic Lupus Erythematosus Patients?
A ~3-kb deletion-type DNA copy number variation (CNV, esv3587290) located at intron 7 of the gene (1p13.1, MIM*610132) has been proposed as a genetic factor in lupus nephritis (LN) development in adult systemic lupus erythematosus (SLE) patients across European-descent populations, but its replication in other ethnicities has been inconsistent and its association with LN in childhood-onset SLE (cSLE) remains unknown. Here, we performed an exploratory association study in a sample of 66 unrelated cSLE Mexican patients (11 males, 55 females; ages 7.8 to 18.6 years). Two stratified groups were compared: cSLE patients with (N = 39) or without (N = 27) LN, as diagnosed by renal biopsy (N = 17), proteinuria (N = 33), urinary protein-creatinine ratio > 0.2 (N = 34), and erythrocyturia and/or granular casts in urinary sediment (N = 16). For esv3587290 CNV genotyping, we performed an end-point PCR assay with breakpoint confirmation using Sanger sequencing. We also determined the allelic frequencies of the esv3587290 CNV in 181 deidentified ethnically matched individuals (reference group). The obtained genotypes were tested for Hardy-Weinberg equilibrium using the χ test. Associations between LN and esv3587290 CNV were tested by calculating the odds ratio (OR) and using Pearson's χ tests, with a 95% confidence interval and ≤ 0.05. The esv3587290 CNV allele (OR 0.108, 95% CI 0.034-0.33, = 0.0003) and the heterozygous genotype (OR 0.04, 95% CI 0.119-0.9811, = 0.002) showed a significant protective effect against LN development. Finally, we characterized the precise breakpoint of the esv3587290 CNV to be NG_016548.1(NM_138959.3):c.1314+1339_1315-897del in our population. This report supports the notion that a broad genetic heterogeneity underlies the susceptibility for developing LN.
PubMed: 38929291
DOI: 10.3390/children11060712 -
International Journal of Molecular... Jun 2024Application of laser-generated electron beams in radiotherapy is a recent development. Accordingly, mechanisms of biological response to radiation damage need to be...
Application of laser-generated electron beams in radiotherapy is a recent development. Accordingly, mechanisms of biological response to radiation damage need to be investigated. In this study, telomere length (TL) as endpoint of genetic damage was analyzed in human blood cells (leukocytes) and K562 leukemic cells irradiated with laser-generated ultrashort electron beam. Metaphases and interphases were analyzed in quantitative fluorescence in situ hybridization (Q-FISH) to assess TL. TLs were shortened compared to non-irradiated controls in both settings (metaphase and interphase) after irradiation with 0.5, 1.5, and 3.0 Gy in blood leukocytes. Radiation also caused a significant TL shortening detectable in the interphase of K562 cells. Overall, a negative correlation between TL and radiation doses was observed in normal and leukemic cells in a dose-dependent manner. K562 cells were more sensitive than normal blood cells to increasing doses of ultrashort electron beam radiation. As telomere shortening leads to genome instability and cell death, the results obtained confirm the suitability of this biomarker for assessing genotoxic effects of accelerated electrons for their further use in radiation therapy. Observed differences in TL shortening between normal and K562 cells provide an opportunity for further development of optimal radiation parameters to reduce side effects in normal cells during radiotherapy.
Topics: Humans; K562 Cells; Leukocytes; Electrons; Telomere; Leukemia; Telomere Homeostasis; In Situ Hybridization, Fluorescence; Telomere Shortening; DNA Damage; Dose-Response Relationship, Radiation
PubMed: 38928414
DOI: 10.3390/ijms25126709 -
International Journal of Molecular... Jun 2024Oncolytic adenoviruses are in development as immunotherapeutic agents for solid tumors. Their efficacy is in part dependent on their ability to replicate in tumors. It...
Oncolytic adenoviruses are in development as immunotherapeutic agents for solid tumors. Their efficacy is in part dependent on their ability to replicate in tumors. It is, however, difficult to obtain evidence for intratumoral oncolytic adenovirus replication if direct access to the tumor is not possible. Detection of systemic adenovirus DNA, which is sometimes used as a proxy, has limited value because it does not distinguish between the product of intratumoral replication and injected virus that did not replicate. Therefore, we investigated if detection of virus-associated RNA (VA RNA) by RT-qPCR on liquid biopsies could be used as an alternative. We found that VA RNA is expressed in adenovirus-infected cells in a replication-dependent manner and is secreted by these cells in association with extracellular vesicles. This allowed VA RNA detection in the peripheral blood of a preclinical in vivo model carrying adenovirus-injected human tumors and on liquid biopsies from a human clinical trial. Our results confirm that VA RNA detection in liquid biopsies can be used for minimally invasive assessment of oncolytic adenovirus replication in solid tumors in vivo.
Topics: Humans; Virus Replication; Oncolytic Viruses; RNA, Viral; Adenoviridae; Animals; Oncolytic Virotherapy; Mice; Cell Line, Tumor; Neoplasms; Female
PubMed: 38928259
DOI: 10.3390/ijms25126551 -
Genes May 2024Protein-DNA complex interactivity plays a crucial role in biological activities such as gene expression, modification, replication and transcription. Understanding the...
Protein-DNA complex interactivity plays a crucial role in biological activities such as gene expression, modification, replication and transcription. Understanding the physiological significance of protein-DNA binding interfacial hot spots, as well as the development of computational biology, depends on the precise identification of these regions. In this paper, a hot spot prediction method called EC-PDH is proposed. First, we extracted features of these hot spots' solid solvent-accessible surface area (ASA) and secondary structure, and then the mean, variance, energy and autocorrelation function values of the first three intrinsic modal components (IMFs) of these conventional features were extracted as new features via the empirical modal decomposition algorithm (EMD). A total of 218 dimensional features were obtained. For feature selection, we used the maximum correlation minimum redundancy sequence forward selection method (mRMR-SFS) to obtain an optimal 11-dimensional-feature subset. To address the issue of data imbalance, we used the SMOTE-Tomek algorithm to balance positive and negative samples and finally used cat gradient boosting (CatBoost) to construct our hot spot prediction model for protein-DNA binding interfaces. Our method performs well on the test set, with AUC, MCC and F1 score values of 0.847, 0.543 and 0.772, respectively. After a comparative evaluation, EC-PDH outperforms the existing state-of-the-art methods in identifying hot spots.
Topics: Machine Learning; DNA; Algorithms; Protein Binding; DNA-Binding Proteins; Computational Biology; Binding Sites
PubMed: 38927611
DOI: 10.3390/genes15060676 -
Antibiotics (Basel, Switzerland) Jun 2024The emergence of carbapenem-resistant Gram-negative pathogens presents a clinical challenge in infection treatment, prompting the repurposing of existing drugs as an...
The emergence of carbapenem-resistant Gram-negative pathogens presents a clinical challenge in infection treatment, prompting the repurposing of existing drugs as an essential strategy to address this crisis. Although the anticancer drug 5-fluorouracil (5-FU) has been recognized for its antibacterial properties, its mechanisms are not fully understood. Here, we found that the minimal inhibitory concentration (MIC) of 5-FU against was 32-64 µg/mL, including strains carrying , which confers resistance to carbapenems. We further elucidated the antibacterial mechanism of 5-FU against by using genetic and biochemical analyses. We revealed that the mutation of uracil phosphoribosyltransferase-encoding gene increased the MIC of 5-FU against by 32-fold, indicating the role of the gene in 5-FU resistance. Additionally, transcriptomic analysis of treated with 5-FU at 8 µg/mL and 32 µg/mL identified 602 and 1082 differentially expressed genes involved in carbon and nucleic acid metabolism, DNA replication, and repair pathways. The biochemical assays showed that 5-FU induced bacterial DNA damage, significantly increased intracellular ATP levels and the NAD/NADH ratio, and promoted reactive oxygen species (ROS) production. These findings suggested that 5-FU may exert antibacterial effects on through multiple pathways, laying the groundwork for its further development as a therapeutic candidate against carbapenem-resistant bacterial infections.
PubMed: 38927194
DOI: 10.3390/antibiotics13060528 -
Biomolecules Jun 2024Clickable nucleosides, most often 5-ethynyl-2'-deoxyuridine (EtU), are widely used in studies of DNA replication in living cells and in DNA functionalization for...
Clickable nucleosides, most often 5-ethynyl-2'-deoxyuridine (EtU), are widely used in studies of DNA replication in living cells and in DNA functionalization for bionanotechology applications. Although clickable dNTPs are easily incorporated by DNA polymerases into the growing chain, afterwards they might become targets for DNA repair systems or interfere with faithful nucleotide insertion. Little is known about the possibility and mechanisms of these post-synthetic events. Here, we investigated the repair and (mis)coding properties of EtU and two bulkier clickable pyrimidine nucleosides, 5-(octa-1,7-diyn-1-yl)-U (C8-AlkU) and 5-(octa-1,7-diyn-1-yl)-C (C8-AlkC). In vitro, EtU and C8-AlkU, but not C8-AlkC, were excised by SMUG1 and MBD4, two DNA glycosylases from the base excision repair pathway. However, when placed into a plasmid encoding a fluorescent reporter inactivated by repair in human cells, EtU and C8-AlkU persisted for much longer than uracil or its poorly repairable phosphorothioate-flanked derivative. DNA polymerases from four different structural families preferentially bypassed EtU, C8-AlkU and C8-AlkC in an error-free manner, but a certain degree of misincorporation was also observed, especially evident for DNA polymerase β. Overall, clickable pyrimidine nucleotides could undergo repair and be a source of mutations, but the frequency of such events in the cell is unlikely to be considerable.
Topics: DNA Repair; Humans; Pyrimidine Nucleotides; Click Chemistry; DNA-Directed DNA Polymerase; Deoxyuridine; DNA; DNA Replication; Uracil-DNA Glycosidase
PubMed: 38927084
DOI: 10.3390/biom14060681 -
Zhongguo Shi Yan Xue Ye Xue Za Zhi Jun 2024Studies have found that 1/3 patients with acquired aplastic anemia have shortened telomere length, and the shorter the telomere, the longer the disease course, the more... (Review)
Review
Studies have found that 1/3 patients with acquired aplastic anemia have shortened telomere length, and the shorter the telomere, the longer the disease course, the more prone to relapse, the lower the overall survival rate, and the higher the probability of clonal evolution. The regulation of telomere length is affected by many factors, including telomerase activity, telomerase-related genes, telomere regulatory proteins and other related factors. Telomere shortening can lead to genetic instability and increases the probability of clonal evolution in patients with acquired aplastic anemia. This article reviews the role of telomere in the clonal evolution of acquired aplastic anemia and factors affecting telomere length.
Topics: Anemia, Aplastic; Telomere Shortening; Clonal Evolution; Survival Rate; Recurrence; Telomere Homeostasis; Telomerase; Genomic Instability; Humans
PubMed: 38926996
DOI: 10.19746/j.cnki.issn.1009-2137.2024.03.048 -
Nature Communications Jun 2024Oncogene-induced senescence (OIS) arrests cell proliferation in response to replication stress (RS) induced by oncogenes. OIS depends on the DNA damage response (DDR),...
Oncogene-induced senescence (OIS) arrests cell proliferation in response to replication stress (RS) induced by oncogenes. OIS depends on the DNA damage response (DDR), but also on the cGAS-STING pathway, which detects cytosolic DNA and induces type I interferons (IFNs). Whether and how RS and IFN responses cooperate to promote OIS remains unknown. Here, we show that the induction of OIS by the H-RAS oncogene in immortalized human fibroblasts depends on the MRE11 nuclease. Indeed, treatment with the MRE11 inhibitor Mirin prevented RS, micronuclei formation and IFN response induced by RAS. Overexpression of the cytosolic nuclease TREX1 also prevented OIS. Conversely, overexpression of a dominant negative mutant of TREX1 or treatment with IFN-β was sufficient to induce RS and DNA damage, independent of RAS induction. These data suggest that the IFN response acts as a positive feedback loop to amplify DDR in OIS through a process regulated by MRE11 and TREX1.
Topics: Humans; Exodeoxyribonucleases; Phosphoproteins; MRE11 Homologue Protein; Signal Transduction; Cellular Senescence; DNA Replication; DNA Damage; Fibroblasts; Interferon-beta
PubMed: 38926338
DOI: 10.1038/s41467-024-49740-w