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BioRxiv : the Preprint Server For... Apr 2024Viruses with double-stranded (ds) DNA genomes in the realm share a conserved structural gene module but show a broad range of variation in their repertoires of DNA...
Viruses with double-stranded (ds) DNA genomes in the realm share a conserved structural gene module but show a broad range of variation in their repertoires of DNA replication proteins. Some of the duplodnaviruses encode (nearly) complete replication systems whereas others lack (almost) all genes required for replication, relying on the host replication machinery. DNA polymerases (DNAPs) comprise the centerpiece of the DNA replication apparatus. The replicative DNAPs are classified into 4 unrelated or distantly related families (A-D), with the protein structures and sequences within each family being, generally, highly conserved. More than half of the duplodnaviruses encode a DNAP of family A, B or C. We showed previously that multiple pairs of closely related viruses in the order encode DNAPs of different families. Here we identify four additional groups of tailed phages in the class in which the DNAPs apparently were swapped on multiple occasions, with replacements occurring both between families A and B, or A and C, or between distinct subfamilies within the same family. The DNAP swapping always occurs "in situ", without changes in the organization of the surrounding genes. In several cases, the DNAP gene is the only region of substantial divergence between closely related phage genomes, whereas in others, the swap apparently involved neighboring genes encoding other proteins involved in phage replication. We hypothesize that DNAP swapping is driven by selection for avoidance of host antiphage mechanisms targeting the phage DNAP that remain to be identified, and/or by selection against replicon incompatibility. In addition, we identified two previously undetected, highly divergent groups of family A DNAPs that are encoded in some phage genomes along with the main DNAP implicated in genome replication.
PubMed: 38903090
DOI: 10.1101/2024.04.26.591309 -
DNA Repair Jun 2024The KU heterodimer (KU70/80) is rapidly recruited to DNA double-strand breaks (DSBs) to regulate their processing and repair. Previous work has revealed that the...
The KU heterodimer (KU70/80) is rapidly recruited to DNA double-strand breaks (DSBs) to regulate their processing and repair. Previous work has revealed that the amino-terminal von Willebrand-like (vWA-like) domain in KU80 harbours a conserved hydrophobic pocket that interacts with a short peptide motif known as the Ku-binding motif (KBM). The KBM is present in a variety of DNA repair proteins such as APLF, CYREN, and Werner protein (WRN). Here, to investigate the importance of KBM-mediated protein-protein interactions for KU80 function, we employed KU80-deficient Chinese Hamster Ovary (Xrs-6) cells transfected with RFP-tagged wild-type human KU80 or KU80 harbouring a mutant vWA-like domain (KU80). Surprisingly, while mutant RFP-KU80 largely or entirely restored NHEJ efficiency and radiation resistance in KU80-deficient Xrs-6 cells, it failed to restore cellular resistance to DNA replication stress induced by camptothecin (CPT) or hydroxyurea (HU). Moreover, KU80-deficient Xrs-6 cells expressing RFP-KU80 accumulated pan-nuclear γH2AX in an S/G2-phase-dependent manner following treatment with CPT or HU, suggesting that the binding of KU80 to one or more KBM-containing proteins is required for the processing and/or repair of DNA ends that arise during DNA replication stress. Consistent with this idea, depletion of WRN helicase/exonuclease recapitulated the CPT-induced γH2AX phenotype, and did so epistatically with mutation of the KU80 vWA-like domain. These data identify a role for the KBM-binding by KU80 in the response and resistance of CHO cells to arrested and/or collapsed DNA replication forks, and implicate the KBM-mediated interaction of KU80 with WRN as a critical effector of this role.
PubMed: 38901287
DOI: 10.1016/j.dnarep.2024.103710 -
Journal of Cellular and Molecular... Jun 2024Bacillus subtilis relies on biofilms for survival in harsh environments. Extracellular polymeric substance (EPS) is a crucial component of biofilms, yet the dynamics of...
Bacillus subtilis relies on biofilms for survival in harsh environments. Extracellular polymeric substance (EPS) is a crucial component of biofilms, yet the dynamics of EPS production in single cells remain elusive. To unveil the modulation of EPS synthesis, we built a minimal network model comprising the SinI-SinR-SlrR module, Spo0A, and EPS. Stochastic simulations revealed that antagonistic interplay between SinI and SinR enables EPS production in bursts. SlrR widens these bursts and increases their frequency by stabilizing SinR-SlrR complexes and depleting free SinR. DNA replication and chromosomal positioning of key genes dictate pulsatile changes in the slrR:sinR gene dosage ratio (g) and Spo0A-P levels, each promoting EPS production in distinct phases of the cell cycle. As the cell cycle lengthens with nutrient stress, the duty cycle of g pulsing decreases, whereas the amplitude of Spo0A-P pulses elevates. This coordinated response facilitates keeping a constant proportion of EPS-secreting cells within colonies across diverse nutrient conditions. Our results suggest that bacteria may 'encode' eps expression through strategic chromosomal organization. This work illuminates how stochastic protein interactions, gene copy number imbalance, and cell-cycle dynamics orchestrate EPS synthesis, offering a deeper understanding of biofilm formation.
Topics: Biofilms; Bacillus subtilis; DNA Replication; Gene Expression Regulation, Bacterial; Bacterial Proteins; Extracellular Polymeric Substance Matrix; Cell Cycle
PubMed: 38899542
DOI: 10.1111/jcmm.18481 -
World Journal of Gastroenterology Jun 2024Obesity is associated with a significantly increased risk for chronic diarrhea, which has been proposed as Linghu's obesity-diarrhea syndrome (ODS); however, its...
BACKGROUND
Obesity is associated with a significantly increased risk for chronic diarrhea, which has been proposed as Linghu's obesity-diarrhea syndrome (ODS); however, its molecular mechanisms are largely unknown.
AIM
To reveal the transcriptomic changes in the jejunum involved in ODS.
METHODS
In a cohort of 6 ODS patients (JOD group), 6 obese people without diarrhea (JO group), and 6 healthy controls (JC group), high-throughput sequencing and bioinformatics analyses were performed to identify jejunal mucosal mRNA expression alterations and dysfunctional biological processes. In another cohort of 16 ODS patients (SOD group), 16 obese people without diarrhea (SO group), and 16 healthy controls (SC group), serum diamine oxidase (DAO) and D-lactate (D-LA) concentrations were detected to assess changes in intestinal barrier function.
RESULTS
The gene expression profiles of jejunal mucosa in the JO and JC groups were similar, with only 1 differentially expressed gene (DEG). The gene expression profile of the JOD group was significantly changed, with 411 DEGs compared with the JO group and 211 DEGs compared with the JC group, 129 of which overlapped. The enrichment analysis of these DEGs showed that the biological processes such as digestion, absorption, and transport of nutrients (especially lipids) tended to be up-regulated in the JOD group, while the biological processes such as rRNA processing, mitochondrial translation, antimicrobial humoral response, DNA replication, and DNA repair tended to be down-regulated in the JOD group. Eight DEGs (, , , , , , , and ) may play a key regulatory role in the pathological process of ODS, and their expression levels were significantly decreased in ODS patients ( < 0.001). In the second cohort, compared with healthy controls, the levels of serum intestinal barrier function markers (DAO and D-LA) were significantly increased in all obese individuals ( < 0.01), but were higher in the SOD group than in the SO group ( < 0.001).
CONCLUSION
Compared with healthy controls and obese individuals without diarrhea, patients with Linghu's ODS had extensive transcriptomic changes in the jejunal mucosa, likely affecting intestinal barrier function and thus contributing to the obesity and chronic diarrhea phenotypes.
Topics: Humans; Jejunum; Male; Pilot Projects; Female; Diarrhea; Adult; Intestinal Mucosa; Obesity; Middle Aged; Gene Expression Profiling; Transcriptome; Case-Control Studies; Syndrome; Amine Oxidase (Copper-Containing); Computational Biology; Lactic Acid; Chronic Disease
PubMed: 38899329
DOI: 10.3748/wjg.v30.i21.2777 -
Nature Communications Jun 2024DNA replication in differentiated cells follows a defined program, but when and how it is established during mammalian development is not known. Here we show using...
DNA replication in differentiated cells follows a defined program, but when and how it is established during mammalian development is not known. Here we show using single-cell sequencing, that late replicating regions are established in association with the B compartment and the nuclear lamina from the first cell cycle after fertilization on both maternal and paternal genomes. Late replicating regions contain a relative paucity of active origins and few but long genes and low G/C content. In both bovine and mouse embryos, replication timing patterns are established prior to embryonic genome activation. Chromosome breaks, which form spontaneously in bovine embryos at sites concordant with human embryos, preferentially locate to late replicating regions. In mice, late replicating regions show enhanced fragility due to a sparsity of dormant origins that can be activated under conditions of replication stress. This pattern predisposes regions with long neuronal genes to fragility and genetic change prior to separation of soma and germ cell lineages. Our studies show that the segregation of early and late replicating regions is among the first layers of genome organization established after fertilization.
Topics: Animals; DNA Replication; Mice; Embryo, Mammalian; Cattle; Nuclear Lamina; Female; Male; Humans; Embryonic Development; Genome; Single-Cell Analysis
PubMed: 38898078
DOI: 10.1038/s41467-024-49565-7 -
Brazilian Journal of Medical and... 2024Innate immune system activation is crucial in the inflammatory response, but uncontrolled activation can lead to autoimmune diseases. Cellular exhaustion and senescence... (Review)
Review
Innate immune system activation is crucial in the inflammatory response, but uncontrolled activation can lead to autoimmune diseases. Cellular exhaustion and senescence are two processes that contribute to innate immune tolerance breakdown. Exhausted immune cells are unable to respond adequately to specific antigens or stimuli, while senescent cells have impaired DNA replication and metabolic changes. These processes can impair immune system function and disrupt homeostasis, leading to the emergence of autoimmunity. However, the influence of innate immune exhaustion and senescence on autoimmune disorders is not well understood. This review aims to describe the current findings on the role of innate immune exhaustion and senescence in autoimmunity, focusing on the cellular and molecular changes involved in each process. Specifically, the article explores the markers and pathways associated with immune exhaustion, such as PD-1 and TIM-3, and senescence, including Β-galactosidase (β-GAL), lamin B1, and p16ink4a, and their impact on autoimmune diseases, namely type 1 diabetes, rheumatoid arthritis, systemic lupus erythematosus, and immune-mediated myopathies. Understanding the mechanisms underlying innate immune exhaustion and senescence in autoimmunity may provide insights for the development of novel therapeutic strategies.
Topics: Humans; Immunity, Innate; Autoimmune Diseases; Cellular Senescence; Autoimmunity; Immune System Exhaustion
PubMed: 38896644
DOI: 10.1590/1414-431X2024e13225 -
ELife Jun 2024ASARs are a family of very-long noncoding RNAs that control replication timing on individual human autosomes, and are essential for chromosome stability. The eight known...
ASARs are a family of very-long noncoding RNAs that control replication timing on individual human autosomes, and are essential for chromosome stability. The eight known ASAR lncRNAs remain closely associated with their parent chromosomes. Analysis of RNA-protein interaction data (from ENCODE) revealed numerous RBPs with significant interactions with multiple ASAR lncRNAs, with several hnRNPs as abundant interactors. An ~7 kb domain within the lncRNA shows a striking density of RBP interaction sites. Genetic deletion and ectopic integration assays indicate that this ~7 kb RNA binding protein domain contains functional sequences for controlling replication timing of entire chromosomes in cis. shRNA-mediated depletion of 10 different RNA binding proteins, including HNRNPA1, HNRNPC, HNRNPL, HNRNPM, HNRNPU, or HNRNPUL1, results in dissociation of ASAR lncRNAs from their chromosome territories, and disrupts the synchronous replication that occurs on all autosome pairs, recapitulating the effect of individual ASAR knockouts on a genome-wide scale. Our results further demonstrate the role that ASARs play during the temporal order of genome-wide replication, and we propose that ASARs function as essential RNA scaffolds for the assembly of hnRNP complexes that help maintain the structural integrity of each mammalian chromosome.
Topics: RNA, Long Noncoding; Humans; Heterogeneous-Nuclear Ribonucleoproteins; DNA Replication Timing; Protein Binding; RNA-Binding Proteins
PubMed: 38896448
DOI: 10.7554/eLife.95898 -
Journal of Clinical Immunology Jun 2024A cell's ability to survive and to evade cancer is contingent on its ability to retain genomic integrity, which can be seriously compromised when nucleic acid...
A cell's ability to survive and to evade cancer is contingent on its ability to retain genomic integrity, which can be seriously compromised when nucleic acid phosphodiester bonds are disrupted. DNA Ligase 1 (LIG1) plays a key role in genome maintenance by sealing single-stranded nicks that are produced during DNA replication and repair. Autosomal recessive mutations in a limited number of individuals have been previously described for this gene. Here we report a homozygous LIG1 mutation (p.A624T), affecting a universally conserved residue, in a patient presenting with leukopenia, neutropenia, lymphopenia, pan-hypogammaglobulinemia, and diminished in vitro response to mitogen stimulation. Patient fibroblasts expressed normal levels of LIG1 protein but exhibited impaired growth, poor viability, high baseline levels of gamma-H2AX foci, and an enhanced susceptibility to DNA-damaging agents. The mutation reduced LIG1 activity by lowering its affinity for magnesium 2.5-fold. Remarkably, it also increased LIG1 fidelity > 50-fold against 3' end 8-Oxoguanine mismatches, exhibiting a marked reduction in its ability to process such nicks. This is expected to yield increased ss- and dsDNA breaks. Molecular dynamic simulations, and Residue Interaction Network studies, predicted an allosteric effect for this mutation on the protein loops associated with the LIG1 high-fidelity magnesium, as well as on DNA binding within the adenylation domain. These dual alterations of suppressed activity and enhanced fidelity, arising from a single mutation, underscore the mechanistic picture of how a LIG1 defect can lead to severe immunological disease.
Topics: Female; Humans; Male; DNA Ligase ATP; Fibroblasts; Homozygote; Molecular Dynamics Simulation; Mutation; Severe Combined Immunodeficiency; Infant
PubMed: 38896336
DOI: 10.1007/s10875-024-01754-1 -
BioRxiv : the Preprint Server For... Jun 2024All bacteria encode a multifunctional DNA-binding protein, DnaA, which initiates chromosomal replication. Despite having the most complex, segmented bacterial genome,...
All bacteria encode a multifunctional DNA-binding protein, DnaA, which initiates chromosomal replication. Despite having the most complex, segmented bacterial genome, little is known about DnaA and its role in maintaining the spirochete's physiology. We utilized inducible CRISPR-interference and overexpression to modulate cellular levels of DnaA to better understand this essential protein. Dysregulation of DnaA, either up or down, significantly slowed replication and increased or decreased cell lengths. Using fluorescent microscopy, we found the DnaA CRISPRi mutants had increased numbers of chromosomes with irregular spacing patterns. The DnaA-depleted spirochetes also exhibited a significant defect in helical morphology. RNA-seq of the conditional mutants showed significant changes in the levels of transcripts involved with flagellar synthesis, elongation, cell division, virulence, and other functions. These findings demonstrate that the DnaA plays a commanding role in maintaining borrelial growth dynamics and protein expression, which are essential for the survival of the Lyme disease spirochete.
PubMed: 38895450
DOI: 10.1101/2024.06.08.598065 -
BioRxiv : the Preprint Server For... Jun 2024In most eukaryotes, mitochondrial organelles contain their own genome, usually circular, which is the remnant of the genome of the ancestral bacterial endosymbiont that...
In most eukaryotes, mitochondrial organelles contain their own genome, usually circular, which is the remnant of the genome of the ancestral bacterial endosymbiont that gave rise to modern mitochondria. Mitochondrial genomes are dramatically reduced in their gene content due to the process of endosymbiotic gene transfer to the nucleus; as a result most mitochondrial proteins are encoded in the nucleus and imported into mitochondria. This includes the components of the dedicated mitochondrial transcription and replication systems and regulatory factors, which are entirely distinct from the information processing systems in the nucleus. However, since the 1990s several nuclear transcription factors have been reported to act in mitochondria, and previously we identified 8 human and 3 mouse transcription factors (TFs) with strong localized enrichment over the mitochondrial genome using ChIP-seq (Chromatin Immunoprecipitation) datasets from the second phase of the ENCODE (Encyclopedia of DNA Elements) Project Consortium. Here, we analyze the greatly expanded in the intervening decade ENCODE compendium of TF ChIP-seq datasets (a total of 6,153 ChIP experiments for 942 proteins, of which 763 are sequence-specific TFs) combined with interpretative deep learning models of TF occupancy to create a comprehensive compendium of nuclear TFs that show evidence of association with the mitochondrial genome. We find some evidence for chrM occupancy for 50 nuclear TFs and two other proteins, with bZIP TFs emerging as most likely to be playing a role in mitochondria. However, we also observe that in cases where the same TF has been assayed with multiple antibodies and ChIP protocols, evidence for its chrM occupancy is not always reproducible. In the light of these findings, we discuss the evidential criteria for establishing chrM occupancy and reevaluate the overall compendium of putative mitochondrial-acting nuclear TFs.
PubMed: 38895386
DOI: 10.1101/2024.06.04.597442