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Trends in Genetics : TIG Aug 2021Cell-free DNA (cfDNA) is a widely used noninvasive biomarker for diagnosis and prognosis of multiple disease states. Emerging evidence suggests that cfDNA might not just... (Review)
Review
Cell-free DNA (cfDNA) is a widely used noninvasive biomarker for diagnosis and prognosis of multiple disease states. Emerging evidence suggests that cfDNA might not just be passive waste products of cell death but could have a physiological and pathological function in inflammation and autoimmunity. The balance of cfDNA generation and clearance may thus be vital in health and disease. In particular, plasma nuclease activity has been linked to multiple pathologies including cancer and systemic lupus erythematosus (SLE) and associated with profound changes in the nonrandom fragmentation of cfDNA. Lastly, in this review, we explore the effects of DNA fragmentation factor B (DFFB), DNASE1L3, and DNASE1 on cfDNA levels and their fragmentomic profiles, and what these recent insights reveal about the biology of cfDNA.
Topics: Autoimmunity; Cell-Free Nucleic Acids; DNA Fragmentation; Deoxyribonuclease I; Deoxyribonucleases; Endodeoxyribonucleases; Humans; Inflammation; Poly-ADP-Ribose Binding Proteins
PubMed: 34006390
DOI: 10.1016/j.tig.2021.04.005 -
Science Advances Sep 2023Programmable RNA-guided DNA nucleases perform numerous roles in prokaryotes, but the extent of their spread outside prokaryotes is unclear. Fanzors, the eukaryotic...
Programmable RNA-guided DNA nucleases perform numerous roles in prokaryotes, but the extent of their spread outside prokaryotes is unclear. Fanzors, the eukaryotic homolog of prokaryotic TnpB proteins, have been detected in genomes of eukaryotes and large viruses, but their activity and functions in eukaryotes remain unknown. Here, we characterize Fanzors as RNA-programmable DNA endonucleases, using biochemical and cellular evidence. We found diverse Fanzors that frequently associate with various eukaryotic transposases. Reconstruction of Fanzors evolution revealed multiple radiations of RuvC-containing TnpB homologs in eukaryotes. Fanzor genes captured introns and proteins acquired nuclear localization signals, indicating extensive, long-term adaptation to functioning in eukaryotic cells. Fanzor nucleases contain a rearranged catalytic site of the RuvC domain, similar to a distinct subset of TnpBs, and lack collateral cleavage activity. We demonstrate that Fanzors can be harnessed for genome editing in human cells, highlighting the potential of these widespread eukaryotic RNA-guided nucleases for biotechnology applications.
Topics: Humans; Eukaryota; Deoxyribonuclease I; RNA; Deoxyribonucleases; Viruses
PubMed: 37756409
DOI: 10.1126/sciadv.adk0171 -
Frontiers in Immunology 2023Neutrophil Extracellular Traps (NETs) are key mediators of immunothrombotic mechanisms and defective clearance of NETs from the circulation underlies an array of...
BACKGROUND
Neutrophil Extracellular Traps (NETs) are key mediators of immunothrombotic mechanisms and defective clearance of NETs from the circulation underlies an array of thrombotic, inflammatory, infectious, and autoimmune diseases. Efficient NET degradation depends on the combined activity of two distinct DNases, DNase1 and DNase1-like 3 (DNase1L3) that preferentially digest double-stranded DNA (dsDNA) and chromatin, respectively.
METHODS
Here, we engineered a dual-active DNase with combined DNase1 and DNase1L3 activities and characterized the enzyme for its NET degrading potential in vitro. Furthermore, we produced a mouse model with transgenic expression of the dual-active DNase and analyzed body fluids of these animals for DNase1 and DNase 1L3 activities. We systematically substituted 20 amino acid stretches in DNase1 that were not conserved among DNase1 and DNase1L3 with homologous DNase1L3 sequences.
RESULTS
We found that the ability of DNase1L3 to degrade chromatin is embedded into three discrete areas of the enzyme's core body, not the C-terminal domain as suggested by the state-of-the-art. Further, combined transfer of the aforementioned areas of DNase1L3 to DNase1 generated a dual-active DNase1 enzyme with additional chromatin degrading activity. The dual-active DNase1 mutant was superior to native DNase1 and DNase1L3 in degrading dsDNA and chromatin, respectively. Transgenic expression of the dual-active DNase1 mutant in hepatocytes of mice lacking endogenous DNases revealed that the engineered enzyme was stable in the circulation, released into serum and filtered to the bile but not into the urine.
CONCLUSION
Therefore, the dual-active DNase1 mutant is a promising tool for neutralization of DNA and NETs with potential therapeutic applications for interference with thromboinflammatory disease states.
Topics: Mice; Animals; Endodeoxyribonucleases; Extracellular Traps; Deoxyribonuclease I; Chromatin; DNA; Deoxyribonucleases
PubMed: 37287977
DOI: 10.3389/fimmu.2023.1181761 -
The Journal of Experimental Medicine Jun 2023Extracellular DNase DNASE1L3 maintains tolerance to self-DNA in humans and mice, whereas the role of its homolog DNASE1 remains controversial, and the overall function...
Extracellular DNase DNASE1L3 maintains tolerance to self-DNA in humans and mice, whereas the role of its homolog DNASE1 remains controversial, and the overall function of secreted DNases in immunity is unclear. We report that deletion of murine DNASE1 neither caused autoreactivity in isolation nor exacerbated lupus-like disease in DNASE1L3-deficient mice. However, combined deficiency of DNASE1 and DNASE1L3 rendered mice susceptible to bloodstream infection with Staphylococcus aureus. DNASE1/DNASE1L3 double-deficient mice mounted a normal innate response to S. aureus and did not accumulate neutrophil extracellular traps (NETs). However, their kidneys manifested severe pathology, increased bacterial burden, and biofilm-like bacterial lesions that contained bacterial DNA and excluded neutrophils. Furthermore, systemic administration of recombinant DNASE1 protein during S. aureus infection rescued the mortality of DNase-deficient mice and ameliorated the disease in wild-type mice. Thus, DNASE1 and DNASE1L3 jointly facilitate the control of bacterial infection by digesting extracellular microbial DNA in biofilms, suggesting the original evolutionary function of secreted DNases as antimicrobial agents.
Topics: Animals; Mice; Deoxyribonuclease I; Deoxyribonucleases; DNA; Endodeoxyribonucleases; Extracellular Traps; Mammals; Sepsis; Staphylococcal Infections; Staphylococcus aureus; Biofilms
PubMed: 36928522
DOI: 10.1084/jem.20221086 -
JCI Insight Apr 2022Cell-free extrachromosomal circular DNA (eccDNA) as a distinct topological form from linear DNA has recently gained increasing research interest, with possible clinical...
Cell-free extrachromosomal circular DNA (eccDNA) as a distinct topological form from linear DNA has recently gained increasing research interest, with possible clinical applications as a class of biomarkers. In this study, we aimed to explore the relationship between nucleases and eccDNA characteristics in plasma. By using knockout mouse models with deficiencies in deoxyribonuclease 1 (DNASE1) or deoxyribonuclease 1 like 3 (DNASE1L3), we found that cell-free eccDNA in Dnase1l3-/- mice exhibited larger size distributions than that in wild-type mice. Such size alterations were not found in tissue eccDNA of either Dnase1-/- or Dnase1l3-/- mice, suggesting that DNASE1L3 could digest eccDNA extracellularly but did not seem to affect intracellular eccDNA. Using a mouse pregnancy model, we observed that in Dnase1l3-/- mice pregnant with Dnase1l3+/- fetuses, the eccDNA in the maternal plasma was shorter compared with that of Dnase1l3-/- mice carrying Dnase1l3-/- fetuses, highlighting the systemic effects of circulating fetal DNASE1L3 degrading the maternal eccDNA extracellularly. Furthermore, plasma eccDNA in patients with DNASE1L3 mutations also exhibited longer size distributions than that in healthy controls. Taken together, this study provided a hitherto missing link between nuclease activity and the biological manifestations of eccDNA in plasma, paving the way for future biomarker development of this special form of DNA molecules.
Topics: Animals; DNA; DNA, Circular; Deoxyribonucleases; Endodeoxyribonucleases; Female; Fetus; Humans; Mice; Mice, Knockout; Pregnancy
PubMed: 35451374
DOI: 10.1172/jci.insight.156070 -
Epigenetics & Chromatin Mar 2021The Hi-C technique is widely employed to study the 3-dimensional chromatin architecture and to assemble genomes. The conventional in situ Hi-C protocol employs...
BACKGROUND
The Hi-C technique is widely employed to study the 3-dimensional chromatin architecture and to assemble genomes. The conventional in situ Hi-C protocol employs restriction enzymes to digest chromatin, which results in nonuniform genomic coverage. Using sequence-agnostic restriction enzymes, such as DNAse I, could help to overcome this limitation.
RESULTS
In this study, we compare different DNAse Hi-C protocols and identify the critical steps that significantly affect the efficiency of the protocol. In particular, we show that the SDS quenching strategy strongly affects subsequent chromatin digestion. The presence of biotinylated oligonucleotide adapters may lead to ligase reaction by-products, which can be avoided by rational design of the adapter sequences. Moreover, the use of nucleotide-exchange enzymes for biotin fill-in enables simultaneous labelling and repair of DNA ends, similar to the conventional Hi-C protocol. These improvements simplify the protocol, making it less expensive and time-consuming.
CONCLUSIONS
We propose a new robust protocol for the preparation of DNAse Hi-C libraries from cultured human cells and blood samples supplemented with experimental controls and computational tools for the evaluation of library quality.
Topics: Chromatin; Chromosomes; Deoxyribonuclease I; Deoxyribonucleases; Genome; Humans
PubMed: 33743768
DOI: 10.1186/s13072-021-00389-5 -
Scientific Reports Jun 2022The human genome contains four DNase1 and two DNase2 genes. The origin and functional specialization of this repertoire are not fully understood. Here we use genomics...
The human genome contains four DNase1 and two DNase2 genes. The origin and functional specialization of this repertoire are not fully understood. Here we use genomics and transcriptomics data to infer the evolutionary history of DNases and investigate their biological significance. Both DNase1 and DNase2 families have expanded in vertebrates since ~ 650 million years ago before the divergence of jawless and jawed vertebrates. DNase1, DNase1L1, and DNase1L3 co-existed in jawless fish, whereas DNase1L2 originated in amniotes by tandem duplication of DNase1. Among the non-human DNases, DNase1L4 and newly identified DNase1L5 derived from early duplications that were lost in terrestrial vertebrates. The ancestral gene of the DNase2 family, DNase2b, has been conserved in synteny with the Uox gene across 700 million years of animal evolution,while DNase2 originated in jawless fish. DNase1L1 acquired a GPI-anchor for plasma membrane attachment in bony fishes, and DNase1L3 acquired a C-terminal basic peptide for the degradation of microparticle DNA in jawed vertebrates. The appearance of DNase1L2, with a distinct low pH optimum and skin localization, is among the amniote adaptations to life on land. The expansion of the DNase repertoire in vertebrates meets the diversified demand for DNA debris removal in complex multicellular organisms.
Topics: Animals; DNA; Deoxyribonuclease I; Deoxyribonucleases; Evolution, Molecular; Fishes; Gene Duplication; Humans; Phylogeny; Synteny; Vertebrates
PubMed: 35725583
DOI: 10.1038/s41598-022-14133-w -
Advanced Science (Weinheim,... Sep 2023Acute lung injury (ALI) is a frequent and serious complication of sepsis with limited therapeutic options. Gaining insights into the inflammatory dysregulation that...
Acute lung injury (ALI) is a frequent and serious complication of sepsis with limited therapeutic options. Gaining insights into the inflammatory dysregulation that causes sepsis-associated ALI can help develop new therapeutic strategies. Herein, the crucial role of cell-free mitochondrial DNA (cf-mtDNA) in the regulation of alveolar macrophage activation during sepsis-associated ALI is identified. Most importantly, a biocompatible hybrid protein nanomotor (NM) composed of recombinant deoxyribonuclease I (DNase-I) and human serum albumin (HSA) via glutaraldehyde-mediated crosslinking is prepared to obtain an inhalable nanotherapeutic platform targeting pulmonary cf-mtDNA clearance. The synthesized DNase-I/HSA NMs are endowed with self-propulsive capability and demonstrate superior performances in stability, DNA hydrolysis, and biosafety. Pulmonary delivery of DNase-I/HSA NMs effectively eliminates cf-mtDNAs in the lungs, and also improves sepsis survival by attenuating pulmonary inflammation and lung injury. Therefore, pulmonary cf-mtDNA clearance strategy using DNase-I/HSA NMs is considered to be an attractive approach for sepsis-associated ALI.
Topics: Humans; DNA, Mitochondrial; Acute Lung Injury; Lung; Sepsis; Deoxyribonucleases
PubMed: 37518854
DOI: 10.1002/advs.202301635 -
The Journal of Investigative Dermatology Jan 2023Hidradenitis suppurativa (HS) is a debilitating inflammatory skin disorder characterized by abscess-like nodules and boils resulting in fistulas and tissue scarring. We...
Hidradenitis suppurativa (HS) is a debilitating inflammatory skin disorder characterized by abscess-like nodules and boils resulting in fistulas and tissue scarring. We previously reported evidence of an autoimmune signature in HS, characterized by enhanced neutrophil extracellular trap (NET) infiltration in HS skin lesions and dysregulation of the adaptive immune system characterized by the presence of autoantibodies. Timely removal of NETs is critical for tissue homeostasis to prevent a dysregulated generation of modified autoantigens and tissue damage. DNases 1 and 1L3 play important roles in proper NET removal. We tested the hypothesis that NETs in patients with HS are not effectively cleared owing to the presence of antibodies against DNase 1 and DNase 1L3. We report that HS serum poorly degraded NETs. Addition of exogenous DNase 1 restored NET degradation capabilities in a subset of HS samples. DNase 1 activity was significantly decreased in HS sera. Anti‒DNase 1 and ‒DNase 1L3 antibodies were detected in serum samples and skin lesions from patients with HS. Purified IgGs from HS decreased DNase 1 activity and NET degradation. Taken together, this identification of neutralizing antibodies against nucleases in HS expands the understanding of the pathogenesis of this disease to support an autoimmune mechanism in its underlying pathogenesis.
Topics: Humans; Extracellular Traps; Hidradenitis Suppurativa; Deoxyribonucleases; Deoxyribonuclease I; Autoantibodies
PubMed: 35934056
DOI: 10.1016/j.jid.2022.06.024 -
Drug Delivery Dec 2023Rheumatoid arthritis (RA), an autoimmune disease, is characterized by inflammatory cell infiltration that damages cartilage, disrupts bone, and impairs joint function....
Rheumatoid arthritis (RA), an autoimmune disease, is characterized by inflammatory cell infiltration that damages cartilage, disrupts bone, and impairs joint function. The therapeutic efficacy of RA treatments with the severely affected side remains unsatisfactory despite current treatment methods that primarily focus on anti-inflammatory activity, largely because of the complicatedly pathological mechanisms. A recently identified mechanism for RA development involves the interaction of RA autoantibodies with various proinflammatory cytokines to facilitate the formation of neutrophil extracellular traps (NETs), which increased inflammatory responses to express inflammatory cytokines and chemokines. Therefore, NETs architecture digestion may inhibit the positive-feedback inflammatory signal pathway and lessen joint damage in RA. In this work, deoxyribonuclease I (DNase) is connected to oxidized hyaluronic acid (OHA) via Schiff base reaction to extend the half-life of DNase. The modification does not influence the DNase activity for plasmid deoxyribonucleic acid hydrolysis and NETs' architecture disruption. Carboxymethyl chitosan is crosslinked with DNase-functionalised OHA (DHA) to form an injectable, degradable, and biocompatible hydrogel (DHY) to further strengthen the adhesive capability of DHA. Importantly, the collagen-induced arthritis model demonstrates that intra-articular injection of DHY can significantly reduce inflammatory cytokine expression and alleviate RA symptoms, which can be significantly improved by combining methotrexate. Here, a DNase-functionalised hydrogel has been developed for RA treatment by constantly degrading the novel drug target of NETs to decrease inflammatory response in RA.
Topics: Humans; Extracellular Traps; Hydrogels; Arthritis, Rheumatoid; Cytokines; Deoxyribonucleases; Neutrophils
PubMed: 36724178
DOI: 10.1080/10717544.2023.2173332