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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 -
International Journal of Molecular... Mar 2023Although many studies have been exploring the mechanisms driving NETs formation, much less attention has been paid to the degradation and elimination of these... (Review)
Review
Although many studies have been exploring the mechanisms driving NETs formation, much less attention has been paid to the degradation and elimination of these structures. The NETs clearance and the effective removal of extracellular DNA, enzymatic proteins (neutrophil elastase, proteinase 3, myeloperoxidase) or histones are necessary to maintain tissue homeostasis, to prevent inflammation and to avoid the presentation of self-antigens. The persistence and overabundance of DNA fibers in the circulation and tissues may have dramatic consequences for a host leading to the development of various systemic and local damage. NETs are cleaved by a concerted action of extracellular and secreted deoxyribonucleases (DNases) followed by intracellular degradation by macrophages. NETs accumulation depends on the ability of DNase I and DNAse II to hydrolyze DNA. Furthermore, the macrophages actively engulf NETs and this event is facilitated by the preprocessing of NETs by DNase I. The purpose of this review is to present and discuss the current knowledge about the mechanisms of NETs degradation and its role in the pathogenesis of thrombosis, autoimmune diseases, cancer and severe infections, as well as to discuss the possibilities for potential therapeutic interventions. Several anti-NETs approaches had therapeutic effects in animal models of cancer and autoimmune diseases; nevertheless, the development of new drugs for patients needs further study for an effective development of clinical compounds that are able to target NETs.
Topics: Animals; Extracellular Traps; Neutrophils; Deoxyribonuclease I; Autoimmune Diseases; DNA
PubMed: 36902325
DOI: 10.3390/ijms24054896 -
American Journal of Human Genetics Feb 2020Cell-free DNA (cf.DNA) is a powerful noninvasive biomarker for cancer and prenatal testing, and it circulates in plasma as short fragments. To elucidate the biology of...
Cell-free DNA (cf.DNA) is a powerful noninvasive biomarker for cancer and prenatal testing, and it circulates in plasma as short fragments. To elucidate the biology of cf.DNA fragmentation, we explored the roles of deoxyribonuclease 1 (DNASE1), deoxyribonuclease 1 like 3 (DNASE1L3), and DNA fragmentation factor subunit beta (DFFB) with mice deficient in each of these nucleases. By analyzing the ends of cf.DNA fragments in each type of nuclease-deficient mice with those in wild-type mice, we show that each nuclease has a specific cutting preference that reveals the stepwise process of cf.DNA fragmentation. Essentially, we demonstrate that cf.DNA is generated first intracellularly with DFFB, intracellular DNASE1L3, and other nucleases. Then, cf.DNA fragmentation continues extracellularly with circulating DNASE1L3 and DNASE1. With the use of heparin to disrupt the nucleosomal structure, we also show that the 10 bp periodicity originates from the cutting of DNA within an intact nucleosomal structure. Altogether, this work establishes a model of cf.DNA fragmentation.
Topics: Animals; Cell-Free Nucleic Acids; Chromatin; DNA Fragmentation; Deoxyribonuclease I; Deoxyribonucleases; Endodeoxyribonucleases; Female; Male; Mice; Mice, Knockout; Nucleosomes; Poly-ADP-Ribose Binding Proteins
PubMed: 32004449
DOI: 10.1016/j.ajhg.2020.01.008 -
Viruses Mar 2023Nucleases are ubiquitous hydrolytic enzymes that cleave phosphodiester bond of DNA (DNases), RNA (RNases), or protein-RNA/DNA (phosphodiesterases), within the strand...
Nucleases are ubiquitous hydrolytic enzymes that cleave phosphodiester bond of DNA (DNases), RNA (RNases), or protein-RNA/DNA (phosphodiesterases), within the strand (endonucleases) or from the end (exonucleases) [...].
Topics: Endonucleases; Deoxyribonucleases; Phosphoric Diester Hydrolases; DNA; RNA
PubMed: 36992449
DOI: 10.3390/v15030740 -
Cell Nov 2021Biofilms are community architectures adopted by bacteria inclusive of a self-formed extracellular matrix that protects resident bacteria from diverse environmental...
Biofilms are community architectures adopted by bacteria inclusive of a self-formed extracellular matrix that protects resident bacteria from diverse environmental stresses and, in many species, incorporates extracellular DNA (eDNA) and DNABII proteins for structural integrity throughout biofilm development. Here, we present evidence that this eDNA-based architecture relies on the rare Z-form. Z-form DNA accumulates as biofilms mature and, through stabilization by the DNABII proteins, confers structural integrity to the biofilm matrix. Indeed, substances known to drive B-DNA into Z-DNA promoted biofilm formation whereas those that drive Z-DNA into B-DNA disrupted extant biofilms. Importantly, we demonstrated that the universal bacterial DNABII family of proteins stabilizes both bacterial- and host-eDNA in the Z-form in situ. A model is proposed that incorporates the role of Z-DNA in biofilm pathogenesis, innate immune response, and immune evasion.
Topics: Animals; Antibody Specificity; Bacteria; Bacterial Proteins; Biofilms; Cell Line; Chinchilla; DNA, Bacterial; DNA, Cruciform; Deoxyribonucleases; Extracellular Matrix; Extracellular Space; Extracellular Traps; Humans; Tetradecanoylphorbol Acetate
PubMed: 34735796
DOI: 10.1016/j.cell.2021.10.010 -
Proceedings of the National Academy of... Apr 2023Cell-free DNA (cfDNA) fragmentation is nonrandom, at least partially mediated by various DNA nucleases, forming characteristic cfDNA end motifs. However, there is a...
Cell-free DNA (cfDNA) fragmentation is nonrandom, at least partially mediated by various DNA nucleases, forming characteristic cfDNA end motifs. However, there is a paucity of tools for deciphering the relative contributions of cfDNA cleavage patterns related to underlying fragmentation factors. In this study, through non-negative matrix factorization algorithm, we used 256 5' 4-mer end motifs to identify distinct types of cfDNA cleavage patterns, referred to as "founder" end-motif profiles (F-profiles). F-profiles were associated with different DNA nucleases based on whether such patterns were disrupted in nuclease-knockout mouse models. Contributions of individual F-profiles in a cfDNA sample could be determined by deconvolutional analysis. We analyzed 93 murine cfDNA samples of different nuclease-deficient mice and identified six types of F-profiles. F-profiles I, II, and III were linked to deoxyribonuclease 1 like 3 (DNASE1L3), deoxyribonuclease 1 (DNASE1), and DNA fragmentation factor subunit beta (DFFB), respectively. We revealed that 42.9% of plasma cfDNA molecules were attributed to DNASE1L3-mediated fragmentation, whereas 43.4% of urinary cfDNA molecules involved DNASE1-mediated fragmentation. We further demonstrated that the relative contributions of F-profiles were useful to inform pathological states, such as autoimmune disorders and cancer. Among the six F-profiles, the use of F-profile I could inform the human patients with systemic lupus erythematosus. F-profile VI could be used to detect individuals with hepatocellular carcinoma, with an area under the receiver operating characteristic curve of 0.97. F-profile VI was more prominent in patients with nasopharyngeal carcinoma undergoing chemoradiotherapy. We proposed that this profile might be related to oxidative stress.
Topics: Humans; Mice; Animals; Cell-Free Nucleic Acids; Deoxyribonucleases; Mice, Knockout; Endonucleases; DNA Fragmentation; Endodeoxyribonucleases
PubMed: 37075072
DOI: 10.1073/pnas.2220982120 -
Nucleic Acids Research Jul 2015Using CRISPR/Cas9, it is possible to target virtually any gene in any organism. A major limitation to its application in gene therapy is the size of Cas9 (>4 kb),...
Using CRISPR/Cas9, it is possible to target virtually any gene in any organism. A major limitation to its application in gene therapy is the size of Cas9 (>4 kb), impeding its efficient delivery via recombinant adeno-associated virus (rAAV). Therefore, we developed a split-Cas9 system, bypassing the packaging limit using split-inteins. Each Cas9 half was fused to the corresponding split-intein moiety and, only upon co-expression, the intein-mediated trans-splicing occurs and the full Cas9 protein is reconstituted. We demonstrated that the nuclease activity of our split-intein system is comparable to wild-type Cas9, shown by a genome-integrated surrogate reporter and by targeting three different endogenous genes. An analogously designed split-Cas9D10A nickase version showed similar activity as Cas9D10A. Moreover, we showed that the double nick strategy increased the homologous directed recombination (HDR). In addition, we explored the possibility of delivering the repair template accommodated on the same dual-plasmid system, by transient transfection, showing an efficient HDR. Most importantly, we revealed for the first time that intein-mediated split-Cas9 can be packaged, delivered and its nuclease activity reconstituted efficiently, in cells via rAAV.
Topics: CRISPR-Associated Proteins; CRISPR-Cas Systems; Cell Line; Deoxyribonucleases; Dependovirus; Gene Targeting; Genetic Therapy; Humans; Inteins; Plasmids; Streptococcus pyogenes; Transfection
PubMed: 26082496
DOI: 10.1093/nar/gkv601 -
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 -
Biomolecules Jul 2020Extracellular DNA, also called cell-free DNA, released from dying cells or activated immune cells can be recognized by the immune system as a danger signal causing or... (Review)
Review
Extracellular DNA, also called cell-free DNA, released from dying cells or activated immune cells can be recognized by the immune system as a danger signal causing or enhancing inflammation. The cleavage of extracellular DNA is crucial for limiting the inflammatory response and maintaining homeostasis. Deoxyribonucleases (DNases) as enzymes that degrade DNA are hypothesized to play a key role in this process as a determinant of the variable concentration of extracellular DNA. DNases are divided into two families-DNase I and DNase II, according to their biochemical and biological properties as well as the tissue-specific production. Studies have shown that low DNase activity is both, a biomarker and a pathogenic factor in systemic lupus erythematosus. Interventional experiments proved that administration of exogenous DNase has beneficial effects in inflammatory diseases. Recombinant human DNase reduces mucus viscosity in lungs and is used for the treatment of patients with cystic fibrosis. This review summarizes the currently available published data about DNases, their activity as a potential biomarker and methods used for their assessment. An overview of the experiments with systemic administration of DNase is also included. Whether low-plasma DNase activity is involved in the etiopathogenesis of diseases remains unknown and needs to be elucidated.
Topics: Biomarkers; Cell-Free Nucleic Acids; Cystic Fibrosis; Deoxyribonucleases; Humans; Lupus Erythematosus, Systemic; Organ Specificity
PubMed: 32664541
DOI: 10.3390/biom10071036