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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 -
Nature Nov 2021Transposition has a key role in reshaping genomes of all living organisms. Insertion sequences of IS200/IS605 and IS607 families are among the simplest mobile genetic...
Transposition has a key role in reshaping genomes of all living organisms. Insertion sequences of IS200/IS605 and IS607 families are among the simplest mobile genetic elements and contain only the genes that are required for their transposition and its regulation. These elements encode tnpA transposase, which is essential for mobilization, and often carry an accessory tnpB gene, which is dispensable for transposition. Although the role of TnpA in transposon mobilization of IS200/IS605 is well documented, the function of TnpB has remained largely unknown. It had been suggested that TnpB has a role in the regulation of transposition, although no mechanism for this has been established. A bioinformatic analysis indicated that TnpB might be a predecessor of the CRISPR-Cas9/Cas12 nucleases. However, no biochemical activities have been ascribed to TnpB. Here we show that TnpB of Deinococcus radiodurans ISDra2 is an RNA-directed nuclease that is guided by an RNA, derived from the right-end element of a transposon, to cleave DNA next to the 5'-TTGAT transposon-associated motif. We also show that TnpB could be reprogrammed to cleave DNA target sites in human cells. Together, this study expands our understanding of transposition mechanisms by highlighting the role of TnpB in transposition, experimentally confirms that TnpB is a functional progenitor of CRISPR-Cas nucleases and establishes TnpB as a prototype of a new system for genome editing.
Topics: Base Sequence; CRISPR-Associated Proteins; CRISPR-Cas Systems; DNA Transposable Elements; Deinococcus; Deoxyribonuclease I; Escherichia coli; Gene Editing; HEK293 Cells; Humans; Nucleotide Motifs; RNA
PubMed: 34619744
DOI: 10.1038/s41586-021-04058-1 -
International Journal of Molecular... Jan 2021Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay is a long-established assay used to detect cell death-associated DNA fragmentation (3'-OH DNA... (Review)
Review
Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay is a long-established assay used to detect cell death-associated DNA fragmentation (3'-OH DNA termini) by endonucleases. Because these enzymes are particularly active in the kidney, TUNEL is widely used to identify and quantify DNA fragmentation and cell death in cultured kidney cells and animal and human kidneys resulting from toxic or hypoxic injury. The early characterization of TUNEL as an apoptotic assay has led to numerous misinterpretations of the mechanisms of kidney cell injury. Nevertheless, TUNEL is becoming increasingly popular for kidney injury assessment because it can be used universally in cultured and tissue cells and for all mechanisms of cell death. Furthermore, it is sensitive, accurate, quantitative, easily linked to particular cells or tissue compartments, and can be combined with immunohistochemistry to allow reliable identification of cell types or likely mechanisms of cell death. Traditionally, TUNEL analysis has been limited to the presence or absence of a TUNEL signal. However, additional information on the mechanism of cell death can be obtained from the analysis of TUNEL patterns.
Topics: Animals; Apoptosis; Cells, Cultured; DNA Fragmentation; Deoxyribonucleases; Endonucleases; Humans; In Situ Nick-End Labeling; Kidney; Kidney Diseases
PubMed: 33401733
DOI: 10.3390/ijms22010412 -
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 -
JAMA Pediatrics Apr 2020Clinical guidelines recommend that children with pleural empyema be treated with chest tube insertion and intrapleural fibrinolytics. The addition of dornase alfa... (Randomized Controlled Trial)
Randomized Controlled Trial
Effectiveness of Intrapleural Tissue Plasminogen Activator and Dornase Alfa vs Tissue Plasminogen Activator Alone in Children with Pleural Empyema: A Randomized Clinical Trial.
IMPORTANCE
Clinical guidelines recommend that children with pleural empyema be treated with chest tube insertion and intrapleural fibrinolytics. The addition of dornase alfa (DNase) has been reported to improve outcomes in adults but remains unproven in children.
OBJECTIVE
To determine if intrapleural tissue plasminogen activator (tPA) and DNase is more effective than tPA and placebo at reducing hospital length of stay in children with pleural empyema.
DESIGN, SETTING, AND PARTICIPANTS
This multicenter, parallel-group, placebo-controlled, superiority randomized clinical trial included children diagnosed as having pleural empyema requiring drainage aged 6 months to 18 years treated at 6 tertiary Canadian children's hospitals. A total of 379 children were assessed for eligibility; 281 were excluded and 98 were randomized. One child was excluded after randomization for not meeting the inclusion criteria. Data were collected from March 4, 2013, to December 13, 2017.
INTERVENTIONS
Participants underwent chest tube insertion and 3 daily administrations of intrapleural tPA, 4 mg, followed by DNase, 5 mg (intervention group), or 5 mL of normal saline (placebo; control group). Participants, families, clinical staff, and members of the study team were blinded to allocation.
MAIN OUTCOMES AND MEASURES
The primary outcome was hospital length of stay from chest tube insertion to discharge. Secondary outcomes included time to meeting discharge criteria, time to chest tube removal, mean fever duration, additional pleural drainage procedures, hospital readmissions, and total health care cost.
RESULTS
Of the 97 analyzed children with pleural empyema, 52 (54%) were male, and the mean (SD) age was 5.1 (3.6) years. A total of 49 children were randomized to tPA and DNase and 48 were randomized to tPA and placebo. Treatment with tPA and DNase was not associated with decreased hospital length of stay compared with tPA and placebo (mean [SD] length of stay, 9.0 [4.9] vs 9.1 [5.3] days; mean difference, -0.1 days; 95% CI, -2.0 to 2.1; P = .96). Similarly, no significant differences were observed for any of the secondary outcomes. Of the 14 adverse events in the tPA and DNase group, 6 (43%) were serious; of the 21 adverse events in the tPA and placebo group, 8 (38%) were serious. There were no deaths.
CONCLUSIONS AND RELEVANCE
The addition of DNase to intrapleural tPA for children with pleural empyema had no effect on hospital length of stay or other outcomes compared with tPA with placebo. Clinical practice guidelines should continue to support the use of chest tube insertion and intrapleural fibrinolytics alone as first-line treatment for pediatric empyema.
TRIAL REGISTRATION
ClinicalTrials.gov identifier: NCT01717742.
Topics: Adolescent; Chest Tubes; Child; Child, Preschool; Deoxyribonuclease I; Empyema, Pleural; Female; Fibrinolytic Agents; Health Care Costs; Humans; Infant; Length of Stay; Male; Patient Readmission; Recombinant Proteins; Tissue Plasminogen Activator
PubMed: 32011642
DOI: 10.1001/jamapediatrics.2019.5863 -
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