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Cold Spring Harbor Protocols Oct 2019RNA samples prepared using monophasic lysis reagents may contain small amounts of contaminating genomic DNA, which must be removed if the RNA will be used in subsequent...
RNA samples prepared using monophasic lysis reagents may contain small amounts of contaminating genomic DNA, which must be removed if the RNA will be used in subsequent analyses such as reverse transcriptase-polymerase chain reaction (RT-PCR) or quantitative real-time RT-PCR. In addition, the presence of contaminating DNA can render the quantitative determination of RNA in a sample inaccurate. The most common and effective method for removing trace to moderate amounts of DNA contamination from RNA samples is digestion with DNase I, as described here.
Topics: DNA; DNA Contamination; Deoxyribonuclease I; Deoxyribonucleases; Edetic Acid; Enzyme Activation; Hydrolysis; RNA; RNA, Messenger; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; Ribonucleases
PubMed: 31575796
DOI: 10.1101/pdb.prot101725 -
European Journal of Medicinal Chemistry Dec 2014Deoxyribonucleases (DNases) are a class of enzymes able to catalyze DNA hydrolysis. DNases play important roles in cell function, while DNase inhibitors control or... (Review)
Review
Deoxyribonucleases (DNases) are a class of enzymes able to catalyze DNA hydrolysis. DNases play important roles in cell function, while DNase inhibitors control or modify their activities. This review focuses on DNase inhibitors. Some DNase inhibitors have been isolated from various natural sources, such as humans, animals (beef, calf, rabbit and rat), plants (Nicotiana tabacum), and microorganisms (some Streptomyces and Adenovirus species, Micromonospora echinospora and Escherichia coli), while others have been obtained by chemical synthesis. They differ in chemical structure (various proteins, nucleotides, anthracycline and aminoglycoside antibiotics, synthetic organic and inorganic compounds) and mechanism of action (forming complexes with DNases or DNA). Some of the inhibitors are specific toward only one type of DNase, while others are active towards two or more. Physico-chemical properties of DNase inhibitors are calculated using the Molinspiration tool and most of them meet all criteria for good solubility and permeability. DNase inhibitors may be used as pharmaceuticals for preventing, monitoring and treating various diseases.
Topics: Animals; Deoxyribonucleases; Enzyme Inhibitors; Humans; Molecular Structure; Structure-Activity Relationship
PubMed: 25042005
DOI: 10.1016/j.ejmech.2014.07.040 -
Circulation Research Mar 2015
Topics: Acute Coronary Syndrome; Coronary Thrombosis; Deoxyribonucleases; Extracellular Traps; Female; Humans; Male; Myocardial Infarction; Neutrophil Infiltration
PubMed: 25814679
DOI: 10.1161/CIRCRESAHA.115.306050 -
Methods in Molecular Biology (Clifton,... 2021DNA double-strand breaks (DSBs) represent the most toxic form of DNA damage and can arise in either physiological or pathological conditions. If left unrepaired, these...
DNA double-strand breaks (DSBs) represent the most toxic form of DNA damage and can arise in either physiological or pathological conditions. If left unrepaired, these DSBs can lead to genome instability which serves as a major driver to tumorigenesis and other pathologies. Consequently, localizing DSBs and understanding the dynamics of break formation and the repair process are of great interest for dissecting underlying mechanisms and in the development of targeted therapies. Here, we describe END-seq, a highly sensitive next-generation sequencing technique for quantitatively mapping DNA double-strand breaks (DSB) at nucleotide resolution across the genome in an unbiased manner. END-seq is based on the direct ligation of a sequencing adapter to the ends of DSBs and provides information about DNA processing (end resection) at DSBs, a critical determinant in the selection of repair pathways. The absence of cell fixation and the use of agarose for embedding cells and exonucleases for blunting the ends of DSBs are key advances that contribute to the technique's increased sensitivity and robustness over previously established methods. Overall, END-seq has provided a major technical advance for mapping DSBs and has also helped inform the biology of complex biological processes including genome organization, replication fork collapse and chromosome fragility, off-target identification of RAG recombinase and gene-editing nucleases, and DNA end resection at sites of DSBs.
Topics: Computational Biology; DNA Breaks, Double-Stranded; DNA Repair; Deoxyribonucleases; Exonucleases; Gene Editing; High-Throughput Nucleotide Sequencing; Humans; Software; Whole Genome Sequencing
PubMed: 32840769
DOI: 10.1007/978-1-0716-0644-5_2 -
Nature Jan 2024Bacteria encode hundreds of diverse defence systems that protect them from viral infection and inhibit phage propagation. Gabija is one of the most prevalent anti-phage...
Bacteria encode hundreds of diverse defence systems that protect them from viral infection and inhibit phage propagation. Gabija is one of the most prevalent anti-phage defence systems, occurring in more than 15% of all sequenced bacterial and archaeal genomes, but the molecular basis of how Gabija defends cells from viral infection remains poorly understood. Here we use X-ray crystallography and cryo-electron microscopy (cryo-EM) to define how Gabija proteins assemble into a supramolecular complex of around 500 kDa that degrades phage DNA. Gabija protein A (GajA) is a DNA endonuclease that tetramerizes to form the core of the anti-phage defence complex. Two sets of Gabija protein B (GajB) dimers dock at opposite sides of the complex and create a 4:4 GajA-GajB assembly (hereafter, GajAB) that is essential for phage resistance in vivo. We show that a phage-encoded protein, Gabija anti-defence 1 (Gad1), directly binds to the Gabija GajAB complex and inactivates defence. A cryo-EM structure of the virally inhibited state shows that Gad1 forms an octameric web that encases the GajAB complex and inhibits DNA recognition and cleavage. Our results reveal the structural basis of assembly of the Gabija anti-phage defence complex and define a unique mechanism of viral immune evasion.
Topics: Bacteria; Bacterial Proteins; Bacteriophages; Cryoelectron Microscopy; Crystallography, X-Ray; Deoxyribonucleases; DNA, Viral; Immune Evasion; Protein Multimerization
PubMed: 37992757
DOI: 10.1038/s41586-023-06855-2 -
Virulence 2015
Topics: Aeromonas hydrophila; Animals; Deoxyribonucleases; Female; Fishes; Gram-Negative Bacterial Infections; Virulence Factors
PubMed: 26055576
DOI: 10.1080/21505594.2015.1058479 -
Developmental Biology Sep 2017DNA degradation is critical to healthy organism development and survival. Two nuclease families that play key roles in development and in disease are the Dnase1 and... (Review)
Review
DNA degradation is critical to healthy organism development and survival. Two nuclease families that play key roles in development and in disease are the Dnase1 and Dnase2 families. While these two families were initially characterized by biochemical function, it is now clear that multiple enzymes in each family perform similar, non-redundant roles in many different tissues. Most Dnase1 and Dnase2 family members are poorly characterized, yet their elimination can lead to a wide range of diseases, including lethal anemia, parakeratosis, cataracts and systemic lupus erythematosus. Therefore, understanding these enzyme families represents a critical field of emerging research. This review explores what is currently known about Dnase1 and Dnase2 family members, highlighting important questions about the structure and function of family members, and how their absence translates to disease.
Topics: Animals; Deoxyribonucleases; Disease; Health; Humans; Organ Specificity
PubMed: 28666955
DOI: 10.1016/j.ydbio.2017.06.028 -
Cells Oct 2021Neutrophil extracellular traps (NETs) are macromolecular structures programmed to trap circulating bacteria and viruses. The accumulation of NETs in the circulation... (Review)
Review
Neutrophil extracellular traps (NETs) are macromolecular structures programmed to trap circulating bacteria and viruses. The accumulation of NETs in the circulation correlates with the formation of anti-double-stranded (ds) DNA antibodies and is considered a causative factor for systemic lupus erythematosus (SLE). The digestion of DNA by DNase1 and DNases1L3 is the rate- limiting factor for NET accumulation. Mutations occurring in one of these two DNase genes determine anti-DNA formation and are associated with severe Lupus-like syndromes and lupus nephritis (LN). A second mechanism that may lead to DNase functional impairment is the presence of circulating DNase inhibitors in patients with low DNase activity, or the generation of anti-DNase antibodies. This phenomenon has been described in a relevant number of patients with SLE and may represent an important mechanism determining autoimmunity flares. On the basis of the reviewed studies, it is tempting to suppose that the blockade or selective depletion of anti-DNase autoantibodies could represent a potential novel therapeutic approach to prevent or halt SLE and LN. In general, strategies aimed at reducing NET formation might have a similar impact on the progression of SLE and LN.
Topics: Animals; Antibodies; Autoimmunity; DNA; Deoxyribonucleases; Extracellular Traps; Humans; Mutation
PubMed: 34685647
DOI: 10.3390/cells10102667 -
Methods in Molecular Biology (Clifton,... 2021Technology advance during the past decade has greatly expanded our understanding of the higher-order structure of the genome. The various chromosome conformation capture...
Technology advance during the past decade has greatly expanded our understanding of the higher-order structure of the genome. The various chromosome conformation capture (3C)-based techniques such as Hi-C have provided the most widely used tools for interrogating three-dimensional (3D) genome organization. We recently developed a Hi-C variant, DNase Hi-C, for characterizing 3D genome organization. DNase Hi-C employs DNase I for chromatin fragmentation, aiming to overcome restriction enzyme digestion-related limitations associated with traditional Hi-C methods. By combining DNase Hi-C with DNA capture technology, we further implemented a high-throughput approach, called targeted DNase Hi-C, which enables to map fine-scale chromatin architecture at exceptionally high resolution and thereby is an ideal tool for mapping the physical landscapes of cis-regulatory networks and for characterizing phenotype-associated chromatin 3D signatures. Here, I describe a detailed protocol of targeted DNase Hi-C library preparation, which covers experimental steps starting from cell cross-linking to library amplification.
Topics: Chromatin; Chromosomes; Deoxyribonucleases; Genome; Saccharomyces cerevisiae
PubMed: 32820399
DOI: 10.1007/978-1-0716-0664-3_5 -
Inflammation Dec 2023Heterotopic ossification (HO) severely affects people's lives; however, its pathological mechanism remains poorly understood. Although extracellular DNA (ecDNA) has been...
Heterotopic ossification (HO) severely affects people's lives; however, its pathological mechanism remains poorly understood. Although extracellular DNA (ecDNA) has been shown to play important roles in pathological calcification, its effects in HO development and progression remain unknown. The in vivo rat Achilles tendon injury model and in vitro collagen I calcification model were used to evaluate the effects of ecDNA in the ectopic calcifications and the main cell types involved in those pathological process. Histology, immunofluorescent staining, reverse transcriptase-polymerase chain reaction analysis and micro-computed tomography were used to identify the distribution of macrophage-derived ecDNA and elucidate their roles in HO. The results showed that the amount of ecDNA and ectopic calcification increased significantly and exhibited a strong correlation in the injured tendons of HO model compared with those of the controls, which was accompanied by a significantly increased number of M2 macrophages in the injured tendon. During in vitro co-culture experiments, M2 macrophages calcified the reconstituted type I collagen and ectopic bone collected from the injured tendons of HO rats, while those effects were inhibited by deoxyribonuclease. More importantly, deoxyribonuclease reversed the pathological calcification in the injured rat tendon HO model. The present study showed that ecDNA from M2 macrophages initiates pathological calcification in HO, and the elimination of ecDNA might be developed into a clinical strategy to prevent ectopic mineralization diseases. The use of deoxyribonuclease for the targeted degradation of ecDNA at affected tissue sites provides a potential solution to treat diseases associated with ectopic mineralization.
Topics: Humans; Rats; Animals; X-Ray Microtomography; Ossification, Heterotopic; Tendons; Macrophages; Deoxyribonucleases; Osteogenesis
PubMed: 37458919
DOI: 10.1007/s10753-023-01873-8