Trashing the genome: the role of nucleases during apoptosis

Two classes of nucleases degrade the cellular DNA during apoptosis. Cell-autonomous nucleases cleave DNA within the dying cell. They are not essential for apoptotic cell death or the life of the organism, but they might affect the efficiency of the process. By contrast, waste-management nucleases are essential for the life of the organism. In post-engulfment DNA degradation, the DNA of apoptotic cells is destroyed in lysosomes of the cells that have phagocytosed the corpses. Waste-management nucleases also destroy DNA that is released into the extracellular compartment. Here, we describe the complex group of nucleases that are involved in DNA destruction during apoptotic cell death.     

Extracellular nuclease of Bacillus mesentericus]

Bacillus mesentericus is found to secrete three type of nucleases: alkaline ribonuclease (EC 2.7.7.17), acidic ribonuclease (EC 2.7.7.17) and Ca2+-activated exonucleease (EC 3.1.4.7). These nucleases are purified and characterized. They are similar to those from Bac. subtilis in main biochemical and physico-chemical properties and in their chromatographical behaviour. Studying physiological functions of Bac. mesentericus extracellular nucleases, it is shown that bacteria, which are capable to produce extracellular nucleases, utilize exogenous RNAs and a bit worse, DNAs as a single and additional source of nitrogen or phosphorus. In view of this it is believed that extracellular nucleases participate in bacteria nutrition.     

CRISPR/Cas9系统在疾病研究和治疗中的应用

基因组编辑技术(Genome editing technology)是一种通过人工手段在基因组水平对DNA序列进行改造的遗传操作技术,包括特定DNA片段的插入、敲除、替换和点突变.其中,依赖核酸酶的基因组编辑技术的基本原理是在基因组的特定位置产生双链DNA断裂(Double-stranded break,DSB)后通过...     

Investigation and improvement of DNA cleavage models of polyamide + Cu(II) nuclease + OOH<Superscript>-</Superscript> ligands bound to DNA

Background  

Copper nucleases as a famous class of artificial metallonucleases have attracted considerable interest in relation to their diverse potentials not only as therapeutic agents but also in genomic researches. Copper nucleases present high efficient oxidative cleavage of DNA, in which DNA strand scission occurs generally after hydrogen atom abstracted from a sugar moiety. In order to achieve the selective cleavage of DNA sequences by copper nucleases, the DNA specific recognition agents of the Dervan-type hairpin and cyclic polyamides can be considered as proper carriers of copper nucleases. Investigation of the DNA cleavage selectivity of copper nucleases assisted by the hairpin and cyclic polyamides at the molecular level has not yet been elucidated.     

Barley aleurone cell death is not apoptotic: characterization of nuclease activities and DNA degradation

Barley aleurone cells undergo programmed cell death (PCD) when exposed to gibberellic acid (GA), but incubation in abscisic acid (ABA) prevent PCD. We tested the hypothesis that PCD in aleurone cells occurs by apoptosis, and show that the hallmark of apoptosis, namely DNA cleavage into 180 bp fragments, plasma membrane blebbing, and the formation of apoptotic bodies do not occur when aleurone cells die. We show that endogenous barley aleurone nucleases and nucleases present in enzymes used for protoplast preparation degrade aleurone DNA and that DNA degradation by these nucleases is rapid and can result in the formation of 180 bp DNA ladders. Methods are described that prevent DNA degradation during isolation from aleurone layers or protoplasts. Barley aleurone cells contain three nucleases whose activities are regulated by GA and ABA. CA induction and ABA repression of nuclease activities correlate with PCD in aleurone cells. Cells incubated in ABA remain alive and do not degrade their DNA, but living aleurone cells treated with GA accumulate nucleases and hydrolyze their nuclear DNA. We propose that barley nucleases play a role in DNA cleavage during aleurone PCD.     

Surrogate reporters for enrichment of cells with nuclease-induced mutations

Zinc-finger nucleases (ZFNs) and TAL-effector nucleases (TALENs) are powerful tools for creating genetic modifications in eukaryotic cells and organisms. But wild-type and mutant cells that contain genetic modifications induced by these programmable nucleases are often phenotypically indistinguishable, hampering isolation of mutant cells. Here we show that transiently transfected episomal reporters encoding fluorescent proteins can be used as surrogate genes for the efficient enrichment of endogenous gene-modified cells by flow cytometry.     

Cuts can kill: the roles of apoptotic nucleases in cell death and animal development

Chromosome fragmentation is one of the major biochemical hallmarks of apoptosis. However, until recently, its roles in apoptosis and mechanisms of action remained elusive. Recent biochemical and genetic studies have shown that chromosome fragmentation is a complex biochemical process that involves a plethora of conserved nucleases with distinct nuclease activities and substrate specificities. These apoptotic nucleases act cooperatively among themselves and with other nonnuclease cofactors to promote stepwise chromosome fragmentation and DNA degradation. Importantly, in addition to its direct contribution to the dismantling of the dying cell, apoptotic DNA degradation can facilitate cell killing and other apoptotic events such as clearance of apoptotic cells. Furthermore, some apoptotic nucleases apparently affect other aspects of animal development, including immune responses. The identification of new apoptotic nucleases and analysis of their functions in apoptosis and animal development should pave the way for future studies to uncover new functions for apoptotic nucleases and shed light on the hidden links between apoptotic DNA degradation and human diseases.     

Nucleases activity in different segments of the human digestive tube compared to the incidence of carcinomas (histochemical study)

Summary The alkaline and acid DNase and RNase activity was histochemically investigated in biopsies from the human digestive tube. Activity of these enzymes in the mucosal epithelium in different segments of the digestive tube was compared to the statistical incidence of malignant tumors deriving from this tissue (carcinomas). It was found that the alkaline and acid nucleases activity was very intense in small intestine precisely in this segment where the incidence of carcinomas was low, whereas the low activity of these enzymes in the stomach and large intestine corresponded to the high incidence of carcinomas. This observation confirmed our previously elaborated hypothesis, according to which the low activity of nucleases in normal tissues appeared to be a predisposing factor for malignant transformation.It could be also supposed that the nucleases constitute some kind of double barrier mechanism protecting the genetical stability of the cell against foreign nucleic acid incorporation or production; alkaline nucleases being an extracellular and acid nucleases an intracellular barrier.     

Activities of nucleases in senescing daylily petals

The activity of nucleases during organ death was investigated using daylily petals (Hemerocallis hybrid cv. Stella d’Oro), in which the processes associated with senescence are rapid and clearly ordered. The number of nuclei with fragmented DNA as well as activities of various nucleases increase before certain other events that are related to senescence. Furthermore, DNA breakage and activities of nucleases occur earlier when senescence is accelerated by abscisic acid and occur later when senescence is retarded by cycloheximide. These results suggest that the activities of nucleases contribute to the senescence of daylily petals. Therefore, studying the regulation of nuclease gene expression may be useful for understanding components of the signal transduction system that leads to the death of these organs.     

Systems of Delivery of CRISPR/Cas9 Ribonucleoprotein Complexes for Genome Editing

Russian Journal of Bioorganic Chemistry - The discovery of RNA-guided nucleases have enabled to leap forward in genome editing of cells and organisms. These nucleases can be delivered into cells as...     

Variations in the enzymatic activity of S1-type nucleases results from differences in their active site structures

BackgroundS1-like nucleases are widespread enzymes commonly used in biotechnology and molecular biology. Although it is commonly believed that they are mainly Zn²⁺-dependent acidic enzymes, we have found that numerous members of this family deviate from this rule. Therefore, in this work, we decided to check how broad is the range of non‑zinc-dependent S1-like nucleases and what is the molecular basis of their activities.MethodsS1-like nucleases chosen for analysis were achieved through heterologous expression in appropriate eukaryotic hosts. To characterize nucleases' active-site properties, point mutations were introduced in selected positions. The enzymatic activities of wild-type and mutant nucleases were tested by in-gel nuclease activity assay.ResultsWe discovered that S1-like nucleases encoded by non-vascular plants and single-celled protozoa, like their higher plant homologues, exhibit a large variety of catalytic properties. We have shown that these individual properties are determined by specific non-conserved active site residues.ConclusionsOur findings demonstrate that mutations that occur during evolution can significantly alter the catalytic properties of S1-like nucleases. As a result, different ions can compete for particular S1-type nucleases' active sites. This phenomenon undermines the existing classification of S1-like nucleases.General significanceOur findings have numerous implications for applications and understanding the S1-like nucleases' biological functions. For example, new biotechnological applications should take into account their unexpected catalytic properties. Moreover, these results demonstrate that the trinuclear zinc-based model commonly used to characterize the catalytic activities of S1-like nucleases is insufficient to explain the actions of non‑zinc-dependent members of this family.     

Properties of enzymes that recognize modified structures in DNA

The properties of the major classes of DNA repair enzymes, such as DNA glycosylases, AP-endonucleases, incision nucleases, and alkyl transferases, are reviewed. With the exceptions of the incision nucleases, the properties of the enzymes are quite similar in prokaryotic and eukaryotic cells. The incision nucleases probably do not recognize the modified base residues as such, but rather helical distortion brought about by the modifying agents. The other classes of enzymes are more or less specific for certain modified structures.