Role Of Nuclease Research Articles

The Role of Nucleases and Nucleic Acid Editing Enzymes in the Regulation of Self-Nucleic Acid Sensing.

Detection of microbial nucleic acids by the innate immune system is mediated by numerous intracellular nucleic acids sensors. Upon the detection of nucleic acids these sensors induce the production of inflammatory cytokines, and thus play a crucial role in the activation of anti-microbial immunity. In addition to microbial genetic material, nucleic acid sensors can also recognize self-nucleic acids exposed extracellularly during turn-over of cells, inefficient efferocytosis, or intracellularly upon mislocalization. Safeguard mechanisms have evolved to dispose of such self-nucleic acids to impede the development of autoinflammatory and autoimmune responses. These safeguard mechanisms involve nucleases that are either specific to DNA (DNases) or RNA (RNases) as well as nucleic acid editing enzymes, whose biochemical properties, expression profiles, functions and mechanisms of action will be detailed in this review. Fully elucidating the role of these enzymes in degrading and/or processing of self-nucleic acids to thwart their immunostimulatory potential is of utmost importance to develop novel therapeutic strategies for patients affected by inflammatory and autoimmune diseases.

Nuclease activity decreases the RNAi response in the sweetpotato weevil Cylas puncticollis

RNA interference (RNAi) refers to the process of suppression of gene expression in eukaryotes, which has a great potential for the control of pest and diseases. Unfortunately, the efficacy of this technology is limited or at best variable in some insects. In the African sweet potato weevil (SPW) Cylas puncticollis, a devastating pest that affects the sweet potato production in Sub-Saharan Africa (SSA), the RNAi response was highly efficient when dsRNA was delivered by injection, but it showed a reduced response by oral feeding. In the present study, the role of nucleases in the reduced RNAi efficiency in C. puncticollis is investigated. Several putative dsRNases were first identified in the transcriptome of the SPW through homology search and were subsequently further characterized. Two of these dsRNases were specifically expressed in the gut tissue of the insect and we could demonstrate through RNAi experiments that these affected dsRNA stability in the gut. Furthermore, RNAi-of-RNAi studies, using snf7 as a reporter gene, demonstrated that silencing one of these nucleases, Cp-dsRNase-3, clearly increases RNAi efficacy. After silencing this nuclease, significantly higher mortality was observed in dssnf7-treated insects 14 days post-feeding as compared to control treatments, and the gene downregulation was confirmed at the transcript level via qPCR analysis. Taken together, our results demonstrate that the RNAi efficiency is certainly impaired by nuclease activity in the gut environment of the SPW Cylas puncticollis.

Arabidopsis Ca2+-dependent nuclease AtCaN2 plays a negative role in plant responses to salt stress

Eukaryotic nucleases are involved in processes such as DNA restriction digestion, repair, recombination, transposition, and programmed cell death (PCD). Studies on the role of nucleases have mostly focused on PCD during plant development, while the information on nucleases involved in responses to different abiotic stress conditions remains limited. Here, we identified a Ca2+-dependent nuclease, AtCaN2, in Arabidopsis thaliana and characterized its activity, expression patterns, and involvement in plant responses to salt stress. AtCaN2 showed a dual endonuclease and exonuclease activity, being able to degrade circular plasmids, RNA, single-stranded DNA, and double-stranded DNA. Expression analysis showed that AtCaN2 was strongly induced in senescent siliques and by salt stress. Overexpression of AtCaN2 decreased the plant tolerance to salt stress conditions, leading to an excessive H2O2 accumulation. However, an atcan2 mutant showed better tolerance to salt stress and a lower level of H2O2 accumulation. Moreover, the expression of several genes (AtAPX1, AtGPX8, and AtSOD1), encoding reactive oxygen species-scavenging enzymes (ascorbate peroxidase 1, glutathione peroxidase 8, and superoxide dismutase 1, respectively), was highly induced in the atcan2 mutant under salt stress conditions. In addition, salt-stress-induced cell death was increased in the AtCaN2-overexpressing transgenic plant but decreased in the atcan2 mutant. On the basis of these findings, we conclude that AtCaN2 plays a negative role in plant tolerance to salt stress. A AtCaN2 knock out could reduce ROS accumulation, decrease ROS-induced PCD, and improve overall plant tolerance.

Activation of Nucleases, PCD, and Mobilization of Reserves in the Araucaria angustifolia Megagametophyte During Germination.

The megagametophyte of mature seeds of Araucaria angustifolia consists of cells with thin walls, one or more nuclei, a central vacuole storing proteins, and a cytoplasm rich in amyloplasts, mitochondria and lipid bodies. In this study, we describe the process of mobilization of reserves and analyzed the dismantling of the tissue during germination, using a range of well-established markers of programmed cell death (PCD), including: morphological changes in nuclei and amyloplasts, DNA degradation, and changes in nuclease profiles. TUNEL reaction and DNA electrophoresis demonstrate that DNA fragmentation in nuclei occurs at early stages of germination, which correlates with induction of specific nucleases. The results of the present study add knowledge on the dismantling of the megagametophyte of genus Araucaria, a storage tissue that stores starch as the main reserve substance, as well as on the PCD pathway, by revealing new insights into the role of nucleases and the expression patterns of putative nuclease genes during germination.

Metabolism of DNA secondary structures at the eukaryotic replication fork

DNA secondary structures are largely advantageous for numerous cellular processes but can pose specific threats to the progression of the replication machinery and therefore genome duplication and cell division. A number of specialized enzymes dismantle these structures to allow replication fork progression to proceed faithfully. In this review, we discuss the in vitro and in vivo data that has lead to the identification of these enzymes in eukaryotes, and the evidence that suggests that they act specifically at replication forks to resolve secondary structures. We focus on the role of helicases, which catalyze the dissociation of nucleotide complexes, and on the role of nucleases, which cleave secondary structures to allow replication fork progression at the expense of local rearrangements. Finally, we discuss outstanding questions in terms of dismantling DNA secondary structures, as well as the interplay between diverse enzymes that act upon specific types of structures.

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.

Purification, Characterization, and Role of Nucleases and Serine Proteases in Streptomyces Differentiation: ANALOGIES WITH THE BIOCHEMICAL PROCESSES DESCRIBED IN LATE STEPS OF EUKARYOTIC APOPTOSIS

Two exocellular nucleases with molecular masses of 18 and 34 kDa, which are nutritionally regulated and reach their maximum activity during aerial mycelium formation and sporulation, have been detected in Streptomyces antibioticus. Their function appears to be DNA degradation in the substrate mycelium, and in agreement with this proposed role the two nucleases cooperate efficiently with a periplasmic nuclease previously described in Streptomyces antibioticus to completely hydrolyze DNA. The nucleases cut DNA nonspecifically, leaving 5'-phosphate mononucleotides as the predominant products. Both proteins require Mg2+, and the additional presence of Ca2+ notably stimulates their activities. The two nucleases are inhibited by Zn2+ and aurin tricarboxylic acid. The 18-kDa nuclease from Streptomyces is reminiscent of NUC-18, a thymocyte nuclease proposed to have a key role in glucocorticoid-stimulated apoptosis. The 18-kDa nuclease was shown, by amino-terminal protein sequencing, to be a member of the cyclophilin family and also to possess peptidylprolyl cis-trans-isomerase activity. NUC-18 has also been shown to be a cyclophilin, and "native" cyclophilins are capable of DNA degradation. The S. antibioticus 18-kDa nuclease is produced by a proteolytic processing from a less active protein precursor. The protease responsible has been identified as a serine protease that is inhibited by Nalpha-p-tosyl-L-lysine chloromethyl ketone and leupeptin. Inhibition of both of the nucleases or the protease impairs aerial mycelium development in S. antibioticus. The biochemical features of cellular DNA degradation during Streptomyces development show significant analogies with the late steps of apoptosis of eukaryotic cells.

Role of nucleases in apoptosis.

The last decade has seen the rapid development of research investigating the mechanisms of apoptosis in a variety of experimental systems. Among the multitude of changes observed in apoptotic cells, chromatin cleavage is considered a biochemical hallmark of apoptosis. Chromatin fragmentation is an enzymatic process which depends on the activity of endogenous nuclease(s) and the susceptibility of chromatin to endonuclease activity. The characteristics of some nucleases of potential importance in apoptosis and their possible role in the regulation of this process are discussed in this paper.

Fluorescence microscopy of plastid nucleoids and a survey of nuclease C in higher plants with respect to mode of plastid inheritance

Plastid nucleoids (pt nucleoids) were observed during pollen formation, or in generative cells of mature pollen grains using fluorescence microscopy after staining with 4′,6-diamidino-2-phenylindole (DAPI). Nuclease C activity was surveyed using SDS-PAGE and agarose gel nuclease assay methods. InMirabilis jalapa, pt nucleoids were observed both in pollen mother cells and the monocellular pollen grains after meiosis, followed by the complete disappearance both in the generative and vegetative cells at the bicellular pollen grain stage. This observation is a direct evidence of maternal plastid inheritance. By contrast, in the generative cells of mature pollen grains fromRhododendron kaempferi, Zygocactus truncatus, Oenothera laciniata, andO. speciosa, pt nucleoids were clearly observed. Thus cytological evidence convinces the mode of biparental plastid inheritance. Nuclease C activity was clearly detected both in the stamen and pistil ofM. jalapa. InR. kaempferi low nuclease C activity was detected in both organs, but the activity in the stamen was much less than in the pistil. InZ. truncatus, O. laciniata, andO. speciosa, the activities were difficult to detect in both organs. These results suggest a significant role of nuclease C for the digestion of pt nucleoids in the generative cells.

Role of nucleases in the isolation of plasmid deoxyribonucleic acid from Pseudomonas cepacia 4G9.

Brij 58-cleared lysates of Pseudomonas cepacia 4G9 contain both exonucleolytic and endonucleolytic activities. Endonuclease activity was unaffected by 125 mM ethylenediaminetetraacetic acid, whereas the exonuclease activity was inhibited. In contrast, Sarkosyl NL97 inhibited only the endonuclease. Sodium dodecyl sulfate inhibited all nuclease activity in in vitro assays, but plasmid deoxyribonucleic acid added to P. cepacia 4G9 spheroplasts during sodium dodecyl sulfate lysis was degraded. Irreproducible plasmid isolation from P. cepacia 4G9 may be due to this nucleolytic activity.

Nucleases and Adenosine 3′,5′-Cyclic Monophosphate Phosphodiesterase Activities in Murine Sarcoma Virus (Moloney)-Infected Mice<xref ref-type="fn" rid="FN1">2</xref>

To provide information on the role of nucleases in oncogenic virus infection, the activities of 3'-nucleotide phosphodiesterase (3'-NPDase), 5'-nucleotide phosphodiesterase (5'-NPDase), acid deoxyribonuclease (DNase II), and 3',5'-cyclic AMP phosphodiesterase (cAMPDase) in spleen extracts of murine sarcoma virus-infected C57BL/6 inbred mice were studied. At the peak of tumor growth and of the cell-mediated cytotoxic response (CMC) against tumor-associated antigens, 3'-NPDase, 5'-NPDase, and DNase II all showed depressed activities in the spleen, whereas the activity of cAMPDase in the spleen increased at the peak of CMC and remained elevated thereafter. Serum enzyme activities of the infected mice were also determined, and only 3'-NPD-ase in serum correlated well with CMC. Inasmuch as the correlation of the tumor growth with CMC was established in this system, further study on tumors with variance between CMC and growth is necessary to determine if serum 3'-NPDase is a useful biochemical marker for CMC in vivo.

Nucleic Acids and Protamine in Salmon Testes

This chapter focuses on the presence of nucleic acids and protamine in Salmon testes. It highlights the approach of the centenaries of the discoveries of deoxyribonucleic acid (nuclein) and of protamine. The subsequent relative lack of research on the biochemistry of the salmon testes is in all probability because of the limited geographic distribution of salmonoid fish together with the small fraction of the year when fish are available in the appropriate physiological condition. The chapter presents information on salmon sperm nucleic acids and protarnine pertinent to current interests in molecular biology. Among areas of considerable interest are the replication of DNA during spermatogenesis and the role of nucleases in replication, together with the studies of possible biochemical differences between the mitotic and meiotic (reduction) divisions. The small size and unusual composition of protamine invite a detailed study of its mechanism of biosynthesis, particularly whether the late “turning-on” of the synthesis is controlled at the transcriptional or translational level. Additionally, the biochemistry of the action of protein and steroid hormones can be studied. The spermatozoon itself offers a unique opportunity to study a free-living, highly differentiated cell from a higher organism, possessing characteristic organelles, but with nuclear genes in an inactive state until the events of fertilization reactivate the male genetic material.