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Radiotherapy is the first line treatment for small cell lung cancer (SCLC); However, radio-resistance accompanies with the treatment and hampers the prognosis for SCLC patients. The underlying mechanisms remains elusive. Here we discovered that self-inflicted DNA breaks exist in SCLC cells after radiation. Moreover, using nuclease siRNA screening combined with high-content ArrayScan™ cell analyzer, we identified that Ribonuclease ZC3H12A is required for the self-inflicted DNA breaks after radiation and for SCLC cell survival after DNA damage. ZC3H12A expression was increased in response to DNA damage and when ZC3H12A was knocked down, the DNA repair ability of the cells was impaired, as evidenced by decreased expression of the DNA damage repair protein BRCA1, and increased γH2AX at DNA damage sites. Colony formation assay demonstrates that ZC3H12A knocked down sensitized small cell lung cancer radiotherapy. Therefore, the Ribonuclease ZC3H12A regulates endogenous secondary breaks in small cell lung cancer and affects DNA damage repair. ZC3H12A may act as an important radiotherapy target in small cell lung cancer. [ABSTRACT FROM AUTHOR]

The use of CRISPR–Cas bacterial adaptive immunity system components for targeted DNA changes has opened broad prospects for programmable genome editing of higher organisms. The most widely used gene editors are based on the Cas9 effectors of the type II CRISPR–Cas systems. In complex with guide RNAs, Cas9 proteins are able to directionally introduce double-stranded breaks into DNA regions that are complementary to guide RNA sequences. Despite the wide range of characterized Cas9s, the search for new Cas9 variants remains an important task, since the available Cas9 editors have several limitations. This paper presents a workflow for the search for and subsequent characterization of new Cas9 nucleases developed in our laboratory. Detailed protocols describing the bioinformatical search, cloning, and isolation of recombinant Cas9 proteins, testing for the presence of nuclease activity in vitro, and determining the PAM sequence, which is required for recognition of DNA targets, are presented. Potential difficulties that may arise, as well as ways to overcome them, are considered. [ABSTRACT FROM AUTHOR]

The use of processed nerve allografts as an alternative to autologous nerve grafts, the gold standard treatment for peripheral nerve defects, is increasing. However, it is not widely used in Korea due to cost and insurance issues. Moreover, the main detergent used in the conventional Hudson method is unavailable. Therefore, a new nerve allograft decellularization process is needed. We aimed to compare the traditional Hudson method with a novel decellularization process that may remove cellular content more efficiently while preserving the extracellular matrix (ECM) structure using low concentration sodium dodecyl sulfate (SDS) and nuclease. After each decellularization process, DNA content was measured in nerve tissue. Masson's trichrome staining and scanning electron microscopy were performed to determine the state of preservation of the ECM. A significantly greater amount of DNA content was removed in the novel method, and the ECM structure was preserved in both methods. For the in vivo study, a 15-mm long sciatic nerve defect was created in two groups of Sprague-Dawley rats, and processed nerve allografts decellularized using the Hudson or novel method were transplanted. Functional and histological recovery results were measured 12 weeks post-transplantation. Ankle contracture angle, maximal isometric tetanic force of the tibialis anterior (TA), and the TA mass were compared between the groups, as well as the percent neural tissue (100 × neural area/intrafascicular area). There was no significant difference in functional and histological nerve recovery between the methods. The novel method is appropriate for developing a processed nerve allograft. [ABSTRACT FROM AUTHOR]

Argonaute proteins (Agos) from thermophiles function as endonucleases via guide-target base-pairing cleavage for host defense. Since guides play a key role in regulating the catalytic specificity of Agos, elucidating its underlying molecular mechanisms would promote the application of Agos in the medical sciences. Here, we reveal that an Ago from Pyrococcus furiosus (PfAgo) showed a stepwise endonuclease activity, which was demonstrated through a double-stranded DNA cleavage directed by a single guide DNA (gDNA) rather than a canonical pair of gDNAs. We validated that the cleavage products with 5'-phosphorylated ends can be used as a new guide to induce a new round of cleavage. Based on the reprogrammable capacity of Ago's stepwise activity, we established a rapid and specific platform for unambiguous multiplex gene detection, termed Renewed-gDNA Assisted DNA cleavage by Argonaute (RADAR). Combined with a pre-amplification step, RADAR achieved sensitivity at the femtomolar level and specificity with at least a di-nucleotide resolution. Furthermore, RADAR simultaneously discriminated among multiple target sequences simply by corresponding multiple guides. We successfully distinguished four human papillomavirus serotypes from patient samples in a single reaction. Our technique, based on the unique properties of Ago, provides a versatile and sensitive method for molecular diagnosis. [ABSTRACT FROM AUTHOR]

The SbcCD complex is an essential component of the DNA double-strand break (DSB) repair system in bacteria. The bacterial SbcCD complex recognizes and cleaves the DNA ends in DSBs by ATP-dependent endo- and exonuclease activities as an early step of the DNA repair process. SbcD consists of nuclease, capping, and helix-loop-helix domains. Here, we present the crystal structure of a SbcD fragment from Staphylococcus aureus, which contained nuclease and capping domains, at a resolution of 2.9 Å. This structure shows a dimeric assembly similar to that of the corresponding domains of SbcD from Escherichia coli. The S. aureus SbcD fragment exhibited endonuclease activities on supercoiled DNA and exonuclease activity on linear and nicked DNA. This study contributes to the understanding of the molecular basis for how bacteria can resist sterilizing treatment, causing DNA damage. [ABSTRACT FROM AUTHOR]

The review summarizes the problems, achievements, and prospects of various approaches related to the delivery of components of DNA editing systems to plant cells, the regeneration of whole plants with an edited genome, and the development of transgene-free or transgene-clean crops. Here, an attempt was made to systematize the results of various studies describing the successful production of genome-edited (GE) plants with various genome modifications/mutations via the application of nuclease DNA–editing systems (ZFN, TALEN, and CRISPR/Cas). We discuss the main directions for the development of nuclease-based genome-editing methods to obtain GE plants free of foreign sequences from genome-editing tools. [ABSTRACT FROM AUTHOR]

Filament formation by non-cytoskeletal enzymes has been known for decades, yet only relatively recently has its wide-spread role in enzyme regulation and biology come to be appreciated. This comprehensive review summarizes what is known for each enzyme confirmed to form filamentous structures in vitro, and for the many that are known only to form large self-assemblies within cells. For some enzymes, studies describing both the in vitro filamentous structures and cellular self-assembly formation are also known and described. Special attention is paid to the detailed structures of each type of enzyme filament, as well as the roles the structures play in enzyme regulation and in biology. Where it is known or hypothesized, the advantages conferred by enzyme filamentation are reviewed. Finally, the similarities, differences, and comparison to the SgrAI endonuclease system are also highlighted. [ABSTRACT FROM AUTHOR]

Nucleases are an important group of hydrolases that degrade nucleic acids, with broad spectrum of applications in science and industry. In this paper, we report the identification and characterization of the nuclease from extremely psychrophilic bacterium Psychromonas ingrahamii that grows exponentially at 5 °C, but may also grow at even lower temperatures (down to − 12 °C). The putative endonuclease I gene, identified in the genome of P. ingrahamii, was cloned and expressed in Pichia pastoris. The recombinant protein was purified and its nucleolytic features were studied. The new enzyme, named by us as PinNuc, displays the features characteristic for the nonselective endonucleases, and has the ability to degrade different forms of nucleic acids. It is very active at room temperature in low ion-strength buffer and in the presence of low concentrations of magnesium ions. The enzyme, which possesses six cysteine residues, the most likely all engaged in disulphide bridges, is active only in oxidized form, and can be efficiently inactivated by the addition of low amounts of a reducing agent. According to our knowledge, it is the first nuclease, belonging to endonuclease I family, isolated from such extremely psychrophilic organism. [ABSTRACT FROM AUTHOR]

ATPase/Helicases and nucleases play important roles in DNA end-resection, a critical step during homologous recombination repair in all organisms. In hyperthermophilic archaea the exo-endonuclease NurA and the ATPase HerA cooperate with the highly conserved Mre11-Rad50 complex in 3′ single-stranded DNA (ssDNA) end processing to coordinate repair of double-stranded DNA breaks. Little is known, however, about the assembly mechanism and activation of the HerA-NurA complex. In this study we demonstrate that the NurA exonuclease activity is inhibited by the Sulfolobus solfataricus RecQ-like Hel112 helicase. Inhibition occurs both in the presence and in the absence of HerA, but is much stronger when NurA is in complex with HerA. In contrast, the endonuclease activity of NurA is not affected by the presence of Hel112. Taken together these results suggest that the functional interaction between NurA/HerA and Hel112 is important for DNA end-resection in archaeal homologous recombination. [ABSTRACT FROM AUTHOR]

Objectives: To reduce unwanted Fab' leakage from an autonucleolytic Escherichia coli strain, which co-expresses OmpA-signalled Staphylococcal nuclease and Fab' fragment in the periplasm, by substituting in Serratial nuclease and the DsbA periplasm translocation signal as alternatives. Results: We attempted to genetically fuse a nuclease from Serratia marcescens to the OmpA signal peptide but plasmid construction failed, possibly due to toxicity of the resultant nuclease. Combining Serratial nuclease to the DsbA signal peptide was successful. The strain co-expressing this nuclease and periplasmic Fab' grew in complex media and exhibited nuclease activity detectable by DNAse agar plate but its growth in defined medium was retarded. Fab' coexpression with Staphylococcal nuclease fused to the DsbA signal peptide resulted in cells exhibiting nuclease activity and growth in defined medium. In cultivation to high cell density in a 5 l bioreactor, DsbA-fused Staphylococcal nuclease co-expression coincided with reduced Fab' leakage relative to the original autonucleolytic Fab' strain with OmpA-fused staphylococcal nuclease. Conclusions: We successfully rescued Fab' leakage back to acceptable levels and established a basis for future investigation of the linkage between periplasmic nuclease expression and leakage of co-expressed periplasmic Fab' fragment to the surrounding growth media. [ABSTRACT FROM AUTHOR]

One of the most promising New Plant Breeding Techniques is genome editing (also called gene editing) with the help of a programmable site-directed nuclease (SDN). In this review, we focus on SDN-1, which is the generation of small deletions or insertions (indels) at a precisely defined location in the genome with zinc finger nucleases (ZFN), TALENs, or CRISPR-Cas9. The programmable nuclease is used to induce a double-strand break in the DNA, while the repair is left to the plant cell itself, and mistakes are introduced, while the cell is repairing the double-strand break using the relatively error-prone NHEJ pathway. From a biological point of view, it could be considered as a form of targeted mutagenesis. We first discuss improvements and new technical variants for SDN-1, in particular employing CRISPR-Cas, and subsequently explore the effectiveness of targeted deletions that eliminate the function of a gene, as an approach to generate novel traits useful for improving agricultural sustainability, including disease resistances. We compare them with examples of deletions that resulted in novel functionality as known from crop domestication and classical mutation breeding (both using radiation and chemical mutagens). Finally, we touch upon regulatory and access and benefit sharing issues regarding the plants produced. [ABSTRACT FROM AUTHOR]

Comparative characterization of the pigmented and nonpigmented Serratia marcescens strains and their extracellular nuclease mutants was carried out. Biomass accumulation by the mutant strains decreased on average by 20%, while proteolytic activity of the culture liquid was 4-5 times lower than in the case of the wild type strains. The mutants with impaired extracellular nuclease genes exhibited higher sensitivity to reactive oxygen species. Comparative analysis of motility of the strains revealed the highest flagellar activity in the wild type nonpigmented strain, while the cells of its mutant completely lost this feature. [ABSTRACT FROM AUTHOR]

Streptococcus pyogenes is a human pathogen which causes a spectrum of diseases ranging from pharyngitis to rheumatic fever, necrotising fasciitis and toxic shock syndrome. Development of a vaccine for S. pyogenes has been confounded both by the diversity of the disease-causing serotypes and the spectre of inadvertently stimulating autoimmunity. The S. pyogenes nuclease A (SpnA) is a recently characterised virulence factor that is highly conserved across strains and expressed during human disease. Deletion of spnA from S. pyogenes results in reduced survival of bacteria in whole human blood and attenuated virulence in a mouse model of infection. Collectively these features suggest that SpnA has potential as a vaccine candidate for S. pyogenes. Mice vaccinated subcutaneously with single or multiple doses of recombinant SpnA emulsified in Incomplete Freund's Adjuvant developed a robust and durable IgG response, including neutralising activity, to this protein. However, vaccination with rSpnA conferred no advantage in terms of lesion development, disease symptoms or colonisation levels after a sub-lethal subcutaneous challenge with S. pyogenes. Anti-SpnA serum IgG responses and neutralising activity were increased in response to challenge, indicating that SpnA is expressed in vivo. SpnA is unlikely to be a suitable antigen for a vaccine against S. pyogenes. [ABSTRACT FROM AUTHOR]

With increasing global food demands in the face of challenging biotic and abiotic pressures on crop production, there is a vital need for good crop improvement strategies. Gene editing and gene targeting using designer nucleases are relatively new, sophisticated approaches that can be used for crop improvement. Designer nucleases are molecules that can be engineered to cleave virtually any endogenous DNA target sequence, making this technology inherently more powerful over current, essentially random mutation strategies. These molecules can also be used to promote targeted DNA insertions and homologous recombination. Further modifications of these molecules can convert them into designer transcription factors that can activate or suppress a gene of choice. Four designer nuclease platforms are currently available: meganucleases, zinc finger nucleases, TALENs and the more recently developed CRISPR/Cas9 system. All four of these systems have been shown to function in crop plants and have been used for site-specific gene targeting and gene editing. Herein, we describe the basis of each designer nuclease platform, highlighting the advantages and disadvantages of each, and give examples of their application in crop improvement. [ABSTRACT FROM AUTHOR]

Additions of enzymes involved in organic phosphorus (P) hydrolysis can be used to characterize the hydrolyzability of molybdate-unreactive P (MUP) in soil water extracts. Our aim was to test the feasibility of enzyme additions to soil water suspensions with respect to (1) suitable enzyme preparations and (2) recovery of molybdate-reactive P (MRP). To this end, we compared the substrate specificity of seven commercially available enzyme preparations (acid and alkaline phosphomonoesterase, phytase, and nuclease preparations) and optimized the assay conditions in microplates. We then measured MRP release after the addition of the enzymes to soil water suspensions and filtrates of two Swiss grassland soils (midland and alpine). In some cases, commercial preparations of the same enzyme differed in their specificity, presumably due to contamination with other enzymes, and also in their efficiency in soil suspensions. Addition of EDTA to the buffer was required to decrease sorption of released P in soil suspensions. Enzymatic release of P was consistently equal or higher in soil suspensions than in soil filtrates. However, also more dissolved MUP was present in soil suspensions than in filtrates, since the buffer interacted with the solid phase. Of the total dissolved MUP in soil suspensions, 94 and 61 % were hydrolyzable in midland and alpine soil, respectively. More specifically, 60 and 17 % of MUP were in nucleic acids, 6 and 39 % in simple monoesters, and 28 and 5 % in inositol hexakisphosphate in midland and alpine soil, respectively. Thus, we show that the characterization of hydrolyzable organic P in soil suspensions with hydrolytic enzyme preparations may be useful to better understand the availability of soil organic P to enzymatic hydrolysis, but that it requires soil-specific adaptation for optimum P recovery. [ABSTRACT FROM AUTHOR]

Nature has illustrated through numerous examples that protein dimerization has structural and functional advantages. We previously reported the design and characterization of an engineered 'metallohomeodomain' protein (C2) based on a chimera of the EF-hand Ca-binding motif and the helix-turn-helix motif of homeodomains (Lim and Franklin in Protein Sci. 15:2159-2165, ). This small metalloprotein binds the hard metal ions Ca(II) and Ln(III) and interacts with DNA with modest sequence preference and affinity, yet exhibits only residual DNA cleavage activity. Here we have achieved substantial improvement in function by constructing a covalent dimer of this C2 module (F2) to create a larger multidomain protein. As assayed via fluorescence spectroscopy, this F2 protein binds Ca(II) more avidly (25-fold) than C2 on a per-domain basis; in gel shift selection experiments, metallated F2 exhibits a specificity toward 5′-TAATTA-3′ sequences. Finally, CaF2 cleaves plasmid DNA and generates a linear product in a Ca(II)-dependent way, unlike the CaC2 monomer. To the best of our knowledge this activation of Ca(II) in the context of an EF-hand binding motif is unique and represents a significant step forward in the design of artificial metallonucleases by utilizing biologically significant metal ions. [ABSTRACT FROM AUTHOR]

In 2009, a total of 113 strains of staphylococci were isolated from the thigh muscles of ten hunted and 20 farmed wild rabbits (Oryctolagus cuniculus) in the Slovak Republic. Only two isolates (1.8%) possessed coagulase activity, the rest of 111 staphylococcal isolates were coagulase-negative. Among them, six isolates (5.4%) showed the production of DNase. In each isolate, resistance to eight antibiotics by means of agar dilution test was tested. Based on these results, 110 isolates were found to be resistant to at least one antibiotic. Only one isolate was susceptible to all eight antibiotics tested. Another two isolates were susceptible, however, they showed intermediate susceptibility to cefoxitin. Resistance to ampicillin (78.8%), erythromycin (58.4%), penicillin (51.3%) and oxacillin (46.0%) was found most frequently. Twenty-six isolates (23.0%) were resistant to novobiocin. On the other hand, resistance to cefoxitin (8.0%) and gentamicin (1.8%) were quite rare. Fifteen percent of isolates were resistant to one antibiotic, simultaneous resistance to two, three, four and five antibiotics was confirmed in 22.1%, 23.9%, 21.2% and 13.3% of isolates, respectively. Except for two coagulase-positive Staphylococcus aureus isolates (1.8%), seven species of coagulase-negative staphylococci were identified using the MALDI BioTyper (TM) sytem as follows: Staphylococcus warneri (45.1%), Staphylococcus epidermidis (21.2%), Staphylococcus pasteuri (13.3%), Staphylococcus xylosus (8.0%), Staphylococcus capitis (7.1%), Staphylococcus haemolyticus (1.8%) and Staphylococcus cohnii ssp cohnii (1.8%). [ABSTRACT FROM AUTHOR]

Previously, we characterized a gene encoding the unique nuclease ( LdNuc) from a Sudanese isolate of the human pathogen Leishmania donovani. This parasite secretory enzyme is involved in the salvage of host-derived purines and is constitutively expressed by both developmental forms of the parasite . Currently, we assessed whether an LdNuc-like nuclease was conserved among other geographically disparate isolates of L. donovani and whether this enzyme was produced by intracellular amastigotes during human infections. Using RT-PCR and Southern blotting, we showed that LdNuc gene homologs were present in each of the viscerotropic Leishmania tested (i.e ., L. donovani isolates from the Sudan , Ethiopia and India as well as L. infantum). Further results of in situ enzyme activity gel analyses showed that each of these parasite isolates also expressed a released/secreted LdNuc-like nuclease activity. In Western blots, our anti- LdNuc (Sudan) peptide-specific antibody reacted with only a single ~35 kDa protein in each of the viscerotropic Leishmania isolates. Further, the ~35 kDa nuclease secreted by each of these isolates was specifically immunoprecipitated by the anti- LdNuc antibody above. In situ gel analyses showed that each of these immunoprecipitates had LdNuc-like nuclease activity. Moreover, sera from acute visceral leishmaniasis patients from India, Sudan and Brazil all immunoprecipitated an LdNuc-HA expressed nuclease demonstrating, that these patients possessed antibodies against this parasite secretory enzyme. Cumulatively, these results showed that the LdNuc homologs were functionally conserved among geographically disparate visceral Leishmania spp. and that amastigotes of these parasites must produce this nuclease enzyme during the course of human disease. [ABSTRACT FROM AUTHOR]

Understanding the roles of metal ions in restriction enzymes has been complicated by both the presence of two metal ions in many active sites and their homodimeric structure. Using a single-chain form of the wild-type restriction enzyme PvuII (scWT) in which subunits are fused with a short polypeptide linker (Simoncsits et al. in J. Mol. Biol. 309:89-97, ), we have characterized metal ion and DNA binding behavior in one subunit and examined the effects of the linker on dimer behavior. scWT exhibits heteronuclear single quantum coherence NMR spectra similar to those of native wild-type PvuII (WT). For scWT, isothermal titration calorimetry data fit to two Ca(II) sites per subunit with low-millimolar Ks. The variant scWT|E68A, in which metal ion binding in one subunit is abolished by mutation, also binds two Ca(II) ions in the WT subunit with low-millimolar Ks. When there are no added metal ions, DNA binding affinity for scWT is tenfold stronger than that of the native WT, but tenfold weaker at saturating Ca(II) concentration. In the presence of Ca(II), scWT|E68A binds target DNA similarly to scWT, indicating that high-affinity substrate binding can be carried energetically by one metal-ion-binding subunit. Global analysis of DNA binding data for scWT|E68A suggests that the metal-ion-dependent behaviors observed for WT are reflective of independent subunit behavior. This characterization provides an understanding of subunit contributions in a homodimeric context. [ABSTRACT FROM AUTHOR]

The archaea possess RNase H proteins that share features of both prokaryotic and eukaryotic forms. Although the Sulfolobus RNase HI has been reported to have unique structural and biochemical properties, its RNase HII has not yet been investigated and its biochemical properties remain unknown. In the present study, we have characterized the ST0519 RNase HII from S. tokodaii as a new form. The enzyme utilized hybrid RNA/DNA as a substrate and had an optimal temperature between 37 and 50°C. The activity of wild-type protein was stimulated by Mn2+, whereas this cation significantly inhibited the activity of C-terminal truncated mutant proteins. A series of mutation assays revealed a regulatory C-terminal tail in the S. tokodaii RNase HII. One mutant, ST0519 (residues 1–195), retained only partial activity, while ST0519 (residues 1–196) completely lost its activity. Based on the presumed structure, the C-terminus might form a short α-helix in which two residues, I195 and L196, are essential for the cleavage activity. Our data suggest that the C-terminal α-helix is likely involved in the Mn2+-dependent substrate cleavage activity through stabilization of a flexible loop structure. Our findings offer important clues for further understanding the structure and function of both archaeal and eukaryotic RNase HII. [ABSTRACT FROM AUTHOR]

The specific activities of acid (pH 5.5) and neutral (pH 7) DNases and RNases were determined in alfalfa ( Medicago sativa L.) seedlings grown in the dark in the presence of 3.7 mM paraquat (PQ) or 1 mM roundup (RD). Seedlings were taken at 0, 1, 3, and 5 days. Plant growth parameters (plant height and fresh weight) were dramatically reduced under these conditions of growth comparing to the control (grown in water). The DNase and RNase specific activities of herbicide-treated seedlings were reduced. The reduction of activities ranged by about 50–90 and 15–70% in PQ- and RD-treated seedlings, respectively. In vitro, PQ- and RD-treated nucleic acids [single-stranded DNA (ssDNA), RNA, and plasmid DNA (pl-DNA)] were incubated with acid and neutral nucleases. Both enzymes were isolated and purified from alfalfa seedlings. Electrophoretic analysis on agarose gel of the above incubated mixtures revealed the following: (a) neutral nuclease (pH 7) was capable of hydrolyzing PQ-treated ssDNA while acid nuclease (pH 5.5) was incapable. This could be due to the fact that acid and neutral nucleases displayed different base linkage specificity toward ssDNA; (b) RD formed strong complexes with ssDNA that were unable to be hydrolyzed by both nucleases; (c) in contrast, both enzymes were capable of hydrolyzing PQ- or RD-treated RNA; (d) neutral nuclease was capable of nicking and linearizing both PQ- and RD-treated pl-DNA while acid nuclease had the same activity only toward the PQ-treated pl-DNA; (e) the enzyme activities were not inhibited in the presence of both herbicides. The data suggest that the complexes of PQ or RD with DNA should not be functional substrates of nucleases, and consequently cell processes (e.g., metabolism of nucleic acids, gene expression, replication), in which DNA and nucleases are involved, could be disturbed. [ABSTRACT FROM AUTHOR]

Mutations in the genes encoding the RNaseH2 and TREX1 nucleases have been identified in patients with Aicardi–Goutieres syndrome (AGS). To determine if the AGS RNaseH2 mutations result in the loss of nuclease activity, the human wild-type RNaseH2 and four mutant complexes that constitute the majority of mutations identified in AGS patients have been prepared and tested for ribonuclease H activity. The heterotrimeric structures of the mutant RNaseH2 complexes are intact. Furthermore, the ribonuclease H activities of the mutant complexes are indistinguishable from the wild-type enzyme with the exception of the RNaseH2 subunit A (Gly37Ser) mutant, which exhibits some evidence of altered nuclease specificity. These data indicate that the mechanism of RNaseH2 dysfunction in AGS cannot be simply explained by loss of ribonuclease H activity and points to a more complex mechanism perhaps mediated through altered interactions with as yet identified nucleic acids or protein partners. [ABSTRACT FROM AUTHOR]

A method for chemico-enzymatic synthesis of (2′-5′)-oligonucleotides with 6- N-benzylaminopurineriboside as the nucleoside units was proposed. The method consisted of enzymatic hydrolysis of the oligonucleotides with mixed (2′-5′)-(3′-5′)-phosphodiesterbonds that were prepared by polymerization of 6- N-benzyladenosine-2′(3′)-monophosphate by using (3′-5′)-specific nuclease and phosphatase contained in the filtrate of culture medium of the mycelial fungus Spicaria violacea. [ABSTRACT FROM AUTHOR]

The genomic region of Prevotella bryantii TC1-1 that conferred an increased nucleolytic activity on Escherichia coli was characterized. It contains two divergent transcriptional units separated by an AT-rich promoter region. One unit is comprised of three genes involved in nucleotide metabolism. nucA, the first gene of this unit, whose product belongs to exonuclease/endonuclease/phosphatase Pfam family, was thought to be required for the increased nucleolytic activity and various expression strategies were employed to confirm its role. The nucA expression was only successful in cell free system where DNase and RNase activity was observed. Two genes downstream of nucA code for a putative uracil DNA glycosylase and uridine kinase which could be involved in the removal of misincorporated uracil from DNA and its reuse. Given that apurinic/apyrimidinic nuclease activity is required after uracil removal from DNA, it was somewhat surprising to find out that nucA, whose product belongs to protein family consisting mostly of apurinic/apyrimidinic nucleases, has no apurinic/apyrimidinic activity. [ABSTRACT FROM AUTHOR]

A fundamental question in DNA repair is how a lesion is detected when embedded in millions to billions of normal base pairs. Extensive structural and functional studies reveal atomic details of DNA repair protein and nucleic acid interactions. This review summarizes seemingly diverse structural motifs used in lesion recognition and suggests a general mechanism to recognize DNA lesion by the poor base stacking. After initial recognition of this shared structural feature of lesions, different DNA repair pathways use unique verification mechanisms to ensure correct lesion identification and removal.Cell Research (2008) 18:184–197. doi: 10.1038/cr.2007.116; published online 24 December 2007 [ABSTRACT FROM AUTHOR]

The hydrolysis of phosphodiester bonds by nucleases is critical to nucleic acid processing. Many nucleases utilize metal ion cofactors, and for a number of these enzymes two active-site metal ions have been detected. Testing proposed mechanistic roles for individual bound metal ions has been hampered by the similarity between the sites and cooperative behavior. In the homodimeric PvuII restriction endonuclease, the metal ion dependence of DNA binding is sigmoidal and consistent with two classes of coupled metal ion binding sites. We reasoned that a conservative active-site mutation would perturb the ligand field sufficiently to observe the titration of individual metal ion binding sites without significantly disturbing enzyme function. Indeed, mutation of a Tyr residue 5.5 Å from both metal ions in the enzyme–substrate crystal structure (Y94F) renders the metal ion dependence of DNA binding biphasic: two classes of metal ion binding sites become distinct in the presence of DNA. The perturbation in metal ion coordination is supported by 1H–15N heteronuclear single quantum coherence spectra of enzyme–Ca(II) and enzyme–Ca(II)–DNA complexes. Metal ion binding by free Y94F is basically unperturbed: through multiple experiments with different metal ions, the data are consistent with two alkaline earth metal ion binding sites per subunit of low millimolar affinity, behavior which is very similar to that of the wild type. The results presented here indicate a role for the hydroxyl group of Tyr94 in the coupling of metal ion binding sites in the presence of DNA. Its removal causes the affinities for the two metal ion binding sites to be resolved in the presence of substrate. Such tuning of metal ion affinities will be invaluable to efforts to ascertain the contributions of individual bound metal ions to metallonuclease function. [ABSTRACT FROM AUTHOR]

DFF40/CAD, the major apoptotic nuclease, is specific for double-stranded DNA. However, RNA and single-stranded DNA, though not substrates for the enzyme, compete with double-stranded DNA and inhibit its cleavage by the nuclease. In addition, other anionic polymers, like poly-glutamic acid and heparin also inhibit DFF40/CAD, the latter one being highly effective at nanomolar concentrations. The inhibitory poly-anions bind to the nuclease and impair its ability to bind double-stranded DNA. We propose that such poly-anions bind to the positively charged surface formed by α4 helices of the DFF40/CAD homodimer. This surface has been proposed recently to bind to either the major groove of DNA or poly (ADP-ribose), another inhibitor of the nuclease. [ABSTRACT FROM AUTHOR]

Expanded bed-ionic exchange chromatography (EB-IEC) was used for the recovery and purification of recombinant staphylococcal nuclease secreted by Lactococcus lactis. At the end of the fermentation process, the nuclease activity reached 39 U ml−1. The EB-IEC performances were firstly evaluated with clarified culture broth. The isocratic elution with 0.5 M NaCl led to approximately 80% of nuclease recovery. Proceeding with 3-fold bed expansion resulted in a reduction of the resin capacity by a factor of 32% compared to the process in a packed bed configuration. Simplification of the early purification steps was reached by loading immediately the unclarified culture broth previously diluted to reduce conductivity. Presence of Cells did not affect the chromatography performances resulting in 55-fold purification with the same yield. [ABSTRACT FROM AUTHOR]

Endonuclease G (EndoG) is a mitochondrial enzyme that becomes an apoptotic nuclease when released from the mitochondrial intermembrane space. EndoG will digest either DNA or RNA, but at physiological ionic strength, RNA is a much more favorable substrate as compared to chromatin. This indicates that EndoG’s major in vivo function(s) may be: (i) an apoptotic RNase, and/or (ii) an apoptotic DNase in the presence of additional co-activators. In the present study we have searched for factors that modulate the activity of human EndoG on DNA substrates. We demonstrate that EndoG forms complexes with AIF and FEN-1 but not with PCNA. Interestingly, heat shock proteins 70 interact with EndoG and are involved in the regulation of its activity. Purified Hsp70 prevented stimulation of EndoG DNase activity by other nuclear factors in the ATP-dependent manner. [ABSTRACT FROM AUTHOR]

Leaf and callus tissues of a creeping bentgrass cultivar (Penn A4) had high nuclease activities that degraded exogenously added plasmid DNA. When callus tissue was incubated for 24 h with heparin, spermidine, aurintricarboxylic acid or polyethylene glycol, only heparin and spermidine were effective as in vitro nuclease inhibitors, protecting exogenously added plasmid DNA from degradation. When β-glucuronidase (GUS) reporter gene activity was evaluated in heparin-treated (0.6%), 14-month old callus following microprojectile bombardment, GUS activity increased 1000-fold compared to equivalent aged untreated Penn A4 callus. Similar enhancement from heparin pretreatment (0.6% or 1.2%) was not observed in 6-month old callus. This is likely due to much higher activities of nuclease in the younger callus. [ABSTRACT FROM AUTHOR]

NM23/NDPk proteins play critical roles in cancer and development; however, our understanding of the underlying biochemical mechanisms is still limited. This large family of highly conserved proteins are known to participate in many events related to DNA metabolism, including nucleotide binding and nucleoside triphosphate synthesis, DNA binding and transcription, and cleavage of DNA strands via covalent protein–DNA complexes. The chemistry of the DNA-cleavage reaction of NM23-H2/NDPk is characteristic of DNA repair enzymes. Both the DNA cleavage and the NDPk reactions are conserved between E. coli and the human enzymes, and several conserved amino acid side chains involved in catalysis are shared by these reactions. It is proposed here that NM23/NDP kinases are important regulators of gene expression during development and cancer via previously unrecognized roles in DNA repair and recombination, and via previously unrecognized pathways and mechanisms of genetic control. [ABSTRACT FROM AUTHOR]

Few biochemical and molecular details are available on microspore growth and development. In this work, a nuclease was partially purified from diffusates of barley (Hordeum vulgare L.) microspores by using concanavalin-A as ligand. The chromatographic preparation contained a 34-kDa protein with nucleolytic activity; the enzyme (called BMN: barley microspore nuclease) was very stable at pH>8.0 and temperatures below 50 °C. Activity was highest at pH 5.6 and increased almost exponentially with temperature until a breakpoint between activity and stability was reached at 70 °C. Although BMN was able to cleave RNA, the enzyme showed a remarkable preference for DNA, especially in the single-stranded form. The best homopolymeric substrates were poly(dA) and poly(A), whereas poly(dC), poly(G) and poly(I) were almost completely uncleaved. When incubated with intact nuclei, BMN caused a nucleosomal DNA ladder of approximately 200 bp. On the basis of DNA laddering, substrate specificity, Mg2+-dependence and best performance at apoplastic pH, BMN can be referred to as a putative apoptotic nuclease involved in pollen development. [ABSTRACT FROM AUTHOR]

The endonuclease DFF40/CAD mediates regulated DNA fragmentation and chromatin condensation in cells undergoing apoptosis. Here we report the enzyme's co-factor requirements, and demonstrate that the ionic changes that occur in apoptotic cells maximize DFF40/CAD activity. The nuclease requires Mg2+, exhibits a trace of activity in the presence of Mn2+, is not co-stimulated by Ca2+, is inhibited by Zn2+ or Cu2+, and has high activity over a rather broad pH range (7.0–8.5). The enzyme is thermally unstable, and is rapidly inactivated at 42°C. Enzyme activity is markedly affected by ionic strength. At the optimal [K+] of 50–125 mM, which is in the range of the cytoplasmic [K+] for cells undergoing apoptosis, the activity of DFF40/CAD for naked DNA cleavage is about 100-fold higher than at 0 or 200 mM [K+]. Although these ranges of ionic strength do not affect DFF40 homo-oligomer formation, at higher ionic strengths the enzyme introduces single-stranded nicks into supercoiled DNA. [ABSTRACT FROM AUTHOR]

The culture liquid and periplasm of Proteus mirabilis contained nuclease, an enzyme with DNase and RNase activities. The nuclease was most actively synthesized in the early exponential and stationary growth phases. Nuclease synthesis was regulated by nucleic acids (induction by substrate) and inorganic phosphate (end-product inhibition). The synthesis and secretion of nuclease by P. mirabilis was induced by mitomycin C, an inducer of the SOS functions of cells. This suggests the involvement of SOS-response proteins in the regulation of nuclease synthesis. [ABSTRACT FROM AUTHOR]

Progress in understanding programmed cell death (PCD) in the cereal aleurone is described. Cereal aleurone cells are specialized endosperm cells that function to synthesize and secrete hydrolytic enzymes that break down reserves in the starchy endosperm. Unlike the cells of the starchy endosperm, aleurone cells are viable in mature grain but undergo PCD when germination is triggered or when isolated aleurone layers or protoplasts are incubated in gibberellic acid (GA). Abscisic acid (ABA) slows down the process of aleurone cell death and isolated aleurone protoplasts can be kept alive in media containing ABA for up to 6 months. Cell death in barley aleurone occurs only after cells become highly vacuolated and is manifested in an abrupt loss of plasma membrane integrity. Aleurone cell death does not follow the apoptotic pathway found in many animal cells. The hallmarks of apoptosis, including internucleosomal DNA cleavage, plasma membrane and nuclear blebbing and formation of apoptotic bodies, are not observed in dying aleurone cells. PCD in barley aleurone cells is accompanied by the accumulation of a spectrum of nuclease and protease activities and the loss of organelles as a result of cellular autolysis. [ABSTRACT FROM AUTHOR]

Plant development involves various programmed cell death (PCD) processes. Among them, cell death occurring during differentiation of procambium into tracheary elements (TEs), which are a major component of vessels or tracheids, has been studied extensively. Recent studies of PCD during TE differentiation mainly using an in vitro differentiation system of Zinnia have revealed that PCD of TEs is a plant-specific one in which the vacuole plays a central role. Furthermore, there are recent findings of several factors that may initiate PCD of TEs and that act at autonomous degradation of cell contents. Herein I summarize the present knowledge about cell death program during TE differentiation as an excellent example of PCD in plants. [ABSTRACT FROM AUTHOR]

The endosperm of cereals functions as a storage tissue in which the majority of starch and seed storage proteins are synthesized. During its development, cereal endosperm initiates a cell death program that eventually affects the entire tissue with the exception of the outermost cells, which differentiate into the aleurone layer and remain living in the mature seed. To date, the cell death program has been described for maize and wheat endosperm, which exhibits common and unique elements for each species. The progression of endosperm programmed cell death (PCD) in both species is accompanied by an increase in nuclease activity and the internucleosomal degradation of nuclear DNA, hallmarks of apoptosis in animals. Moreover, ethylene and abscisic acid are key to mediating PCD in cereal endosperm. The progression of the cell death program in developing maize endosperm follows a highly organized pattern whereas in wheat endosperm, PCD initiates stochastically. Although the essential characteristics of cereal endosperm PCD are now known, the molecular mechanisms responsible for its execution remain to be identified. [ABSTRACT FROM AUTHOR]

Qualitative enzyme assays were used as a tool to investigate the stability of freeze-dried mycorrhizal fungi. Both lyophilized (L) and non-lyophilized (NL) mycelia of individual isolates showed identical response for all the enzymes tested (nitrate reductase, protease, pectinase, and nuclease). All the isolates showed positive nitrate reductase activity, except two isolates of Thelephora terrestris (both L and NL). Both L and NL cultures of individual isolates showed substrate specificity (between gelatin and casein) for protease activity. Though both L and NL mycelia of all the culture isolates grew upon pectin substrate, there was no pectinase activity expressed. RNAase activity was variously exhibited (little activity, little growth–no activity, and no growth–no activity) by individual test cultures. The consistencies in growth and enzyme activity of the cultures before and after lyophilization imply the stability of the freeze-dried vegetative mycelium. [ABSTRACT FROM AUTHOR]

Single-strand DNase and poly rAase, activities characteristic of endo-exonuclease, were co-activated in nuclear fractions of HL-60 cells by caspase-3. Activation was accompanied by cleavages of large soluble polypeptides (130–185 kDa) and a 65 kDa inactive chromatin-associated polypeptide related to the endo-exonuclease of Neurospora crassa as detected on immunoblots. The major products seen in vitro were a 77 kDa soluble polypeptide and an active chromatin-associated 34 kDa polypeptide. When HL-60 cells were induced to undergo apoptosis by treating with 50 μM etoposide (VP-16) for 4 hours, 77 kDa and 40 kDa polypeptides accumulated in nuclear fractions. Chromatin DNA fragmentation activity was also activated in cytosol and nuclear extract either by pre-treating the cells in vivo with VP-16 or by treating the cytosol in vitro with caspase-3 or dATP and cytochrome c. Endo-exonuclease activated by caspase-3 in cytosol-derived fractions augmented chromatin DNA fragmentation activity in vitro. Endo-exonuclease is proposed to act in vivo in conjunction with the caspase-activated DNase (CAD) to degrade chromatin DNA during apoptosis of HL-60 cells. [ABSTRACT FROM AUTHOR]

Apoptosis induced by etoposide (VP-16) in HL-60 cells was confirmed to be caspase-dependent. It was fully inhibited by the broad-spectrum caspase inhibitor Z-VAD-fmk. However, the caspase-3-specific inhibitor Z-DEVD-fmk only partially inhibited apoptosis. This indicated that a second caspase is required in vivo for full activation of the apoptotic nucease CAD. Aurin tricarboxylic acid (ATA) did not inhibit VP-16-induced apoptosis. In contrast, apoptosis induced by hydroxychloroquine (HCQ) in HL-60 cells was caspase-3 independent and was fully inhibited by ATA. Thus, CAD does not appear to be involved in chromatin DNA degradation in this case. A second apoptotic nuclease is postulated to degrade the DNA, likely endo-exonuclease, an abundant nuclear enzyme that acts on both DNA and RNA and is present in latent form. HCQ, but not VP-16, stimulated DNA degradation (“laddering”) in isolated nuclei. This indicates that the drug can act directly in the nuclei to trigger activation of the second latent apoptotic nuclease. [ABSTRACT FROM AUTHOR]

Since mitochondrial factors have been implicated in apoptosis, experiments were designed to assess whether or not the potent mitochondrial nuclease could be one of these factors. Nuclei isolated by two different methods were found to contain mitochondrial nuclease in masked form. This nuclease was released by treatment with the non-ionic detergent NP-40 and rendered trypsin-sensitive. It was not removed appreciably from the nuclei by washing and sedimentation of the nuclei through a sucrose cushion. Levels of the mitochondrial nuclease were followed during drug-induced apoptosis. Time courses of apoptosis in cultures of HL-60 cells were monitored by flow cytometry of propidium iodide-stained cells and by agarose gel electrophoresis of extracted DNA. Changes in the inner mitochondrial transmembrane potential were monitored by flow cytometry of chloromethyl-X-Rosamine-stained cells. Apoptosis was induced by treatment with either the chemotherapeutic agent etoposide (VP-16 at 10 μM) over an 8 h period or with the anti-rheumatic agent hydroxychloroquine (HCQ at 0.28 mM) over a 24 h period. These two drugs likely act in different pathways of apoptosis. VP-16 caused loss of the mitochondrial transmembrane potential 1.0–1.5 h before apoptosis was detected. On the other hand, treatment with HCQ caused these processes to occur in parallel possibly indicating that the mitochondrial changes are secondary events. No losses of masked mitochondrial nuclease were detected with either drug treatment during the course of apoptosis. HL-60 mitochondrial DNA was also not degraded during apoptosis induced by either agent. These observations likely explain why the mitochondrial DNA is not degraded and make it unlikely that mitochondrial nuclease plays any role in vivo in chromatin DNA fragmentation. [ABSTRACT FROM AUTHOR]

The primary structure and physical chemical properties were determined of a nuclease expressed and secreted by Escherichia coli. The plasmid p403-SD2 carried a DNA sequence isolated from Serratia marcescens encoding the enzyme. During cultivation of the E. coli cells, 85% of the enzyme was released to the growth medium. The enzyme was purified and exhibited a single band with a molecular weight about 30,600 daltons on SDS-PAGE similar to nuclease isolated from S. marcescens. The amino acid composition and the amino acid sequence determined directly confirmed the primary structure of 245 amino acids predicted from the DNA sequence, and, in addition, the two disulfide bridges were assigned. Several physical chemical properties were examined. The ability of the enzyme to cross the outer membrane is proposed to depend upon the formation of the proper structures during the folding process. [ABSTRACT FROM AUTHOR]

Programmed cell death (PCD) is activated during the response of multicellular organisms to some invading pathogens. One of the key aspects of this process is the degradation of nuclear DNA which is thought to facilitate the recycling of DNA from dead cells. The PCD of tobacco plants (genotype NN) infected with tobacco mosaic virus (TMV) is accompanied by the induction of nuclease activities and the cleavage of nuclear DNA to fragments of about 50 kb. We examined the correlation between the increase in nuclease activities and the fragmentation of nuclear DNA during TMV- and bacteria-induced PCD in tobacco. We found that the increase in nuclease activities did not always correlate with fragmentation of nuclear DNA. Thus, in addition to pathogens that induce PCD, mechanical injury and infiltration of leaves with 1 M sucrose or bacteria that did not induce PCD also resulted in an increase in nuclease activities. Analysis of nuclease activities in total leaf extracts, nuclear extracts, and intercellular fluid (i.e., apoplast) revealed that at least four different nuclease activities are induced during PCD in tobacco; of these at least three appear to be secreted into the intercellular fluid. Although the latter were also induced in response to treatments that did not result in DNA fragmentation, they may function in the recycling of plant DNA during late stages of PCD when the integrity of the plasma membrane is compromised. This suggestion is supported by the finding that DNA degradation occurred late during TMV-induced PCD in tobacco. In addition, the finding of induced nuclease activities in the intercellular fluid raises the possibility that they may serve a protective function by degrading the DNA of invading pathogens. [ABSTRACT FROM AUTHOR]

We have previously reported that the ferredoxin I gene from Synechococcus sp. PCC 7942 is regulated by iron at the level of differential mRNA stability. To identify iron-responsive elements in the Synechococcus ferredoxin transcript, we have tested chimaeric constructs containing translational fusions between the Synechococcus and the Anabaena sp. PCC 7937 ferredoxin genes for iron-dependent expression in transgenic Synechococcus strains. This strategy was based on the observation that the level of the Anabaena ferredoxin mRNA did not increase upon iron addition in Synechococcus. Our results show that the presence of the first 207 nucleotides of the Synechococcus ferredoxin transcript is sufficient to confer iron responsiveness to the chimaeric transcripts. This iron responsiveness was accomplished by an increased stability of the chimaeric transcript in the presence of iron, as was found for the intact Synechococcus ferredoxin gene. Addition of the translation inhibitor chloramphenicol to the cultures led to a rapid stabilization, in low- and high-iron conditions, of the wild-type Synechococcus ferredoxin transcript as well as all chimaeric ferredoxin transcripts tested. These results suggest the existence of a constitutively expressed nuclease capable of degrading the ferredoxin transcripts. They further support the suggestion that the first 207 nucleotides of the Synechococcus transcript contain a specific sequence that is recognized by an iron-responsive factor and that this interaction leads to protection against degradation. [ABSTRACT FROM AUTHOR]

The biolistic particle delivery system was used to transfer the uidA gene into different garlic tissues, including regenerable calli. These tissues showed a high endogenous nuclease activity preventing exogenous DNA expression. After submerging the tissues for 24 h in a 2 m m solution of the nuclease inhibitor aurintricarboxylic acid, transient expression of the uidA gene was detected. Furthermore, vacuum infiltration of aurintricarboxylic acid into the garlic tissues significantly increased the percentage of explants showing transient expression. Among four plasmids tested, pDE4 containing the CaMV35S- uidA construct produced the best results. [ABSTRACT FROM AUTHOR]

Metal-mediated hydrolysis of phosphate esters is a common catalytic pathway in nucleic acid biochemistry. Two distinct models are principally invoked in mechanistic discussions of these reactions for magnesium-dependent nuclease activation, namely, the one- versus two-metal-ion pathways. By careful consideration of the available experimental data on a variety of enzymes (including the Klenow fragment of DNA pol I, exonuclease III, ribonuclease H domains of HIV reverse transcriptase and the homologous E scherichia coli enzyme, avian sarcoma virus integrase, Eco RI and Eco RV restriction endonucleases, and ribozymes), it is demonstrated that the two-metal-ion paradigm cannot be substantiated as a bona fide mechanism on the basis of the published structural data. The origins of the problems with this model are traced to fundamental issues in solution chemistry. These factors help to explain and resolve the often dichotomous results obtained from X-ray crystallography versus solution studies. [ABSTRACT FROM AUTHOR]

The proteins essential to homologous recombination in E. coli have been purified and their individual activities have been identified, permitting biochemical reconstitution of steps that comprise the cellular recombination process. This review focuses on the biochemical events responsible for the initiation and homologous pairing steps of genetic recombination. The properties of an in vitro recombination reaction that requires the concerted action of recA, recBCD, and SSB proteins and that is stimulated by the recombination hotspot, Chi(χ), are described. The recBCD enzyme serves as the initiator of this reaction; its DNA helicase activity produces single-stranded DNA that is used by the recA protein to promote homologous pairing and DNA strand invasion of supercoiled (recipient) DNA. The SSB protein acts to trap the single-stranded DNA produced by recBCD enzyme and to facilitate pairing by the recA protein. The χ regulatory sequence acts in cis by attenuating the nuclease, but not the helicase, activity of recBCD enzyme. This attenuation assures the preservation of ssDNA produced by the DNA helicase activity and is responsible for the simulation in vitro and, presumably, in vivo. The attenuation of nuclease activity by χ results in the loss or functional inactivation of the recD subunit. [ABSTRACT FROM AUTHOR]

This is the last in a series of four articles in which the chemical, enzymological and crystallographic work on Ribonucleate (deoxyribonucleate)-3′-nucleotidohydrolase, EC 3.1.4.4 (staphylococcal nuclease, micrococcal nuclease) will be reviewed and correlated. This article discusses the use of the nuclease as a model system for the study of the mechanisms and energetics of the folding-unfolding reaction in proteins and for the study of the interrelationships between amino acid sequence and three-dimensional structure. [ABSTRACT FROM AUTHOR]