Combination of antiviral drugs inhibits SARS-CoV-2 polymerase and exonuclease and demonstrates COVID-19 therapeutic potential in viral cell culture.

SARS-CoV-2 has an exonuclease-based proofreader, which removes nucleotide inhibitors such as Remdesivir that are incorporated into the viral RNA during replication, reducing the efficacy of these drugs for treating COVID-19. Combinations of inhibitors of both the viral RNA-dependent RNA polymerase and the exonuclease could overcome this deficiency. Here we report the identification of hepatitis C virus NS5A inhibitors Pibrentasvir and Ombitasvir as SARS-CoV-2 exonuclease inhibitors. In the presence of Pibrentasvir, RNAs terminated with the active forms of the prodrugs Sofosbuvir, Remdesivir, Favipiravir, Molnupiravir and AT-527 were largely protected from excision by the exonuclease, while in the absence of Pibrentasvir, there was rapid excision. Due to its unique structure, Tenofovir-terminated RNA was highly resistant to exonuclease excision even in the absence of Pibrentasvir. Viral cell culture studies also demonstrate significant synergy using this combination strategy. This study supports the use of combination drugs that inhibit both the SARS-CoV-2 polymerase and exonuclease for effective COVID-19 treatment.

Design, synthesis, and biological evaluation of novel urolithins derivatives as potential phosphodiesterase II inhibitors.

A series of urolithins derivatives were designed and synthesized, and their structures have been confirmed by 1H NMR, 13C NMR, and HR-MS. The inhibitory activity of these derivatives on phosphodiesterase II (PDE2) was thoroughly studied with 3-hydroxy-8-methyl-6H-benzo[C]chromen-6-one and 3-hydroxy-7,8,9,10-tetrahydro-6H-benzo[C] chromen-6-one as the lead compounds. The biological activity test showed that compound 2e had the best inhibitory activity on PDE2 with an IC50 of 33.95 µM. This study provides a foundation for further structural modification and transformation of urolithins to obtain PDE2 inhibitor small molecules with better inhibitory activity.

Design, Synthesis, and Biological Evaluation of Novel 6H-Benzo[c]chromen-6-one Derivatives as Potential Phosphodiesterase II Inhibitors.

Urolithins (hydroxylated 6H-benzo[c]chromen-6-ones) are the main bioavailable metabolites of ellagic acid (EA), which was shown to be a cognitive enhancer in the treatment of neurodegenerative diseases. As part of this research, a series of alkoxylated 6H-benzo[c]chromen-6-one derivatives were designed and synthesized. Furthermore, their biological activities were evaluated as potential PDE2 inhibitors, and the alkoxylated 6H-benzo[c]chromen-6-one derivative 1f was found to have the optimal inhibitory potential (IC50: 3.67 ± 0.47 µM). It also exhibited comparable activity in comparison to that of BAY 60-7550 in vitro cell level studies.

Label-Free Screening of SARS-CoV-2 NSP14 Exonuclease Activity Using SAMDI Mass Spectrometry.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus responsible for the global COVID-19 pandemic. Nonstructural protein 14 (NSP14), which features exonuclease (ExoN) and guanine N7 methyltransferase activity, is a critical player in SARS-CoV-2 replication and fidelity and represents an attractive antiviral target. Initiating drug discovery efforts for nucleases such as NSP14 remains a challenge due to a lack of suitable high-throughput assay methodologies. This report describes the combination of self-assembled monolayers and matrix-assisted laser desorption ionization mass spectrometry to enable the first label-free and high-throughput assay for NSP14 ExoN activity. The assay was used to measure NSP14 activity and gain insight into substrate specificity and the reaction mechanism. Next, the assay was optimized for kinetically balanced conditions and miniaturized, while achieving a robust assay (Z factor > 0.8) and a significant assay window (signal-to-background ratio > 200). Screening 10,240 small molecules from a diverse library revealed candidate inhibitors, which were counterscreened for NSP14 selectivity and RNA intercalation. The assay methodology described here will enable, for the first time, a label-free and high-throughput assay for NSP14 ExoN activity to accelerate drug discovery efforts and, due to the assay flexibility, can be more broadly applicable for measuring other enzyme activities from other viruses or implicated in various pathologies.

Synthesis of 2'-O-alkylcarbamoylethyl-modified oligonucleotides with enhanced nuclease resistance that form isostable duplexes with complementary RNA.

To expand the variety of 2'-O-modified oligonucleotides, we synthesized 2'-O-carbamoylethyl-modified oligonucleotides bearing ethyl, n-propyl, n-butyl, n-pentyl, and n-octyl groups on their nitrogen atoms. The corresponding nucleosides were synthesized using 2'-O-benzyloxycarbonylethylthymidine, which was easily converted into the carboxylic acid through hydrogeneration; subsequent condensation with the appropriate amine gave the desired nucleoside. We evaluated the effect of the 2'-O-alkylcarbamoylethyl modifications on duplex stability by analyzing melting temperature, which revealed the formation of isostable duplexes. In addition, we also revealed that these modifications, especially octylcarbamoylethyl, endowed these oligonucleotides with resistance toward a 3'-exonuclease. These results highlight the usefulness of the 2'-O-alkylcarbamoylethyl modification for various biological applications.

New Mechanism of Gemcitabine and Its Phosphates: DNA Polymerization Disruption via 3'-5' Exonuclease Inhibition.

Gemcitabine (dFdC), a modified deoxycytidine (dC) widely used in tumor treatment, is a prodrug that is phosphorylated to generate mono-, di-, and triphosphates. The triphosphate (dFdCTP) is incorporated into DNA to terminate DNA synthesis in cancer. Some incorporated dFdC nucleotides can be partially removed by the 3'-5' exonuclease activity, namely its editing function, and the others escape the editing. However, whether there is an active mechanism for dFdC to escape the editing remains unclear. We have first discovered that unlike dFdC, its mono-, di-, and triphosphates can inhibit the 3'-5' exonuclease of DNA polymerase I, suppress editing, and allow the active escaping mechanism, whereas its polymerase activity is not remarkably affected. As such, these phosphates can prevent the removal of the incorporated dFdC residue, thereby actively blocking DNA extension and synthesis. The inhibition efficiency of these phosphates follows the increased order of the mono-, di-, and triphosphates of gemcitabine (dFdC < dFdCMP < dFdCDP < dFdCTP). In addition, after the deletion of the 3'-5' exonuclease of cellular DNA polymerase I, the Escherichia coli mutant is more sensitive to dFdCTP than is wild-type E. coli. Our new discovery of the ability of these dFdC phosphates to inhibit exonuclease activity suggests a novel anticancer mechanism of gemcitabine and its phosphate derivatives.

Protecting Linear DNA Templates in Cell-Free Expression Systems from Diverse Bacteria.

Recent advances in cell-free systems have opened up new capabilities in synthetic biology from rapid prototyping of genetic circuits and metabolic pathways to portable diagnostics and biomanufacturing. A current bottleneck in cell-free systems, especially those employing non-E. coli bacterial species, is the required use of plasmid DNA, which can be laborious to construct, clone, and verify. Linear DNA templates offer a faster and more direct route for many cell-free applications, but they are often rapidly degraded in cell-free reactions. In this study, we evaluated GamS from λ-phage, DNA fragments containing Chi-sites, and Ku from Mycobacterium tuberculosis for their ability to protect linear DNA templates in diverse bacterial cell-free systems. We show that these nuclease inhibitors exhibit differential protective activities against endogenous exonucleases in five different cell-free lysates, highlighting their utility for diverse bacterial species. We expect these linear DNA protection strategies will accelerate high-throughput approaches in cell-free synthetic biology.

A FRET assay for the quantitation of inhibitors of exonuclease EcoRV by using parchment paper inkjet-printed with graphene oxide and FAM-labelled DNA.

A graphene oxide (GO)-based cost-effective, automatted strip test has developed for screening of inhibitors of endonuclease EcoRV. The method involves the use of GO and a DNA substrate for EcoRV that contains both an ssDNA region for binding of GO and a fluorescein amidite (FAM)-labelled dsDNA. All the components were inkjet printed on a piece of parchment paper. The ssDNA region binds to the surface of GO and anchors so that the fluorescence of FAM is quenched. The parchment paper strip is then incubated with a sample containing EcoRV which causes enzymatic hydrolysis, and dsDNA was separated from the GO. As a result, green fluorescence is generated at the reaction spot. Enzyme activity can be measured in the presence and absence of aurintricarboxy acid acting as an EcoRV inhibitor. This method excels by its need for 2-3 orders less reagents compared to the standard well plate assay. Thus, it is an efficient platform for GO-based screening of EcoRV enzyme inhibitors. Graphical abstract A graphene oxide (GO)-based endonuclease EcoRV inhibition FRET assay using inkjet printing was developed. Printing of GO along with assay reagents has a beneficial effect on the enzymatic reaction on paper. This method was successfully applied to evaluate EcoRV inhibitor activity.

Targeting Allostery with Avatars to Design Inhibitors Assessed by Cell Activity: Dissecting MRE11 Endo- and Exonuclease Activities.

For inhibitor design, as in most research, the best system is question dependent. We suggest structurally defined allostery to design specific inhibitors that target regions beyond active sites. We choose systems allowing efficient quality structures with conformational changes as optimal for structure-based design to optimize inhibitors. We maintain that evolutionarily related targets logically provide molecular avatars, where this Sanskrit term for descent includes ideas of functional relationships and of being a physical embodiment of the target's essential features without requiring high sequence identity. Appropriate biochemical and cell assays provide quantitative measurements, and for biomedical impacts, any inhibitor's activity should be validated in human cells. Specificity is effectively shown empirically by testing if mutations blocking target activity remove cellular inhibitor impact. We propose this approach to be superior to experiments testing for lack of cross-reactivity among possible related enzymes, which is a challenging negative experiment. As an exemplary avatar system for protein and DNA allosteric conformational controls, we focus here on developing separation-of-function inhibitors for meiotic recombination 11 nuclease activities. This was achieved not by targeting the active site but rather by geometrically impacting loop motifs analogously to ribosome antibiotics. These loops are neighboring the dimer interface and active site act in sculpting dsDNA and ssDNA into catalytically competent complexes. One of our design constraints is to preserve DNA substrate binding to geometrically block competing enzymes and pathways from the damaged site. We validate our allosteric approach to controlling outcomes in human cells by reversing the radiation sensitivity and genomic instability in BRCA mutant cells.

The mitochondrial outer-membrane location of the EXD2 exonuclease contradicts its direct role in nuclear DNA repair.

EXD2 is a recently identified exonuclease that has been implicated in nuclear double-strand break repair. Given our long standing interest in mitochondrial DNA maintenance and indications that EXD2 could also be a mitochondrial protein we sought to determine its cellular localization and possible mitochondrial associated functions. Our results show that EXD2 indeed shows mitochondrial localization, but, surprisingly, is found predominantly associated with the mitochondrial outer-membrane. Gradient purified nuclei show only the faintest hint of EXD2 presence while overexpression of the predicted full-length protein shows exclusive mitochondrial localization. Importantly, induction of double-strand DNA breaks via X-irradiation or Zeocin treatment does not support the notion that EXD2 re-locates to the nucleus following double-strand breaks and thus is unlikely to have a direct role in nuclear DNA repair. Knockdown or overexpression of EXD2 affects the cellular distribution of mitochondria. These results suggest that the reported defects in nuclear DNA repair following EXD2 depletion are likely an indirect consequence of altered mitochondrial dynamics and/or function.

Enhancing multiplex genome editing by natural transformation (MuGENT) via inactivation of ssDNA exonucleases.

Recently, we described a method for multiplex genome editing by natural transformation (MuGENT). Mutant constructs for MuGENT require large arms of homology (>2000 bp) surrounding each genome edit, which necessitates laborious in vitro DNA splicing. In Vibrio cholerae, we uncover that this requirement is due to cytoplasmic ssDNA exonucleases, which inhibit natural transformation. In ssDNA exonuclease mutants, one arm of homology can be reduced to as little as 40 bp while still promoting integration of genome edits at rates of ∼50% without selection in cis. Consequently, editing constructs are generated in a single polymerase chain reaction where one homology arm is oligonucleotide encoded. To further enhance editing efficiencies, we also developed a strain for transient inactivation of the mismatch repair system. As a proof-of-concept, we used these advances to rapidly mutate 10 high-affinity binding sites for the nucleoid occlusion protein SlmA and generated a duodecuple mutant of 12 diguanylate cyclases in V. cholerae. Whole genome sequencing revealed little to no off-target mutations in these strains. Finally, we show that ssDNA exonucleases inhibit natural transformation in Acinetobacter baylyi. Thus, rational removal of ssDNA exonucleases may be broadly applicable for enhancing the efficacy and ease of MuGENT in diverse naturally transformable species.

The phosphodiesterase type 2 inhibitor BAY 60-7550 reverses functional impairments induced by brain ischemia by decreasing hippocampal neurodegeneration and enhancing hippocampal neuronal plasticity.

Cognitive and affective impairments are the most characterized consequences following cerebral ischemia. BAY 60-7550, a selective phosphodiesterase type 2 inhibitor (PDE2-I), presents memory-enhancing and anxiolytic-like properties. The behavioral effects of BAY 60-7550 have been associated with its ability to prevent hydrolysis of both cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) thereby interfering with neuronal plasticity. Here, we hypothesize that PDE2-I treatment could promote functional recovery after brain ischemia. Mice C57Bl/6 were submitted to bilateral common carotid artery occlusion (BCCAO), an experimental model of transient brain ischemia, for 20 min. During 21 days after reperfusion, the animals were tested in a battery of behavioral tests including the elevated zero maze (EZM), object location task (OLT) and forced swim test (FST). The effects of BAY 60-7550 were evaluated on neuronal nuclei (NeuN), caspase-9, cAMP response element-binding protein (CREB), phosphorylated CREB (pCREB) and brain-derived neurotrophic factor (BDNF) expression in the hippocampus. BCCAO increased anxiety levels, impaired hippocampus-dependent cognitive function and induced despair-like behavior in mice. Hippocampal neurodegeneration was evidenced by a decrease in NeuN and increase incaspase-9 protein levels in BCCAO mice. Ischemic mice also showed low BDNF protein levels in the hippocampus. Repeated treatment with BAY 60-7550 attenuated the behavioral impairments induced by BCCAO in mice. Concomitantly, BAY 60-7550 enhanced expression of pCREB and BDNF protein levels in the hippocampus of ischemic mice. The present findings suggest that chronic inhibition of PDE2 provides functional recovery in BCCAO mice possibly by augmenting hippocampal neuronal plasticity.

Identification of Inhibitors for the DEDDh Family of Exonucleases and a Unique Inhibition Mechanism by Crystal Structure Analysis of CRN-4 Bound with 2-Morpholin-4-ylethanesulfonate (MES).

The DEDDh family of exonucleases plays essential roles in DNA and RNA metabolism in all kingdoms of life. Several viral and human DEDDh exonucleases can serve as antiviral drug targets due to their critical roles in virus replication. Here using RNase T and CRN-4 as the model systems, we identify potential inhibitors for DEDDh exonucleases. We further show that two of the inhibitors, ATA and PV6R, indeed inhibit the exonuclease activity of the viral protein NP exonuclease of Lassa fever virus in vitro. Moreover, we determine the crystal structure of CRN-4 in complex with MES that reveals a unique inhibition mechanism by inducing the general base His179 to shift out of the active site. Our results not only provide the structural basis for the inhibition mechanism but also suggest potential lead inhibitors for the DEDDh exonucleases that may pave the way for designing nuclease inhibitors for biochemical and biomedical applications.

Precursors of tRNAs are stabilized by methylguanosine cap structures.

Efficient maturation of transfer RNAs (tRNAs) is required for rapid cell growth. However, the precise timing of tRNA processing in coordination with the order of tRNA modifications has not been thoroughly elucidated. To analyze the modification status of tRNA precursors (pre-tRNAs) during maturation, we isolated pre-tRNAs at various stages from Saccharomyces cerevisiae and subjected them to MS analysis. We detected methylated guanosine cap structures at the 5' termini of pre-tRNAs bearing 5' leader sequences. These capped pre-tRNAs accumulated substantially after inhibition of RNase P activity. Upon depletion of the capping enzyme Ceg1p, the steady state level of capped pre-tRNA was markedly reduced. In addition, a population of capped pre-tRNAs accumulated in strains in which 5' exonucleases were inhibited, indicating that the 5' cap structures protect pre-tRNAs from 5'-exonucleolytic degradation during maturation.

Hyperthermia inhibits recombination repair of gemcitabine-stalled replication forks.

BACKGROUND: Gemcitabine is a potent nucleoside analogue against solid tumors, but development of drug resistance is a substantial problem. Removal of gemcitabine incorporated into DNA by repair mechanisms may contribute to resistance in chemo-refractory solid tumors. Human hepatocellular carcinoma (HCC) is usually very chemoresistant to gemcitabine. METHODS: We treated HCC in vitro and in vivo (orthotopic murine model with human Hep3B or HepG2 xenografts, 7-10 CB17SCID mice per group) with gemcitabine. The role of homologous recombination repair proteins in repairing stalled replication forks was evaluated with hyperthermia exposure and cell-cycle analysis. The Student t-test was used for two-sample comparisons. Multiple group data were analyzed using one-way analysis of variance. All statistical tests were two-sided. RESULTS: We demonstrated that Mre11-mediated homologous recombination repair of gemcitabine-stalled replication forks is crucial to survival of HCC cells. Furthermore, we demonstrated inhibition of Mre11 by an exonuclease inhibitor or concomitant hyperthermia. In orthotopic murine models of chemoresistant HCC, the Hep3B tumor mass with radiofrequency plus gemcitabine treatment (mean ± SD, 180±91mg) was statistically significantly smaller compared with gemcitabine alone (661±419mg, P = .0063). CONCLUSIONS: This study provides mechanistic understanding of homologous recombination inhibiting-strategies, such as noninvasive radiofrequency field-induced hyperthermia, to overcome resistance to gemcitabine in refractory human solid tumors.

KREX2 is not essential for either procyclic or bloodstream form Trypanosoma brucei.

BACKGROUND: Most mitochondrial mRNAs in Trypanosoma brucei require RNA editing for maturation and translation. The edited RNAs primarily encode proteins of the oxidative phosphorylation system. These parasites undergo extensive changes in energy metabolism between the insect and bloodstream stages which are mirrored by alterations in RNA editing. Two U-specific exonucleases, KREX1 and KREX2, are both present in protein complexes (editosomes) that catalyze RNA editing but the relative roles of each protein are not known. METHODOLOGY/PRINCIPAL FINDINGS: The requirement for KREX2 for RNA editing in vivo was assessed in both procyclic (insect) and bloodstream form parasites by methods that use homologous recombination for gene elimination. These studies resulted in null mutant cells in which both alleles were eliminated. The viability of these cells demonstrates that KREX2 is not essential in either life cycle stage, despite certain defects in RNA editing in vivo. Furthermore, editosomes isolated from KREX2 null cells require KREX1 for in vitro U-specific exonuclease activity. CONCLUSIONS: KREX2 is a U-specific exonuclease that is dispensable for RNA editing in vivo in T. brucei BFs and PFs. This result suggests that the U deletion activity, which is required for RNA editing, is primarily mediated in vivo by KREX1 which is normally found associated with only one type of editosome. The retention of the KREX2 gene implies a non-essential role or a role that is essential in other life cycle stages or conditions.

Sensitive and rapid screening of T4 polynucleotide kinase activity and inhibition based on coupled exonuclease reaction and graphene oxide platform.

Phosphorylation of DNA with 5'-hydroxyl termini plays a critical role in a majority of normal cellular events, including DNA recombination, DNA replication, and repair of DNA during strand interruption. Determination of nucleotide kinase activity and inhibition is under intense development due to its importance in regulating nucleic acid metabolism. Here, by using T4 polynucleotide kinase (PNK) as a model, which plays an essential role in cellular nucleic acid metabolism, particularly in the cellular responses to DNA damage, we describe a strategy for simply and accurately determining nucleotide kinase activity and inhibition by means of a coupled λ exonuclease cleavage reaction and graphene oxide (GO) based platform. The dye attached dsDNA preserves most of the fluorescence when mixed with GO. While dsDNA is phosphorylated by PNK and then immediately cleaved by λ exonuclease, fluorescence is greatly quenched. Because of the super quenching ability and the high specific surface area of GO, the as-proposed platform presents an excellent performance with wide linear range and low detection limit in the cell extracts environment. Additionally, inhibition effects of adenosine diphosphate, ammonium sulfate, and sodium hydrogen phosphate have also been investigated. The method not only provides a universal platform for monitoring activity and inhibition of nucleotide kinase but also shows great potential in biological process researches, drug discovery, and clinic diagnostics.

Computational techniques are valuable tools for the discovery of protein-protein interaction inhibitors: the 14-3-3σ case.

Targeting the binding site of 14-3-3 proteins lets the release of partner proteins involved in cell cycle progression, apoptosis, cytoskeletal rearrangement and transcriptional regulation and may therefore be regarded as an alternative strategy to integrate conventional therapeutic approaches against cancer. In the present work, we report the identification of two new small molecule inhibitors of 14-3-3σ/c-Abl protein-protein interaction (BV01 and BV101) discovered by means of computational methods. The most interesting compound (BV01) showed a lethal dose (LD(50)) in the low micromolar range against Ba/F3 murine cell lines expressing the Imatinib (IM)-sensitive wild type Bcr-Abl construct and the IM-resistant Bcr-Abl mutation T315I. BV01 interaction with 14-3-3σ was demonstrated by NMR studies and elucidated by docking. It blocked the binding domain of 14-3-3σ, hence promoting the release of the partner protein c-Abl (the one not involved in Bcr rearrangement), and its translocation to both the nuclear compartment and mitochondrial membranes to induce a pro-apoptotic response. Our results advance BV01 as a confirmed hit compound capable of eliciting apoptotic death of Bcr-Abl-expressing cells by interfering with 14-3-3σ/c-Abl protein-protein interaction.

Anxiolytic effects of phosphodiesterase-2 inhibitors associated with increased cGMP signaling.

Phosphodiesterase (PDE)-2 is a component of the nitric-oxide synthase (NOS)/guanylyl cyclase signaling pathway in the brain. Given recent evidence that pharmacologically induced changes in NO-cGMP signaling can affect anxiety-related behaviors, the effects of the PDE2 inhibitors (2-(3,4-dimethoxybenzyl)-7-det-5-methylimidazo-[5,1-f][1,2,4]triazin-4(3H)-one) (Bay 60-7550) and 3-(8-methoxy-1-methyl-2-oxo-7-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-5-yl)benzamide (ND7001), as well as modulators of NO, were assessed on cGMP signaling in neurons and on the behavior of mice in the elevated plus-maze, hole-board, and open-field tests, well established procedures for the evaluation of anxiolytics. Bay 60-7550 (1 microM) and ND7001 (10 microM) increased basal and N-methyl-d-aspartate- or detanonoate-stimulated cGMP in primary cultures of rat cerebral cortical neurons; Bay 60-7550, but not ND7001, also increased cAMP. Increased cGMP signaling, either by administration of the PDE2 inhibitors Bay 60-7550 (0.5, 1, and 3 mg/kg) or ND7001 (1 mg/kg), or the NO donor detanonoate (0.5 mg/kg), antagonized the anxiogenic effects of restraint stress on behavior in the three tests. These drugs also produced anxiolytic effects on behavior in nonstressed mice in the elevated plus-maze and hole-board tests; these effects were antagonized by the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (20 mg/kg). By contrast, the NOS inhibitor N(omega)-nitro-l-arginine methyl ester (50 mg/kg), which reduces cGMP signaling, produced anxiogenic effects similar to restraint stress. Overall, the present behavioral and neurochemical data suggest that PDE2 may be a novel pharmacological target for the development of drugs for the treatment of anxiety disorders.

Characterization of a novel non-specific nuclease from thermophilic bacteriophage GBSV1.

BACKGROUND: Thermostable enzymes from thermophiles have attracted extensive studies. In this investigation, a nuclease-encoding gene (designated as GBSV1-NSN) was obtained from a thermophilic bacteriophage GBSV1 for the first time. RESULTS: After recombinant expression in Escherichia coli, the purified GBSV1-NSN exhibited non-specific nuclease activity, being able to degrade various nucleic acids, including RNA, single-stranded DNA and double-stranded DNA that was circular or linear. Based on sequence analysis, the nuclease shared no homology with any known nucleases, suggesting that it was a novel nuclease. The characterization of the recombinant GBSV1-NSN showed that its optimal temperature and pH were 60 degrees C and 7.5, respectively. The results indicated that the enzymatic activity was inhibited by enzyme inhibitors or detergents, such as ethylene diamine tetraacetic acid, citrate, dithiothreitol, beta-mercaptoethanol, guanidine hydrochloride, urea and SDS. In contrast, the nuclease activity was enhanced by TritonX-100, Tween-20 or chaps to approximately 124.5% - 141.6%. The Km of GBSV1-NSN nuclease was 231, 61 and 92 microM, while its kcat was 1278, 241 and 300 s-1 for the cleavage of dsDNA, ssDNA and RNA, respectively. CONCLUSION: Our study, therefore, presented a novel thermostable non-specific nuclease from thermophilic bacteriophage and its overexpression and purification for scientific research and applications.