Characterization of TK1646, a highly thermostable 3'-5' single strand specific exonuclease from Thermococcus kodakarensis.

Exonucleases catalyze the hydrolysis of terminal phosphodiester bond in nucleic acid. They play important role in maintaining the integrity of DNA in eukaryotes, prokaryotes and archaea. Limited studies have been done on archaeal exonucleases. Here we report molecular cloning of TK1646, a putative exonuclease from the hyperthermophilic archaeon Thermococcus kodakarensis, and expression of the gene in Escherichia coli. Recombinant TK1646, produced in soluble and active form, was purified to apparent homogeneity. Characterization of the recombinant enzyme indicated that it was single strand specific 3'-5' exonuclease which cleaved the substrate DNA after every two nucleotides. It exhibited highest activity at 85-100 °C and pH 9.0. Unique property of TK1646 was its thermostability as it maintained its activity even at 100 °C with a half-life of 180 min. Recombinant TK1646 followed Michaelis-Menten kinetics and exhibited apparent Km and Vmax values of 33 ±â€¯4 µM and 812 ±â€¯48 nmol/min/mg, respectively. To the best of our knowledge this is the most thermostable single strand specific 3'-5' exonuclease characterized to date.

Realizing directional cloning using sticky ends produced by 3'-5' exonuclease of Klenow fragment.

The Klenow fragment (KF) has been used to make the blunt end as a tool enzyme. Its 5'-3' polymerase activity can extend the 5' overhanging sticky end to the blunt end, and 3'-5' exonuclease activity can cleave the 3' overhanging sticky end to the blunt end. The blunt end is useful for cloning. Here, we for the first time determined that a sticky end can be made by using the 3'-5' exonuclease activity of KF. We found that KF can cleave the blunt end into certain sticky ends under controlled conditions. We optimized enzyme cleavage conditions, and characterized the cleaved sticky ends to be mainly 2 nt 5' overhang. By using these sticky ends, we realized ligation reaction in vitro, and accomplished cloning short oligonucleotides directionally with high cloning efficiency. In some cases, this method can provide sticky end fragments in large scale for subsequent convenient cloning at low cost.

A novel single-strand specific 3'-5' exonuclease found in the hyperthermophilic archaeon, Pyrococcus furiosus.

Nucleases play important roles in all DNA transactions, including replication, repair, and recombination. Many different nucleases from bacterial and eukaryotic organisms have been identified and functionally characterized. However, our knowledge about the nucleases from Archaea, the third domain of life, is still limited. We searched for 3'-5' exonuclease activity in the hyperthermophilic archaeon, Pyrococcus furiosus, and identified a protein with the target activity. The purified protein, encoded by PF2046, is composed of 229 amino acids with a molecular weight of 25,596, and displayed single-strand specific 3'-5' exonuclease activity. The protein, designated as PfuExo I, forms a stable trimeric complex in solution and excises the DNA at every two nucleotides from the 3' to 5' direction. The amino acid sequence of this protein is conserved only in Thermococci, one of the hyperthermophilic classes in the Euryarchaeota subdomain in Archaea. The newly discovered exonuclease lacks similarity to any other proteins with known function, including hitherto reported 3'-5' exonucleases. This novel nuclease may be involved in a DNA repair pathway conserved in the living organisms as a specific member for some hyperthermophilic archaea.

Streptomyces nucleases.

Streptomyces nucleases are widely distributed and multifunctional enzymes acting on both DNA and RNA. They occur extra as well as intracellularly and can be classified under sugar specific and sugar non-specific nucleases. Nucleases play different roles like analytical, biological, and nutritional. They are also used in programmed cell death. Although more than 20 nucleases are reported to date, very little information is available regarding their structure-function relationship, active site based sequence homology, and the probable mechanism of action. This review describes the history, occurrence, localization, production, purification, properties, and applications of Streptomyces nucleases.

Yeast exonuclease 5 is essential for mitochondrial genome maintenance.

Yeast exonuclease 5 is encoded by the YBR163w (DEM1) gene, and this gene has been renamed EXO5. It is distantly related to the Escherichia coli RecB exonuclease class. Exo5 is localized to the mitochondria, and EXO5 deletions or nuclease-defective EXO5 mutants invariably yield petites, amplifying either the ori3 or ori5 region of the mitochondrial genome. These petites remain unstable and undergo continuous rearrangement. The mitochondrial phenotype of exo5Delta strains suggests an essential role for the enzyme in DNA replication and recombination. No nuclear phenotype associated with EXO5 deletions has been detected. Exo5 is a monomeric 5' exonuclease that releases dinucleotides as products. It is specific for single-stranded DNA and does not hydrolyze RNA. However, Exo5 has the capacity to slide across 5' double-stranded DNA or 5' RNA sequences and resumes cutting two nucleotides downstream of the double-stranded-to-single-stranded junction or RNA-to-DNA junction, respectively.

Characterization and disruption of exonuclease genes from Streptomyces aureofaciens B96 and S. coelicolor A3(2).

Streptomyces aureofaciens B96 produces several intra- and extracellular enzymes with deoxyribonuclease activity. According to the sequence of the previously published gene exoSc from S. coelicolor A3(2), the DNA sequence from S. aureofaciens B96 was amplified, cloned and expressed in E. coli. The protein product of exoSa gene, recExoSa, was also an exonuclease with DNAase and 5'-phosphomonoesterase activities at optimum temperature 37 degrees C and pH 8.0. It degraded only linear DNA (chromosomal, double-stranded and single-stranded) and linear plasmid DNA from both ends, with a preference to blunt ends in comparison with overhang ends. The purified enzyme exhibited no RNAase activity. Both exoSc and exoSa genes were interrupted by the apramycin resistance gene; constructed fragments were transformed into particular streptomyces protoplasts. Mutation caused by exoSa disruption in S. aureofaciens chromosome and mutation by interrupted exoSc in S. coelicolor were lethal.

Reconstitution of RNA exosomes from human and Saccharomyces cerevisiae cloning, expression, purification, and activity assays.

Eukaryotic RNA exosomes participate in 3' to 5'-processing and degradation of RNA in the nucleus and cytoplasm. RNA exosomes are multisubunit complexes composed of at least nine distinct proteins that form the exosome core. Although the eukaryotic exosome core shares structural and sequence similarity to phosphorolytic archaeal exosomes and bacterial PNPase, the eukaryotic exosome core has diverged from its archaeal and bacterial cousins and appears devoid of phosphorolytic activity. In yeast, the processive hydrolytic 3' to 5'-exoribonuclease Rrp44 associates with exosomes in the nucleus and cytoplasm. Although human Rrp44 appears homologous to yeast Rrp44, it has not yet been shown to associate with human exosomes. In the nucleus, eukaryotic exosomes interact with Rrp6, a distributive hydrolytic 3' to 5'-exoribonuclease. To facilitate analysis of eukaryotic RNA exosomes, we will describe procedures used to clone, express, purify, and reconstitute the nine-subunit human exosome and nine-, ten-, and eleven-subunit yeast exosomes. We will also discuss procedures to assess exoribonuclease activity for reconstituted exosomes.

Determination of DNA polymerase and nuclease activities of DNA-dependent polymerases using fluorescence detection under real-time conditions.

A new method is proposed for estimation of polymerase activities using fluorescence detection during isothermal reaction. The method allows simultaneous determination of DNA-dependent DNA polymerase and 5'-3'-exonuclease activities using amplifiers supplied with an optical module for fluorescence detection under real-time conditions. Different primer-template combinations used as polymerase substrates were compared. Primer elongation (polymerase reaction) is detected by changes in SYBR Green I fluorescence upon binding to dsDNA during reaction; nuclease activities are detected by changes in fluorescence due to cleavage of the probe, containing the reporter fluorophore and fluorescence quencher, and hybridized in advance to the template single-stranded region. It was also shown that the method can be used for determination of relative activities of DNA polymerase preparations, estimation of temperature-time dissociation parameters of polymerase complexes with specific antibodies to its active center, and analysis of effects of inhibitors and activators of different nature on reaction rates of dsDNA polymerization and 5'-3'-exonuclease cleavage by polymerase. The method can be also used for estimation of endonuclease activities of DNA polymerases.

The human homolog of fission yeast Rad17 is implicated in tumor growth.

The Schizosaccharomyces pombe rad17 is a checkpoint protein critical for maintenance of genomic stability. Since the loss of checkpoint control is a common feature of tumor cells, we investigated the biological function of the human homolog hRAD17. Expression of hRAD17 in a fission yeast rad17 deleted strain reduced growth of yeast colonies and caused slower progression through cell cycle. Immunoprecipitated hRad17 exhibited exonuclease activity. hRAD17 delayed growth of NIH3T3 fibroblasts transformed by the H-ras oncogene in nude mice. Our results support that hRAD17, similarly to other human genes involved in checkpoint mechanisms, plays a role in control of tumor growth.

Tri-cistronic cloning, overexpression and purification of human Rad9, Rad1, Hus1 protein complex.

The least understood components of the DNA damage checkpoint are the DNA damage sensors. Genetic studies of Schizosaccharomyces pombe identified six yeast genes, Rad3, Rad17, Rad9, Rad1, Hus1, and Rad26, which encode proteins thought to sense DNA damage and activate the checkpoint-signaling cascade. It has been suggested that Rad9, Rad1 and Hus1 make a heterotrimeric complex forming a PCNA-like structure. In order to carry out structural and biophysical studies of the complex and its associated proteins, the cDNAs encoding full length human Rad9, Rad1 and Hus1 were cloned together into the pET28a vector using a one-step ligation procedure. Here we report successful tri-cistronic cloning, overexpression and purification of this three-protein complex using a single hexa-histidine tag. The trimeric protein complex of Rad9, Rad1 and Hus1 was purified to near homogeneity, yielding approximately 10mg of protein from one liter of Escherichia coli culture.

Mycoplasma hyopneumoniae mhp379 is a Ca2+-dependent, sugar-nonspecific exonuclease exposed on the cell surface.

Mycoplasma hyopneumoniae mhp379 is a putative lipoprotein that shares significant amino acid sequence similarity with a family of bacterial thermostable nucleases. To examine the nuclease activity of mhp379, the gene was cloned and expressed in Escherichia coli following the deletion of the amino-terminal signal sequence and prokaryotic lipoprotein cleavage site and mutagenesis of the mycoplasma TGA tryptophan codons to TGG. The recombinant fusion protein yielded a 33-kDa thrombin cleavage product, corresponding in size to the mature mhp379 protein. Exonuclease activity was indicated by agarose gel electrophoresis analysis of the reaction products that were released when different nucleic acid substrates were used. Endonuclease activity was also indicated by the digestion of closed circular plasmid DNA. The recombinant mhp379 fusion protein completely digested single-stranded DNA, double-stranded DNA (dsDNA), and RNA. The optimal reaction conditions were determined with a novel nuclease assay based on the enhancement of fluorescence of SYBR green I bound to dsDNA. Optimal activity was observed in the presence of calcium ions at a concentration of 15 mM and a pH of 9.5. No nuclease activity was observed in the absence of calcium ions. Mycoplasmas do not have the ability to synthesize nucleic acid precursors, and thus, nucleases are likely to be important in the acquisition of precursors for the synthesis of nucleic acids. Homologs of an ATP-binding cassette (ABC) transport system were identified immediately downstream of the gene encoding mhp379, and two homologs of M. pneumoniae lipoprotein multigene family 2 were also identified immediately upstream. Homologs of mhp379 were identified in the sequenced genomes of a number of mycoplasma species, and in most cases the homologous ABC transport system was identified immediately downstream of the homologous gene; in several cases a homolog of M. pneumoniae lipoprotein multigene family 2 was also identified immediately upstream. These observations suggest that mhp379 comprises part of a conserved ABC transport operon in mycoplasmas and that the exonuclease activity of mhp379 may be associated with the conserved function of the ABC transport system in the import of nucleic acid precursors. This is the first study to identify the gene and characterize the activity of a mycoplasma exonuclease.

A versatile endonuclease IV from Thermus thermophilus has uracil-excising and 3'-5' exonuclease activity.

Apurinic/apyrimidinic (AP) sites arise in DNA through the spontaneous loss of bases or through the release of damaged bases from DNA by DNA glycosylases. AP sites in DNA can be catalyzed by AP endonucleases such as exonuclease III and endonuclease IV, generating a 3'-hydroxyl group and a 5'-terminal sugar phosphate. Here, we have identified and characterized a novel endonuclease IV from a hyperthermophilic bacterium Thermus thermophilus designated as TthNfo. TthNfo efficiently removed AP site from double-stranded oligonucleotide substrate. No significant difference was observed in the rate of reaction of four bases opposite AP site with TthNfo. In addition, TthNfo possesses a 3'-5' exonuclease activity similar to that of Escherichia coli exonuclease III. Surprisingly, we found that TthNfo also catalyzes the excision of uracil from DNA. In comparison with other endonuclease IV proteins, the removal of uracil residue was unique to TthNfo. Based on these observations and the absence of exonuclease III in T. thermophilus, we suggest that versatile enzyme activities of TthNfo play an important role in counteracting DNA base damage in vivo.

Fibroblast extracellular matrix gene expression in response to keratinocyte-releasable stratifin.

Termination of wound-healing process requires a fine balance between connective tissue deposition and its hydrolysis. Previously, we have demonstrated that keratinocyte-releasable stratifin, also known as 14-3-3 sigma protein, stimulates collagenase (MMP-1) expression in dermal fibroblasts. However, role of extracellular stratifin in regulation of extracellular matrix (ECM) factors and other matrix metalloproteinases (MMPs) in dermal fibroblast remains unexplored. To address this question, large-scale ECM gene expression profile were analyzed in human dermal fibroblasts co-cultured with keratinocytes or treated with recombinant stratifin. Superarray pathway-specific microarrays were utilized to identify upregulation or downregulation of 96 human ECM and adhesion molecule genes. RT-PCR and Western blot were used to validate microarray expression profiles of selected genes. Comparison of gene profiles with the appropriate controls showed a significant (more than twofold) increase in expression of collagenase-1, stromelysin-1 and -2, neutrophil collagenase, and membrane type 5 MMP in dermal fibroblasts treated with stratifin or co-cultured with keratinocytes. Expression of type I collagen and fibronectin genes decreased in the same fibroblasts. The results of a dose-response experiment showed that stratifin stimulates the expression of stromelysin-1 (MMP-3) mRNA by dermal fibroblasts in a concentration-dependent fashion. Furthermore, Western blot analysis of fibroblast-conditioned medium showed a peak in MMP-3 protein levels 48 h following treatment with recombinant stratifin. In a lasting-effect study, MMP-3 protein was detected in fibroblast-condition medium for up to 72 h post removal of stratifin. In conclusion, our results suggest that keratinocyte-releasable stratifin plays a major role in induction of ECM degradation by dermal fibroblasts through stimulation of key MMPs, such as MMP-1 and MMP-3. Therefore, stratifin protein may prove to be a useful target for clinical intervention in controlling excessive wound healing in fibrotic conditions.

Purification and characterization of an endo-exonuclease from Podospora anserina mitochondria.

The senescence phenotype of Podospora anserina wild-type strains depends on mitochondrial (mt) genome stability. Characterization of activities implicated in the maintenance of the mt DNA is therefore essential for a better understanding of these degenerative processes. To address this question we looked for a nuclease activity in this fungal mitochondria. Here we describe the purification of an endo-exonuclease active on single-stranded, double-stranded and flap DNA. The Podospora nuclease also possesses an RNase H activity. Gel filtration chromatography showed a native molecular mass of 90 kDa for the P. anserina enzyme. The highly purified fraction shows a single polypeptide chain of 49 kDa on SDS-PAGE, indicating that the Podospora enzyme is probably active as a dimer. Purification and sequencing of the endolysine digestion peptides of the Podospora mt nuclease suggested that this enzyme could belong to the 5' structure-specific endo-exonuclease family. The possible involvement of this nuclease in mt DNA recombination during the senescence process is evoked.

Purification and characterization of human DNA damage checkpoint Rad complexes.

Checkpoint Rad proteins function early in the DNA damage checkpoint signaling cascade to arrest cell cycle progression in response to DNA damage. This checkpoint ensures the transmission of an intact genetic complement to daughter cells. To learn about the damage sensor function of the human checkpoint Rad proteins, we purified a heteropentameric complex composed of hRad17-RFCp36-RFCp37-RFCp38-RFCp40 (hRad17-RFC) and a heterotrimeric complex composed of hRad9-hHus1-hRad1 (checkpoint 9-1-1 complex). hRad17-RFC binds to DNA, with a preference for primed DNA and possesses weak ATPase activity that is stimulated by primed DNA and single-stranded DNA. hRad17-RFC forms a complex with the 9-1-1 heterotrimer reminiscent of the replication factor C/proliferating cell nuclear antigen clamp loader/sliding clamp complex of the replication machinery. These findings constitute biochemical support for models regarding the roles of checkpoint Rads as damage sensors in the DNA damage checkpoint response of human cells.

Cloning, expression, and characterization of DNA polymerase I from the hyperthermophilic archaea Thermococcus fumicolans.

The DNA polymerase I gene of a newly described deep-sea hydrothermal vent Archaea species, Thermococcus fumicolans, from IFREMERS's collection of hyperthermophiles has been cloned in Escherichia coli. As in Thermococcus litoralis, the gene is split by two intervening sequences (IVS) encoding inteins inserted in sites A and C of family B DNA polymerases. The entire DNA polymerase gene, containing both inteins, was expressed at 30 degrees C in E. coli strain BL21(DE3)pLysS using the pARHS2 expression vector. The native polypeptide precursor of 170kDa was obtained, and intein splicing as well as ligation of the three exteins was observed in vitro after heat exposure. The recombinant enzyme was purified and some of its activities were characterized: polymerization, thermostability, exonuclease activities, and fidelity.

Rabbit corpus cavernosum smooth muscle shows a different phosphodiesterase profile than human corpus cavernosum.

PURPOSE: Cyclic nucleotide phosphodiesterases (PDEs) are important regulators of cAMP/cGMP secondary messenger systems. Fluctuations in the level of cyclic nucleotides control the smooth muscle tone of corpus cavernosum. It had been shown that milrinone, a PDE3 inhibitor, was as potent as sildenafil, a PDE5 inhibitor, in relaxing human corpus cavernosum. However, milrinone is much less effective in relaxing rabbit corpus cavernosum than sildenafil. PDEs in rabbit corpus cavernosum were characterized and organ bath experiments were carried out in an attempt to search for the biochemical basis of this species difference. MATERIALS AND METHODS: In a biochemical study, PDE isozymes from rabbit corpus cavernosum were isolated by FPLC and characterized by PDE assay. In organ bath experiments, rabbit corpus cavernous tissue strips were precontracted and increasing doses of various inhibitors were added. RESULTS: The major PDE in rabbit corpus cavernosum is PDE5. There are small amounts of PDE2 and PDE1. PDE3, which contributes significantly to the total PDE activity in human corpus cavernosum, is apparently lacking in rabbit corpus cavernosum. Organ bath experiments with isotype-specific inhibitors confirm this conclusion. CONCLUSION: The distribution of PDE isozymes in corpus cavernosum is different in human and in rabbit. This could be the biochemical basis for the differential effects of milrinone in relaxing rabbit and human corpus cavernosum. Our study emphasizes the importance of a more complete understanding of the tissue distribution of targeted proteins in an animal model before applying the results to humans.

Purification and characterization of the single-strand-specific and guanylic-acid-preferential deoxyribonuclease activity of the extracellular nuclease from Basidiobolus haptosporus.

An extracellular nuclease from Basidiobolus haptosporus (designated as nuclease Bh1) was purified to homogeneity by ammonium sulfate precipitation, heat treatment, negative adsorption on DEAE-cellulose, and chromatography on phenyl-Sepharose followed by FPLC on phenyl-Superose. The overall yield was 26%. The Mr of the purified enzyme, determined by gel filtration, was 41 000 whereas by SDS/PAGE (after deglycosylation) it was 30 000. It is a glycoprotein with a pI of 6.8. The optimum pH and temperature for DNA hydrolysis were 8. 5 and 60 degrees C, respectively. Nuclease Bh1 is a metalloprotein but has no obligate requirement for metal ions to be active, nor is its activity stimulated in the presence of metal ions. The enzyme was inhibited by Zn2+, Ag2+, Hg2+, Fe3+ and Al3+, inorganic phosphate, pyrophosphate, dithiothreitol, 2-mercaptoethanol, NaCl and KCl. It was stable to high concentrations of organic solvents and urea but susceptible to low concentrations of SDS and guanidine hydrochloride. Nuclease Bh1 is a multifunctional enzyme and its substrate specificity is in the order of ssDNA approximately 3'AMP >> RNA > dsDNA. Studies on its mode of action showed that it cleaved supercoiled pUC 18 DNA and phage M13 DNA, endonucleolytically, generating single base nicks. The enzyme hydrolyzed DNA with preferential liberation of 5'dGMP, suggesting it to be a guanylic acid preferential endoexonuclease. 5'dGMP, the end product of hydrolysis, was a competitive inhibitor of the enzyme. The absence of 5'dCMP as a hydrolytic product, coupled with the resistance of (dC)10 and deoxyribodinucleoside monophosphates having cytosine either at the 3' or the 5' end, indicates that C-linkages are resistant to cleavage by nuclease Bh1.

Purification, characterization and cDNA cloning of an endo-exonuclease from the basidiomycete fungus Armillaria mellea.

We have purified an endo-exonuclease from the fruiting body of the basidiomycete fungus Armillaria mellea by using an ethanol fractionation step, followed by two rounds of column chromatography. The enzyme had an apparent molecular mass of 17500 Da and was shown to exist as a monomer by gel-filtration analysis. The nuclease was active on both double-stranded and single-stranded DNA but not on RNA. It was optimally active at pH8.5 and also exhibited a significant degree of thermostability. Three bivalent metal ions, Mg2+, Co2+ and Mn2+, acted as cofactors in the catalysis. It was also inhibited by high salt concentrations: activity was completely abolished at 150 mM NaCl. The nuclease possessed both endonuclease activity on supercoiled DNA and a 3'-5' (but not a 5'-3') exonuclease activity. It generated 5'-phosphomonoesters on its products that, after a prolonged incubation, were hydrolysed to a mixture of free mononucleotides and small oligonucleotides ranging in size from two to eight bases. Elucidation of its N-terminal amino acid sequence permitted the cDNA cloning of the A. mellea nuclease via a PCR-based approach. Peptide mapping of the purified enzyme generated patterns consistent with the amino acid sequence coded for by the cloned cDNA. A BLAST search of the SwissProt database revealed that A. mellea nuclease shared significant amino acid similarity with two nucleases from Bacillus subtilis, suggesting that the three might constitute a distinct class of nucleolytic enzymes.

Overexpression, purification, and characterization of the SbcCD protein from Escherichia coli.

The sbcC and sbcD genes mediate palindrome inviability in Escherichia coli. The sbcCD operon has been cloned into the plasmid pTrc99A under the control of the strong trc promoter and introduced into a strain carrying a chromosomal deletion of sbcCD. The SbcC and SbcD polypeptides were overexpressed to 6% of total cell protein, and both polypeptides copurified in a four-step purification procedure. Purified SbcCD is a processive double-strand exonuclease that has an absolute requirement for Mn2+ and uses ATP as a preferred energy source. Gel filtration chromatography and sedimentation equilibrium analyses were used to show that the SbcC and SbcD polypeptides dissociate at some stage after purification and that this dissociation is reversed by the addition of Mn2+. We demonstrate that SbcD has the potential to form a secondary structural motif found in a number of protein phosphatases and suggest that it is a metalloprotein that contains the catalytic center of the SbcCD exonuclease.