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1.
Acta Biochim Biophys Sin (Shanghai) ; 52(12): 1413-1419, 2020 Dec 29.
Article in English | MEDLINE | ID: mdl-33201182

ABSTRACT

The first case of African swine fever (ASF) outbreak in China was reported in a suburban pig farm in Shenyang in 2018. Since then, the rapid spread and extension of ASF has become the most serious threat for the swine industry. Therefore, rapid and accurate detection of African swine fever virus (ASFV) is essential to provide effective strategies to control the disease. In this study, we developed a rapid and accurate ASFV-detection method based on the DNA endonuclease-targeted CRISPR trans reporter (DETECTR) assay. By combining recombinase polymerase amplification with CRISPR-Cas12a proteins, the DETECTR assay demonstrated a minimum detection limit of eight copies with no cross reactivity with other swine viruses. Clinical blood samples were detected by DETECTR assay and showed 100% (30/30) agreement with real-time polymerase chain reaction assay. The rapid and accurate detection of ASFV may facilitate timely eradication measures and strict sanitary procedures to control and prevent the spread of ASF.


Subject(s)
African Swine Fever Virus/genetics , African Swine Fever/diagnosis , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , Swine/blood , African Swine Fever/blood , African Swine Fever/virology , Animals , Bacterial Proteins/biosynthesis , Bacterial Proteins/isolation & purification , CRISPR-Associated Proteins/biosynthesis , CRISPR-Associated Proteins/isolation & purification , CRISPR-Cas Systems , China , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , DNA, Viral/genetics , Deoxyribonuclease I/genetics , Endodeoxyribonucleases/biosynthesis , Endodeoxyribonucleases/isolation & purification , Fluorescence , Limit of Detection , Real-Time Polymerase Chain Reaction , Recombinases/metabolism , Sensitivity and Specificity
2.
CRISPR J ; 3(6): 454-461, 2020 12.
Article in English | MEDLINE | ID: mdl-33146573

ABSTRACT

Cas12a enzymes are quickly being adopted for use in a variety of genome-editing applications. These programmable nucleases are part of adaptive microbial immune systems, the natural diversity of which has been largely unexplored. Here, we identified novel families of Type V-A CRISPR nucleases through a large-scale analysis of metagenomes collected from a variety of complex environments, and developed representatives of these systems into gene-editing platforms. The nucleases display extensive protein variation and can be programmed by a single-guide RNA with specific motifs. The majority of these enzymes are part of systems recovered from uncultivated organisms, some of which also encode a divergent Type V effector. Biochemical analysis uncovered unexpected protospacer adjacent motif diversity, indicating that these systems will facilitate a variety of genome-engineering applications. The simplicity of guide sequences and activity in human cell lines suggest utility in gene and cell therapies.


Subject(s)
Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , CRISPR-Associated Proteins/isolation & purification , CRISPR-Associated Proteins/metabolism , Endodeoxyribonucleases/isolation & purification , Endodeoxyribonucleases/metabolism , Gene Editing/methods , Bacteria/genetics , Bacterial Proteins/genetics , CRISPR-Associated Protein 9/genetics , CRISPR-Associated Proteins/genetics , CRISPR-Cas Systems/genetics , Clustered Regularly Interspaced Short Palindromic Repeats , Endodeoxyribonucleases/genetics , Endonucleases/genetics , Gene Editing/trends , Humans , Metagenomics/methods , Phylogeny , RNA, Guide, Kinetoplastida/genetics
3.
J Am Chem Soc ; 142(25): 10964-10977, 2020 06 24.
Article in English | MEDLINE | ID: mdl-32470300

ABSTRACT

Recent advances have led to numerous landmark discoveries of [4Fe4S] clusters coordinated by essential enzymes in repair, replication, and transcription across all domains of life. The cofactor has notably been challenging to observe for many nucleic acid processing enzymes due to several factors, including a weak bioinformatic signature of the coordinating cysteines and lability of the metal cofactor. To overcome these challenges, we have used sequence alignments, an anaerobic purification method, iron quantification, and UV-visible and electron paramagnetic resonance spectroscopies to investigate UvrC, the dual-incision endonuclease in the bacterial nucleotide excision repair (NER) pathway. The characteristics of UvrC are consistent with [4Fe4S] coordination with 60-70% cofactor incorporation, and additionally, we show that, bound to UvrC, the [4Fe4S] cofactor is susceptible to oxidative degradation with aggregation of apo species. Importantly, in its holo form with the cofactor bound, UvrC forms high affinity complexes with duplexed DNA substrates; the apparent dissociation constants to well-matched and damaged duplex substrates are 100 ± 20 nM and 80 ± 30 nM, respectively. This high affinity DNA binding contrasts reports made for isolated protein lacking the cofactor. Moreover, using DNA electrochemistry, we find that the cluster coordinated by UvrC is redox-active and participates in DNA-mediated charge transport chemistry with a DNA-bound midpoint potential of 90 mV vs NHE. This work highlights that the [4Fe4S] center is critical to UvrC.


Subject(s)
Endodeoxyribonucleases/chemistry , Escherichia coli Proteins/chemistry , Iron-Sulfur Proteins/chemistry , Oxygen/chemistry , Amino Acid Sequence , Cysteine/chemistry , DNA/metabolism , Endodeoxyribonucleases/isolation & purification , Endodeoxyribonucleases/metabolism , Escherichia coli/enzymology , Escherichia coli Proteins/isolation & purification , Escherichia coli Proteins/metabolism , Iron-Sulfur Proteins/isolation & purification , Iron-Sulfur Proteins/metabolism , Mutation , Oxidation-Reduction , Protein Binding
4.
Protein Expr Purif ; 160: 45-55, 2019 08.
Article in English | MEDLINE | ID: mdl-30954531

ABSTRACT

Packaging the viral genome in the head of double-stranded DNA viruses, such as bacteriophages, requires the activity of a terminase. The bacteriophage terminase consists of a small terminase subunit (TerS), which binds the viral DNA, and a large terminase subunit (TerL) that possesses the ATPase and nuclease activities for packaging the DNA in the phage head. Some phages require additional components for DNA packaging, such as the HNH endonuclease gp74 in the bacteriophage HK97. Gp74 enhances the activity of terminase-mediated digestion of the cohesive (cos) site that connects individual genomes in phage concatemeric DNA, a pre-requisite to DNA packaging, and this enhancement requires an intact HNH motif in gp74. Testing of whether gp74 alters the terminase DNA binding or enzymatic activities requires obtaining isolated samples of pure TerS and TerL, which has been challenging owing to the poor solubility of these proteins. To this end, we developed methods to obtain purified TerS and TerL proteins that are active. TerS is expressed solubly in E. coli as a fusion with SUMO, which can be removed during purification to yield a TerS nonamer (TerS9). Homogenous samples of a TerL monomer are also obtained, but the homogeneity of the sample depends on the solution conditions, as seen for other terminases. DNA binding, ATPase, and nuclease assays demonstrate that our preparations of TerS9 and TerL are functional, and that they also function with gp74. Purified TerS9 and TerL enable studies into the molecular basis by which gp74 regulates terminase activity in phage maturation.


Subject(s)
Bacteriophages/enzymology , Endodeoxyribonucleases/chemistry , Endodeoxyribonucleases/isolation & purification , Viral Proteins/chemistry , Viral Proteins/isolation & purification , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/isolation & purification , Adenosine Triphosphatases/metabolism , Bacteriophages/chemistry , Bacteriophages/genetics , Bacteriophages/physiology , DNA Packaging , Endodeoxyribonucleases/genetics , Endodeoxyribonucleases/metabolism , Escherichia coli/virology , Protein Subunits/chemistry , Protein Subunits/genetics , Protein Subunits/isolation & purification , Protein Subunits/metabolism , Viral Proteins/genetics , Viral Proteins/metabolism , Virus Assembly
5.
Acta Crystallogr F Struct Biol Commun ; 74(Pt 4): 198-204, 2018 04 01.
Article in English | MEDLINE | ID: mdl-29633967

ABSTRACT

Genome packaging is a critical step in the assembly of dsDNA bacteriophages and is carried out by a powerful molecular motor known as the large terminase. To date, wild-type structures of only two large terminase proteins are available, and more structural information is needed to understand the genome-packaging mechanism. Towards this goal, the large and small terminase proteins from bacteriophage N4, which infects the Escherichia coli K12 strain, have been cloned, expressed and purified. The purified putative large terminase protein hydrolyzes ATP, and this is enhanced in the presence of the small terminase. The large terminase protein was crystallized using the sitting-drop vapour-diffusion method and the crystal diffracted to 2.8 Šresolution using a home X-ray source. Analysis of the X-ray diffraction data showed that the crystal belonged to space group P212121, with unit-cell parameters a = 53.7, b = 93.6, c = 124.9 Å, α = ß = γ = 90°. The crystal had a solvent content of 50.2% and contained one molecule in the asymmetric unit.


Subject(s)
Bacteriophage N4/enzymology , Endodeoxyribonucleases/chemistry , Endodeoxyribonucleases/metabolism , Viral Proteins/chemistry , Viral Proteins/metabolism , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Crystallization , Crystallography, X-Ray , Endodeoxyribonucleases/isolation & purification , Models, Molecular , Protein Conformation , Sequence Homology , Viral Proteins/isolation & purification
6.
Methods Enzymol ; 600: 569-590, 2018.
Article in English | MEDLINE | ID: mdl-29458775

ABSTRACT

Holliday junctions provide a covalent link between recombining DNA molecules and need to be removed prior to chromosome segregation at mitosis. Defects in their resolution lead to mitotic catastrophe, characterized by the formation of DNA breaks and chromosome aberrations. Enzymes that resolve recombination intermediates have been identified in all forms of life, from bacteriophage, to bacteria, yeast, and humans. In higher eukaryotes, Holliday junctions are resolved by GEN1, a nuclease that is mechanistically similar to the prototypic resolvase Escherichia coli RuvC, and by the SMX trinuclease complex. Studies of these enzymes have been facilitated by the use of plasmid-sized DNA recombination intermediates made by RecA-mediated strand exchange. Here, we detail the preparation of these recombination intermediates, which resemble α-structures, and their resolution by RuvC and GEN1.


Subject(s)
DNA, Cruciform/chemistry , DNA, Single-Stranded/chemistry , Endodeoxyribonucleases/chemistry , Escherichia coli Proteins/chemistry , Holliday Junction Resolvases/chemistry , Recombinational DNA Repair , DNA, Single-Stranded/isolation & purification , Endodeoxyribonucleases/isolation & purification , Escherichia coli Proteins/isolation & purification , Holliday Junction Resolvases/isolation & purification , Isotope Labeling/instrumentation , Isotope Labeling/methods , Phosphorus Radioisotopes/chemistry
7.
PLoS Genet ; 13(2): e1006586, 2017 02.
Article in English | MEDLINE | ID: mdl-28207825

ABSTRACT

Antibiotic resistance is a serious global problem. Antibiotic resistance genes (ARG), which are widespread in environmental bacteria, can be transferred to pathogenic bacteria via horizontal gene transfer (HGT). Gut microbiomes are especially apt for the emergence and dissemination of ARG. Conjugation is the HGT route that is predominantly responsible for the spread of ARG. Little is known about conjugative elements of Gram-positive bacteria, including those of the phylum Firmicutes, which are abundantly present in gut microbiomes. A critical step in the conjugation process is the relaxase-mediated site- and strand-specific nick in the oriT region of the conjugative element. This generates a single-stranded DNA molecule that is transferred from the donor to the recipient cell via a connecting channel. Here we identified and characterized the relaxosome components oriT and the relaxase of the conjugative plasmid pLS20 of the Firmicute Bacillus subtilis. We show that the relaxase gene, named relLS20, is essential for conjugation, that it can function in trans and provide evidence that Tyr26 constitutes the active site residue. In vivo and in vitro analyses revealed that the oriT is located far upstream of the relaxase gene and that the nick site within oriT is located on the template strand of the conjugation genes. Surprisingly, the RelLS20 shows very limited similarity to known relaxases. However, more than 800 genes to which no function had been attributed so far are predicted to encode proteins showing significant similarity to RelLS20. Interestingly, these putative relaxases are encoded almost exclusively in Firmicutes bacteria. Thus, RelLS20 constitutes the prototype of a new family of relaxases. The identification of this novel relaxase family will have an important impact in different aspects of future research in the field of HGT in Gram-positive bacteria in general, and specifically in the phylum of Firmicutes, and in gut microbiome research.


Subject(s)
Bacterial Proteins/genetics , Conjugation, Genetic , Drug Resistance, Bacterial/genetics , Endodeoxyribonucleases/genetics , Firmicutes/enzymology , Amino Acid Sequence , Bacillus subtilis/enzymology , DNA, Single-Stranded/genetics , Endodeoxyribonucleases/isolation & purification , Firmicutes/genetics , Gastrointestinal Microbiome/genetics , Gene Transfer, Horizontal , Humans , Plasmids/genetics
8.
Virology ; 496: 67-76, 2016 09.
Article in English | MEDLINE | ID: mdl-27261891

ABSTRACT

We demonstrate that the nucleocapsid protein of Sin Nombre hantavirus (SNV-N) has a DNA-specific endonuclease activity. Upon incubation of SNV-N with DNA in the presence of magnesium or manganese, we observed DNA digestion in sequence-unspecific manner. In contrast, RNA was not affected under the same conditions. Moreover, pre-treatment of SNV-N with RNase before DNA cleavage increased the endonucleolytic activity. Structure-based protein fold prediction using known structures from the PDB database revealed that Asp residues in positions 88 and 103 of SNV-N show sequence similarity with the active site of the restriction endonuclease HindIII. Crystal structure of HindIII predicts that residues Asp93 and Asp108 are essential for coordination of the metal ions required for HindIII DNA cleavage. Therefore, we hypothesized that homologous residues in SNV-N, Asp88 and Asp103, may have a similar function. Replacing Asp88 and Asp103 by alanine led to an SNV-N protein almost completely abrogated for endonuclease activity.


Subject(s)
DNA-Binding Proteins/metabolism , Endodeoxyribonucleases/metabolism , Metals/metabolism , Nucleocapsid Proteins/metabolism , Sin Nombre virus/physiology , Amino Acid Sequence , Cations/metabolism , Cloning, Molecular , Consensus Sequence , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , DNA-Binding Proteins/isolation & purification , Endodeoxyribonucleases/chemistry , Endodeoxyribonucleases/genetics , Endodeoxyribonucleases/isolation & purification , Enzyme Activation , Gene Expression , Models, Molecular , Molecular Conformation , Mutation , Nucleocapsid Proteins/chemistry , Nucleocapsid Proteins/genetics , Nucleocapsid Proteins/isolation & purification , Protein Binding , Recombinant Fusion Proteins , Ribonucleases/metabolism , Substrate Specificity
9.
Nucleic Acids Res ; 44(7): 2977-86, 2016 Apr 20.
Article in English | MEDLINE | ID: mdl-27001046

ABSTRACT

The common mismatch repair system processed by MutS and MutL and their homologs was identified in Bacteria and Eukarya. However, no evidence of a functional MutS/L homolog has been reported for archaeal organisms, and it is not known whether the mismatch repair system is conserved in Archaea. Here, we describe an endonuclease that cleaves double-stranded DNA containing a mismatched base pair, from the hyperthermophilic archaeon Pyrococcus furiosus The corresponding gene revealed that the activity originates from PF0012, and we named this enzyme Endonuclease MS (EndoMS) as the mismatch-specific Endonuclease. The sequence similarity suggested that EndoMS is the ortholog of NucS isolated from Pyrococcus abyssi, published previously. Biochemical characterizations of the EndoMS homolog from Thermococcus kodakarensis clearly showed that EndoMS specifically cleaves both strands of double-stranded DNA into 5'-protruding forms, with the mismatched base pair in the central position. EndoMS cleaves G/T, G/G, T/T, T/C and A/G mismatches, with a more preference for G/T, G/G and T/T, but has very little or no effect on C/C, A/C and A/A mismatches. The discovery of this endonuclease suggests the existence of a novel mismatch repair process, initiated by the double-strand break generated by the EndoMS endonuclease, in Archaea and some Bacteria.


Subject(s)
Archaeal Proteins/metabolism , Base Pair Mismatch , Endodeoxyribonucleases/metabolism , Thermococcus/enzymology , Archaeal Proteins/genetics , Archaeal Proteins/isolation & purification , DNA Cleavage , Endodeoxyribonucleases/genetics , Endodeoxyribonucleases/isolation & purification , Hot Temperature , Mutation , Proliferating Cell Nuclear Antigen/metabolism , Pyrococcus furiosus/enzymology , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Substrate Specificity
10.
Mol Microbiol ; 99(4): 700-18, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26507882

ABSTRACT

Bacterial cells are fortified against osmotic lysis by a cell wall made of peptidoglycan (PG). Synthases called penicillin-binding proteins (PBPs), the targets of penicillin and related antibiotics, polymerize the glycan strands of PG and crosslink them into the cell wall meshwork via attached peptides. The average length of glycan chains inserted into the matrix by the PBPs is thought to play an important role in bacterial morphogenesis, but polymerization termination factors controlling this process have yet to be discovered. Here, we report the identification of Escherichia coli MltG (YceG) as a potential terminase for glycan polymerization that is broadly conserved in bacteria. A clone containing mltG was initially isolated in a screen for multicopy plasmids generating a lethal phenotype in cells defective for the PG synthase PBP1b. Biochemical studies revealed that MltG is an inner membrane enzyme with endolytic transglycosylase activity capable of cleaving at internal positions within a glycan polymer. Radiolabeling experiments further demonstrated MltG-dependent nascent PG processing in vivo, and bacterial two-hybrid analysis identified an MltG-PBP1b interaction. Mutants lacking MltG were also shown to have longer glycans in their PG relative to wild-type cells. Our combined results are thus consistent with a model in which MltG associates with PG synthetic complexes to cleave nascent polymers and terminate their elongation.


Subject(s)
Endodeoxyribonucleases/isolation & purification , Endodeoxyribonucleases/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/enzymology , Penicillin-Binding Proteins/genetics , Peptidoglycan/metabolism , Cell Wall/metabolism , Endodeoxyribonucleases/genetics , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Models, Molecular , Mutation , Penicillin-Binding Proteins/metabolism , Peptidoglycan/chemistry , Peptidoglycan Glycosyltransferase/metabolism , Peptidyl Transferases/genetics , Peptidyl Transferases/metabolism , Phenotype , Plasmids , Polymerization , Polysaccharides/metabolism , Serine-Type D-Ala-D-Ala Carboxypeptidase
11.
FEMS Microbiol Lett ; 362(24): fnv204, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26500089

ABSTRACT

The HNH endonuclease superfamily usually contains a conserved HNH motif in the sequence, and the second subfamily of it uses N to replace the second H in the HNH motif. A bacterium with extracellular thermostable DNase was isolated and identified as Exiguobacterium sp. yc3. A 20 kDa putative DNase was later purified and the encoding gene of it was amplified and sequenced, the deduced amino acid sequence analysis showed that the protein belongs to the HNH endonuclease superfamily, and therefore it was named as EheA ( E: xiguobacterium H: NH E: ndonuclease). Characterization of the recombinant EheA confirmed that EheA is a DNase. By site-directed mutation method, H116, N141 and N156 were proved to be essential for the DNase activity. EheA is the first experimentally determined bacterial source endonuclease belonging to the second subfamily of HNH superfamily. Further bioinformatic analysis showed that EheA homologue genes are conserved in the Exiguobacterium species, which suggests their possible important functions for Exiguobacterium species. And as a thermostable DNase, EheA also has a promising future in many application fields.


Subject(s)
Bacillales/enzymology , Endodeoxyribonucleases/genetics , Endodeoxyribonucleases/isolation & purification , Amino Acid Sequence , Bacillales/genetics , Computational Biology , Endodeoxyribonucleases/chemistry , Endodeoxyribonucleases/metabolism , Enzyme Stability , Genome, Bacterial , Hydrogen-Ion Concentration , Molecular Sequence Data , Mutagenesis, Site-Directed , Phylogeny , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Analysis, DNA
12.
Methods Mol Biol ; 1311: 251-64, 2015.
Article in English | MEDLINE | ID: mdl-25981478

ABSTRACT

Cas1 genes encode the signature protein of the CRISPR/Cas system, which is present in all CRISPR-containing organisms. Recently, Cas1 proteins (together with Cas2) have been shown to be essential for the formation of new spacers in Escherichia coli, and purified Cas1 proteins from Pseudomonas aeruginosa and E. coli have been shown to possess a metal-dependent endonuclease activity. Here we describe the protocols for the analysis of nuclease activity of purified Cas1 proteins against various DNA substrates including Holliday junctions and other intermediates of DNA recombination and repair.


Subject(s)
CRISPR-Associated Proteins/metabolism , DNA/metabolism , Endodeoxyribonucleases/metabolism , Enzyme Assays/methods , Escherichia coli Proteins/metabolism , CRISPR-Associated Proteins/isolation & purification , DNA/genetics , DNA, Cruciform/metabolism , Electrophoresis, Polyacrylamide Gel , Endodeoxyribonucleases/isolation & purification , Escherichia coli/enzymology , Escherichia coli Proteins/isolation & purification , Oligonucleotides/metabolism
13.
Protein Expr Purif ; 112: 1-7, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25888783

ABSTRACT

Engineered zinc-finger nucleases (ZFNs) have been widely used for precise genome editing. ZFNs can induce DNA double-strand breaks at specific genomic locations and drive the introduction of an insertion or deletion of base pairs at the targeted region, consequently resulting in a loss-of-function mutation. In this study, we investigated the cloning, expression and purification of ZFN fusion proteins targeting the goat beta-lactoglobulin (BLG) gene and detected the cleavage activities of these ZFN proteins in vitro and in cells, respectively. The results showed that the pET-BLG-LFN and pET-BLG-RFN prokaryotic expression plasmids can be constructed correctly and expressed efficiently in Escherichia coli BL21 (DE3) cells to produce the 6× His-tagged ZFN proteins that can be purified by Ni-IDA-Sefinose Column. The predetermined sequence of BLG can be recognized and excised both in vitro and in goat fibroblasts by the purified ZFN fusion proteins, which demonstrated that the purified ZFN fusion proteins can be used as gene modification tools to knock out the BLG gene. Furthermore, these results lay the foundation for eliminating allergen BLG from goat milk and improving the quality of goat milk products.


Subject(s)
Endodeoxyribonucleases/genetics , Gene Knockout Techniques/methods , Goats/genetics , Lactoglobulins/genetics , Protein Engineering , Zinc Fingers , Animals , Cells, Cultured , Endodeoxyribonucleases/isolation & purification , Endodeoxyribonucleases/metabolism , Escherichia coli/genetics , Nuclear Localization Signals , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism
14.
BMC Genomics ; 16: 126, 2015 Feb 25.
Article in English | MEDLINE | ID: mdl-25766859

ABSTRACT

BACKGROUND: Recent advances in studies of the Schistosoma japonicum genome have opened new avenues for the elucidation of parasite biology and the identification of novel targets for vaccines, drug development and early diagnostic tools. RESULTS: In this study, we surveyed the S. japonicum genome database for genes encoding nucleases. A total of 130 nucleases of 3 classes were found. Transcriptional analysis of these genes using a genomic DNA microarray revealed that the majority of the nucleases were differentially expressed in parasites of different developmental stages or different genders, whereas no obvious transcriptional variation was detected in parasites from different hosts. Further analysis of the putative DNases of S. japonicum revealed a novel DNase II homologue (Sjda) that contained a highly conserved catalytic domain. A recombinant Sjda-GST protein efficiently hydrolysed genomic DNA in the absence of divalent iron. Western-blot and immunofluorescence assays showed that Sjda was mainly expressed on the teguments of female adult parasites and induced early humoral immune responses in infected mice. CONCLUSIONS: A novel DNase II homologue, Sjda, was identified in S. japonicum. Sjda was mainly distributed on the teguments of adult female parasites and possessed a typical divalent iron-independent DNA catalytic activity. This protein may play an important role in the host-parasite interaction.


Subject(s)
Endodeoxyribonucleases/genetics , Host-Parasite Interactions , Schistosoma japonicum/enzymology , Schistosomiasis japonica/genetics , Animals , Endodeoxyribonucleases/biosynthesis , Endodeoxyribonucleases/isolation & purification , Female , Gene Expression Regulation , Humans , Mice , Oligonucleotide Array Sequence Analysis , Schistosoma japonicum/pathogenicity , Schistosomiasis japonica/parasitology
15.
Biologicals ; 42(5): 271-6, 2014 Sep.
Article in English | MEDLINE | ID: mdl-25108516

ABSTRACT

The clearance of host cell DNA is a critical indicator for Vero-cell culture-derived rabies vaccine. In this study, we evaluated the clearance of DNA in Vero-cell culture-derived rabies vaccine by purification process utilizing ultrafiltration, nuclease digestion, and gel filtration chromatography. The results showed that the bioprocess of using nuclease decreased residual DNA. Dot-blot hybridization analysis showed that the residual host cell DNA was <100 pg/ml in the final product. The residual nuclease in rabies vaccine was less than 0.1 ng/ml protein. The residual nuclease could not paly the biologically active role of digestion of DNA. Experiments of stability showed that the freeze-drying rabies virus vaccine was stable and titers were >5.0 IU/ml. Immunogenicity test and protection experiments indicated mice were greatly induced generation of neutralizing antibodies and invoked protective effects immunized with intraperitoneal injections of the rabies vaccine. These results demonstrated that the residual DNA was removed from virus particles and nuclease was removed by gel filtration chromatography. The date indicated that technology was an efficient method to produce rabies vaccine for human use by using nuclease.


Subject(s)
DNA/isolation & purification , Endodeoxyribonucleases , Endoribonucleases , Rabies Vaccines/isolation & purification , Animals , Antibodies, Neutralizing/biosynthesis , Antibodies, Viral/biosynthesis , Chlorocebus aethiops , Chromatography, Gel , Drug Contamination/prevention & control , Drug Stability , Endodeoxyribonucleases/isolation & purification , Endoribonucleases/isolation & purification , Freeze Drying , Humans , Mice , Rabies/immunology , Rabies/prevention & control , Rabies Vaccines/immunology , Rabies virus/immunology , Vero Cells
16.
J Virol ; 88(1): 225-36, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24155374

ABSTRACT

During herpes simplex virus 1 (HSV-1) infection, empty procapsids are assembled and subsequently filled with the viral genome by means of a protein complex called the terminase, which is comprised of the HSV-1 UL15, UL28, and UL33 proteins. Biochemical studies of the terminase proteins have been hampered by the inability to purify the intact terminase complex. In this study, terminase complexes were isolated by tandem-affinity purification (TAP) using recombinant viruses expressing either a full-length NTAP-UL28 fusion protein (vFH476) or a C-terminally truncated NTAP-UL28 fusion protein (vFH499). TAP of the UL28 protein from vFH476-infected cells, followed by silver staining, Western blotting, and mass spectrometry, identified the UL15, UL28, and UL33 subunits, while TAP of vFH499-infected cells confirmed previous findings that the C terminus of UL28 is required for UL28 interaction with UL33 and UL15. Analysis of the oligomeric state of the purified complexes by sucrose density gradient ultracentrifugation revealed that the three proteins formed a complex with a molecular mass that is consistent with the formation of a UL15-UL28-UL33 heterotrimer. In order to assess the importance of conserved regions of the UL15 and UL28 proteins, recombinant NTAP-UL28 viruses with mutations of the putative UL28 metal-binding domain or within the UL15 nuclease domain were generated. TAP of UL28 complexes from cells infected with each domain mutant demonstrated that the conserved cysteine residues of the putative UL28 metal-binding domain and conserved amino acids within the UL15 nuclease domain are required for the cleavage and packaging functions of the viral terminase, but not for terminase complex assembly.


Subject(s)
Endodeoxyribonucleases/isolation & purification , Herpesvirus 1, Human/enzymology , Animals , Base Sequence , Blotting, Southern , Blotting, Western , Chlorocebus aethiops , Chromatography, Affinity , DNA, Viral/chemistry , Endodeoxyribonucleases/chemistry , Mass Spectrometry , Mutation , Vero Cells
17.
Oncogene ; 32(37): 4448-56, 2013 Sep 12.
Article in English | MEDLINE | ID: mdl-23146902

ABSTRACT

In response to ionizing radiation, the MRE11/RAD50/NBN complex re-distributes to the sites of DNA double-strand breaks (DSBs) where each of its individual components is phosphorylated by the serine-threonine kinase, ATM. ATM phosphorylation of NBN is required for the activation of the S-phase checkpoint, but the mechanism whereby these phosphorylation events signal the checkpoint machinery remains unexplained. Here, we describe the use of direct protein transduction of the homing endonuclease, I-PpoI, into human cells to generate site-specific DSBs. Direct transduction of I-PpoI protein results in rapid accumulation and turnover of the endonuclease in live cells, facilitating comparisons across multiple cell lines. We demonstrate the utility of this system by introducing I-PpoI into isogenic cell lines carrying mutations at the ATM phosphorylation sites in NBN and assaying the effects of these mutations on the spatial distribution and temporal accumulation of NBN and ATM at DSBs by chromatin immunoprecipitation, as well as timing and extent of DSB repair. Although the spatial distribution of NBN and ATM recruited to the sites of DSBs was comparable between control cells and those expressing phosphorylation mutants of NBN, the timing of accumulation of NBN and ATM was altered. Serine-to-alanine mutations that blocked phosphorylation resulted in delayed recruitment of both NBN and ATM to DSBs. Serine-to-glutamic acid substitutions that mimicked the phosphorylation event resulted in both increased and prolonged accumulation of both NBN and ATM at DSBs. The repair of DSBs in cells lacking full-length NBN was significantly delayed compared with control cells, whereas blocking phosphorylation of NBN resulted in a more modest delay in repair. These data indicate that following the induction of DSBs, phosphorylation of NBN regulates its accumulation, and that of ATM, at sites of DNA DSB as well as the timing of the repair of these sites.


Subject(s)
Cell Cycle Proteins/metabolism , DNA Breaks, Double-Stranded , DNA-Binding Proteins/metabolism , Nuclear Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Tumor Suppressor Proteins/metabolism , Ataxia Telangiectasia Mutated Proteins , DNA/metabolism , Endodeoxyribonucleases/isolation & purification , Endodeoxyribonucleases/metabolism , Humans , Hydrolysis , Phosphorylation
18.
Protein Expr Purif ; 84(1): 130-9, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22569481

ABSTRACT

Endonuclease VIII-like 3 (Neil3) is one of the five DNA glycosylases found in mammals that recognize and remove oxidized bases, and initiate the base excision repair (BER) pathway. Previous attempts to express and purify the mouse and human orthologs of Neil3 in their active form have not been successful. Here we report the construction of bicistronic expression vectors for expressing in Escherichia coli the full-length mouse Neil3 (MmuNeil3), its glycosylase domain (MmuNeil3Δ324), as well as the glycosylase domain of human Neil3 (NEIL3Δ324). The purified Neil3 proteins are all active, and NEIL3Δ324 exhibits similar glycosylase/lyase activity as MmuNeil3Δ324 on both single-stranded and double-stranded substrates containing thymine glycol (Tg), spiroiminodihydantoin (Sp) or an abasic site (AP). We show that N-terminal initiator methionine processing is critical for the activity of both mouse and human Neil3 proteins. Co-expressing an E. coli methionine aminopeptidase (EcoMap) Y168A variant with MmuNeil3, MmuNeil3Δ324 and NEIL3Δ324 improves the N-terminal methionine processing and increases the percentage of active Neil3 proteins in the preparation. The purified Neil3 proteins are suitable for biochemical, structural and functional studies.


Subject(s)
Endodeoxyribonucleases/biosynthesis , Endodeoxyribonucleases/isolation & purification , N-Glycosyl Hydrolases/biosynthesis , N-Glycosyl Hydrolases/isolation & purification , Recombinant Fusion Proteins/isolation & purification , Animals , Cloning, Molecular , Codon , Electrophoresis, Polyacrylamide Gel , Endodeoxyribonucleases/chemistry , Endodeoxyribonucleases/genetics , Enzyme Stability , Escherichia coli/genetics , Escherichia coli/metabolism , Histidine/chemistry , Histidine/genetics , Histidine/metabolism , Humans , Methionine/metabolism , Mice , N-Glycosyl Hydrolases/chemistry , N-Glycosyl Hydrolases/genetics , Oligopeptides/chemistry , Oligopeptides/genetics , Oligopeptides/metabolism , Protein Structure, Tertiary , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics
19.
Article in English | MEDLINE | ID: mdl-19407388

ABSTRACT

Endonuclease G (EndoG) is a mitochondrial enzyme that responds to apoptotic stimuli by translocating to the nucleus and cleaving chromosomal DNA. EndoG is the main apoptotic endonuclease in the caspase-independent pathway. Mouse EndoG without the mitochondrial localization signal (amino-acid residues 1-43) was successfully overexpressed, purified and crystallized using a microbatch method under oil. The initial crystal (type I) was grown in the presence of the detergent CTAB and diffracted to 2.8 A resolution, with unit-cell parameters a = 72.20, b = 81.88, c = 88.66 A, beta = 97.59 degrees in a monoclinic space group. The crystal contained two monomers in the asymmetric unit, with a predicted solvent content of 46.6%. Subsequent mutation of Cys110 improved the stability of the protein significantly and produced further crystals of types II, III and IV with space groups C2, P4(1)2(1)2 (or P4(3)2(1)2) and P2(1)2(1)2(1), respectively, in various conditions. This suggests the critical involvement of this conserved cysteine residue in the crystallization process.


Subject(s)
Apoptosis , Endodeoxyribonucleases/chemistry , Endodeoxyribonucleases/metabolism , Animals , Crystallization , Crystallography, X-Ray , Endodeoxyribonucleases/genetics , Endodeoxyribonucleases/isolation & purification , Mice
20.
Cell ; 135(2): 250-60, 2008 Oct 17.
Article in English | MEDLINE | ID: mdl-18957200

ABSTRACT

The Mre11/Rad50 complex has been implicated in the early steps of DNA double-strand break (DSB) repair through homologous recombination in several organisms. However, the enzymatic properties of this complex are incompatible with the generation of 3' single-stranded DNA for recombinase loading and strand exchange. In thermophilic archaea, the Mre11 and Rad50 genes cluster in an operon with genes encoding a helicase, HerA, and a 5' to 3' exonuclease, NurA, suggesting a common function. Here we show that purified Mre11 and Rad50 from Pyrococcus furiosus act cooperatively with HerA and NurA to resect the 5' strand at a DNA end under physiological conditions in vitro. The 3' single-stranded DNA generated by these enzymes can be utilized by the archaeal RecA homolog RadA to catalyze strand exchange. This work elucidates how the conserved Mre11/Rad50 complex promotes DNA end resection in archaea and may serve as a model for DSB processing in eukaryotes.


Subject(s)
Archaeal Proteins/metabolism , DNA Breaks, Double-Stranded , DNA Repair , Endodeoxyribonucleases/metabolism , Exodeoxyribonucleases/metabolism , Pyrococcus furiosus/metabolism , Archaeal Proteins/isolation & purification , DNA/metabolism , Endodeoxyribonucleases/isolation & purification , Exodeoxyribonucleases/isolation & purification , Multienzyme Complexes/isolation & purification , Multienzyme Complexes/metabolism , Protein Interaction Mapping , Pyrococcus furiosus/enzymology , Pyrococcus furiosus/genetics
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