Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Epstein-Barr virus (EBV) DNase possesses both endonuclease and exonuclease activities and accepts both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) as substrates. To map regions of EBV DNase responsible for nuclease and DNA binding activities, a series of mutant DNase polypeptides was expressed using a bacterial system for the nuclease assay and in an in vitro transcription/translation system to assay binding activity to dsDNA or ssDNA cellulose. The results indicated that the C-terminus of EBV DNase, residues 450-460, is essential for nuclease activity but dispensable for DNA binding. However, deletion of residues 441-470 resulted in the loss of both nuclease and DNA binding activities. Substitution of Phe452 and Val458 led to inactive enzymes. In the N-terminus, deletion of residues 23-28 and residues 7-61 resulted in the loss of nuclease activity but the DNA binding activities of the deleted enzymes were intermediate and low, respectively. Mutation of Leu23 to Gly showed drastically reduced nuclease activity but its DNA binding ability was not affected. Based on the amino acid sequence alignment of various herpesvirus DNases, we chose four highly conserved and two less well conserved regions as controls for mutagenesis studies. These six internal deletion (ID) mutants were prepared using a recombinant PCR method. Each of the polypeptides was expressed in a bacterial system for the nuclease assay and using an in vitro transcription/translation system for the DNA binding assay. DNA binding and nuclease activities of all six internal deletion mutants were abolished, except that mutant ID2, with deletion of residues 138-152, retained an intermediate ability to bind DNA. These data indicate that since mutations at distinct regions within EBV DNase resulted in the loss of nuclease and/or DNA binding activities, it is suggested that these distinct regions are required for maintenance of an intact and highly ordered structure(s) for both activities.
...
PMID:Distinct regions of EBV DNase are required for nuclease and DNA binding activities. 950 Oct 34

We report the first detailed comparison of two immunity proteins which, in conjunction with recent protein engineering data, begins to explain how these structurally similar proteins are able to bind and inhibit the endonuclease domain of colicin E9 (E9 DNase) with affinities that differ by 12 orders of magnitude. In the present work, we have determined the X-ray structure of the Escherichia coli colicin E7 immunity protein Im7 to 2.0 A resolution by molecular replacement, using as a trial model the recently determined NMR solution structure of Im9. Whereas the two proteins adopt similar four-helix structures, subtle structural differences, in particular involving a conserved tyrosine residue critical for E9 DNase binding, and the identity of key residues in the specificity helix, lie at the heart of their markedly different ability to bind the E9 DNase. Two other crystal structures were reported recently for Im7; in one, Im7 was a monomer and was very similar to the structure reported here, whereas in the other it was a dimer to which functional significance was assigned. Since this previous work suggested that Im7 could exist either as a monomer or a dimer, we used analytical ultracentrifugation to investigate this question further. Under a variety of solution conditions, we found that Im7 only ever exists in solution as a monomer, even up to protein concentrations of 15 mg/ml, casting doubt on the functional significance of the crystallographically observed dimer. This work provides a structural framework with which we can understand immunity-protein specificity, and in addition we believe it to be the first successfully refined crystal structure solved by molecular replacement using an NMR trial model with less than 100% sequence identity.
...
PMID:A structural comparison of the colicin immunity proteins Im7 and Im9 gives new insights into the molecular determinants of immunity-protein specificity. 963 78

The DNase of Epstein-Barr virus (EBV) is a 470-amino-acid protein which possesses both endonuclease and exonuclease activities and accepts both double-stranded DNA and single-stranded DNA as substrates. It has been reported that this protein may be found in the nucleus and/or cytoplasm of infected cells. In this study, using cell fractionation and immunoblotting to determine the distribution of EBV DNase in Akata cells stimulated with anti-human immunoglobulin G antibody (anti-IgG), the DNase was found to be located predominantly in the nucleus. To map the signals in DNase which mediate its nuclear localization, we monitored the nuclear transport of fusion proteins consisting of various fragments of EBV DNase linked to a cytoplasmic protein, beta-galactosidase (beta-Gal). The results demonstrated that two regions of the DNase with nuclear localization signal (NLS) activity, designated NLS-A (amino acids 239-266) and NLS-B (amino acids 291-306), were able independently to localize the beta-Gal to the nuclei of HEp-2 and HeLa cells. Five basic residues (R or K) were found in each NLS and distributed differently in primary structure. The basic domains and flanking residues of NLS-A and NLS-B are 250YKRPCKRSFIRFI262 and 294LKDVRKRKLGPGH306, respectively. Further examination of these sequences revealed that NLS-A contains bulky aromatic amino acids (Y and F) which may diminish its capacity to act as a strong NLS and lacks the typical proline and glycine helix-breakers. However, NLS-B contains typical proline and glycine helix-breakers and the histidine residue at amino acid 306 is required for NLS activity. In addition, two hydrophobic regions within the DNase were found to inhibit the function of NLS-A but not NLS-B, suggesting that these two domains are different types of NLSs and differ in their sensitivity to hydrophobic regions in the context of protein structure.
...
PMID:Epstein-Barr virus DNase contains two nuclear localization signals, which are different in sensitivity to the hydrophobic regions. 968 72

The immunity protein Im2 can bind and inhibit the noncognate endonuclease domain of the bacterial toxin colicin E9 with a Kd of 19 nM, 6 orders of magnitude weaker than that of the cognate immunity protein Im9 with which it shares 68% sequence identity. Previous work from our laboratory has shown that the specificity differences of these four-helix immunity proteins is due almost entirely to helix II which is largely variable in sequence in the immunity protein family. From alanine scanning mutagenesis of Im9 in conjunction with high-field NMR data, a dual recognition model for colicin-immunity protein specificity has been proposed whereby the conserved residues of helix III of the immunity protein act as the anchor of the endonuclease binding site while the variable residues of helix II control the specificity of the protein-protein interaction. In this work, we identify three residues (at positions 33, 34, and 38) in helix II which define the specificity differences of Im2 and Im9 for colicin E9 and, using alanine mutagenesis of the putative endonuclease binding surface of Im2, compare the distribution of binding energies for conserved and nonconserved sites in both immunity proteins. This comparison highlights the conserved residues of both Im2 and Im9 as the major determinants of E9 DNase binding energy. Conversely, the nonconserved, specificity-determining residues only contribute to the E9 DNase binding energy in the cognate Im9 protein, while in the noncognate immunity protein Im2, they either destabilize the complex or do not contribute to the binding energy. This comparative alanine scan of two immunity proteins therefore supports the dual recognition mechanism of selectivity in colicin-immunity protein interactions and provides a basis for understanding specificity in other protein-protein interaction systems involving structurally conserved protein families.
...
PMID:Dual recognition and the role of specificity-determining residues in colicin E9 DNase-immunity protein interactions. 971 99

Herpes simplex virus type 1 DNase (HSV-1 DNase) was expressed in insect cells by recombinant baculovirus (NPVUL12) and purified by a combination of anionic exchanger chromatography and gel filtration. Two polypeptides of 85 and 75 kD, whose ratio varied during purification, were induced 24 h after infection. The 75-kD protein was isolated and shown to possess catalytic activity. Gel filtration analysis indicated that the active form of the enzyme at an ionic strength of I = 0.3 is a dimeric protein with an apparent molecular weight of 130,000. The recombinant enzyme exhibited the overall characteristics of the native enzyme such as 5'-3' exonuclease and endonuclease activities with a preferred degradation of DNA. In the absence of extraneously added Mg2+, the enzyme was capable of removing mononucleotides from 5'-end-labeled DNA, but not from RNA and 3'-end-labeled DNA. The peculiar mechanism of double-strand DNA degradation suggests a specific role of HSV-1 DNase in DNA recombination processes during viral replication.
...
PMID:Herpes simplex virus type 1 DNase: functional analysis of the enzyme expressed by recombinant baculovirus. 982 Aug 45

A nuclease activity has been purified from the nuclei-kinetoplast fraction of Leishmania. This enzyme, termed endonuclease M (Endo M), is shown by electrophoresis in a denaturing polyacrylamide gel to be associated with a single polypeptide of molecular mass 52 kDa. Physical analysis of the enzyme indicates that it has a sedimentation coefficient S20,w of 4.5S, a Stoke's radius of 32.5 A, and a native molecular mass of 53 kDa. The final Mono Q purified Endo M possesses both DNase and RNase activities. It acts as an endonuclease by introducing random single-stranded nicks into the supercoiled DNA molecules, that often leads to its linearization due to nicking at the opposite strands, and subsequent degradation of the DNA with further incubation. Single-stranded DNA is twice preferred to double-stranded DNA as substrate. Single-stranded RNA is also degraded rapidly and is competitive as a substrate with single-stranded DNA. RNA:DNA hybrids, however, are largely resistant to the Endo M digestion.
...
PMID:A novel endonuclease from kinetoplastid hemoflagellated protozoan parasite Leishmania. 983 25

The stem cell leukaemia (SCL) gene is a member of the basic helix-loop-helix family of transcription factors and is essential for the development of all haematopoietic lineages. SCL is expressed in pluripotent haematopoietic stem cells and also following commitment to the erythroid, mast and megakaryocytic lineages. The mechanisms responsible for this pattern of expression are poorly understood, but are likely to illuminate the molecular basis for stem cell development and lineage commitment. Here we present the first description of the regulation of the SCL gene in mast cells. In this study we systematically analysed the chromatin structure of a 45 kb region of the murine SCL locus in mast cells. The pattern of DNase 1 and restriction endonuclease hypersensitive sites in mast cells was distinct from, but overlapped with, the pattern previously described in erythroid and primitive myeloid cells. Each potential regulatory element was tested using transient reporter assays to assess their functional significance in mast cells. These studies identified two potent enhancers, one of which was downstream of the SCL gene. Further characterisation of this 3' enhancer demonstrated that it required the presence of two distinct DNase 1 hypersensitive sites for full activity, and that it was capable of stimulating transcription from both promoter 1a and 1b. Since the 3' enhancer is active in both erythroid and mast cells, it will now be important to see whether it is independently activated in these lineages, or whether it is also active in haematopoietic stem cells.
...
PMID:Chromatin structure and transcriptional regulation of the stem cell leukaemia (SCL) gene in mast cells. 1037 80

Ni2+ affinity columns are widely used for protein purification, but they carry the risk that Ni2+ ions may bind to the protein, either adventitiously or at a physiologically important site. Dialysis against ethylenediaminetetraacetic acid (EDTA) is normally used to remove metal ions bound adventitiously to proteins; however, this approach does not always work. Here we report that a bacterial endonuclease, the DNase domain of colicin E9, binds Ni2+ acquired from Ni2+ affinity columns, and appears to bind [Ni(EDTA)(H2O)n]2- at low ionic strength. NMR was used to detect the presence of both Ni2+ coordinated to amino acid side chains and [Ni(EDTA)(H2O)N]2-. Dialysis against > or =0.2 M NaCl was required to remove the [Ni(EDTA)(H2O)n]2-. The NMR procedure we have used to characterize the presence of Ni2+ and [Ni(EDTA)(H2O)n]2- should be applicable to other proteins where there is the possibility of binding paramagnetic metal ions that are present to expedite protein purification. In the present case, the binding of Ni2+ seems likely to be physiologically relevant, and the NMR data complement recent X-ray crystallographic evidence concerning the number of histidine ligands to bound Ni2+.
...
PMID:NMR study of Ni2+ binding to the H-N-H endonuclease domain of colicin E9. 1045 17

The cytotoxic domain of the bacteriocin colicin E9 (the E9 DNase) is a nonspecific endonuclease that must traverse two membranes to reach its cellular target, bacterial DNA. Recent structural studies revealed that the active site of colicin DNases encompasses the HNH motif found in homing endonucleases, and bound within this motif a single transition metal ion (either Zn(2+) or Ni(2+)) the role of which is unknown. In the present work we find that neither Zn(2+) nor Ni(2+) is required for DNase activity, which instead requires Mg(2+) ions, but binding transition metals to the E9 DNase causes subtle changes to both secondary and tertiary structure. Spectroscopic, proteolytic, and calorimetric data show that, accompanying the binding of 1 eq of Zn(2+), Ni(2+), or Co(2+), the thermodynamic stability of the domain increased substantially, and that the equilibrium dissociation constant for Zn(2+) was less than or equal to nanomolar, while that for Co(2+) and Ni (2+) was micromolar. Our data demonstrate that the transition metal is not essential for colicin DNase activity but rather serves a structural role. We speculate that the HNH motif has been adapted for use by endonuclease colicins because of its involvement in DNA recognition and because removal of the bound metal ion destabilizes the DNase domain, a likely prerequisite for its translocation across bacterial membranes.
...
PMID:Homing in on the role of transition metals in the HNH motif of colicin endonucleases. 1048 Sep 31

: From the coral Galaxea fascicularis, a crude mucus-like extract (MS) and subsequently its purified component (P6) appear to contain a DNase-like activity that indiscriminately digested lambdaDNA, as well as naked genomic DNAs isolated from a multiple-drug-resistant murine leukemia cell line, P388/VCR, and a nontransformed liver cell line, BL8L. However, MS and P6 specifically induced in situ DNA digestion in cultured P388/VCR cells from 30 minutes onward. After 3 days of incubation with MS or P6, DNA degradation coincided with complete killing of P388/VCR. In situ fluorescent labeling of fragmented DNA revealed that P6 induced apoptosis of P388/VCR cells, occurring as early at 1.5 hours. By day 3, all the P6-treated leukemia cells were apoptotic. In contrast, P6 caused neither in situ DNA digestion, nor apoptosis in the untransformed BL8L cells. Whether the DNase-like action of P6 is independent of or responsible for triggering the intrinsic endonuclease activity in the leukemia cell, thus leading to apoptosis, remains an object for further research. Nevertheless, the specificity of the apoptotic action of P6 on P388/VCR cells indicates its potential role in the development of an anticancer agent.
...
PMID:Novel Bioactivities from a Coral, Galaxea fascicularis: DNase-like Activity and Apoptotic Activity Against a Multiple-Drug-Resistant Leukemia Cell Line. 1048 7


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---