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

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

A unique subset of anti-DNA antibodies enters living cells, interacts with DNase 1, and inhibits endonuclease activity, before their nuclear localization and subsequent attenuation of apoptosis. We now report that endocytosis of these immunoglobulins is mediated by cell surface binding to brush border myosin (myosin 1). Cellular entry and internalization via this unique receptor provides initial contact for entry and sorting these immunoglobulins to translocate to the nuclear pore and enter the nucleus, interact with DNase 1 within the cytoplasm, or recycle back to the cell surface. This internalization pathway provides clues to the translocation of large proteins across cell membranes and the functional effects of intracellular antibodies on cytopathology. This is the first demonstration that brush border myosin functions as a specific cell surface receptor for internalization of large proteins.
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PMID:Receptor-mediated cellular entry of nuclear localizing anti-DNA antibodies via myosin 1. 920 53

The gene encoding a mitogenic factor, termed MF, was cloned from Streptococcus pyogenes and the recombinant MF was overexpressed in Escherichia coli. Both the natural and recombinant MF had heat-resistant nuclease activity. The nuclease activity of MF was characterized using the recombinant protein. MF showed endonuclease activity, digesting ssDNA, dsDNA and tRNA. The optimal pH for the DNase activity of MF was 9.5. The DNase activity was enhanced approximately tenfold by the simultaneous presence of two divalent cations, Mg2+ and Ca2+, compared to either alone and was inhibited by EDTA or NaCl. The heat stability of MF was biphasic; the DNase activity was heat-stable from 0 to 50 degrees C over 80 degrees C but very unstable at around 60 degrees C. DNA digested by MF possessed 5'-phosphorylated and 3'-hydroxylated termini, identical to those obtained by digestion of DNA by pancreatic deoxyribonuclease I. A mutant clone revealed that His122 was a residue essential to the nuclease activity.
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PMID:Mitogenic factor secreted by Streptococcus pyogenes is a heat-stable nuclease requiring His122 for activity. 924 25

The colicin DNase-specific immunity proteins interact with the endonuclease domain of the bacterial toxin colicin E9 with dissociation constants that span the millimolar to femtomolar affinity range. Among the non-cognate interactions Im2 shows the strongest binding toward the E9 DNase domain with a Kd of 10(-8) M, 6 orders of magnitude weaker than that of the cognate immunity protein Im9. Based on a NMR structure of Im9 that shows it to be a 4-helix protein, we have conducted a mutagenic scan in which elements of Im9 secondary structure were substituted into Im2 to precisely delineate regions that define specificity. Eleven chimeras were constructed, and their biological cross-reactivity toward colicins E2 and E9 was evaluated. From this set of mutants seven proteins were purified, and the Kd for their interaction with the E9 DNase domain was measured by a combination of stopped-flow fluorescence and subunit exchange kinetics. Our results show that immunity specificity is dominated by residues on helix II, accounting for 5 orders of magnitude binding specificity relative to Im2, and that packing interactions of helix II with its neighbor helix I and the loop connecting helix III with helix IV play minor roles. The conformational stability of these chimeric proteins was also determined. Proteins displaying an Im9 phenotype were all more stable than the parent Im2 protein, and surprisingly some chimeras were significantly more stable than either Im2 or Im9.
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PMID:Protein-protein interaction specificity of Im9 for the endonuclease toxin colicin E9 defined by homologue-scanning mutagenesis. 926 73

A new method for isolation of prawn baculovirus and subsequent extraction of viral DNA was developed. No density gradient centrifugation, ultracentrifugation or phenol-chloroform extraction steps were involved. Phenylmethylsulfonyl fluoride (PMSF) was used to prevent proteinase degradation, DNase and RNase were used to degrade prawn DNA and RNA respectively. The nucleocapsid was a bacilliform virion, about 58 62 nm in width and 300-350 nm in length as observed by transmission electron microscopy. Intact viral DNA was obtained by lysing nucleocapsids with guanidine hydrochloride and degrading protein with proteinase K. As the viral DNA was digested with restriction endonuclease and separated by electrophoresis, restriction fragments were clearly shown on the agarose gel. The size of the DNA was estimated approximately to be 290 kb. The virus which appeared to be a prawn baculovirus was named prawn white spot baculovirus (PWSBV) due to the white spots which appeared on the inside surface of the crust of infected prawns.
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PMID:A simple and efficient method for purification of prawn baculovirus DNA. 927 11

We previously identified three distinct DNA endonucleases, DNases alpha, beta and gamma, present in rat thymocyte nuclei. On the basis of their enzymic and biochemical properties, gamma-type DNase was regarded as a candidate for the apoptotic endonuclease. Here we purified DNase gamma to apparent homogeneity from apoptotic rat thymocyte nuclei induced by X-irradiation and characterized its properties in detail. The purified DNase gamma exhibited one predominant protein band on SDS/PAGE and an endonuclease activity in a zymography with an estimated molecular mass of 33 kDa. The molecular mass of the native form determined by G2000SW gel-filtration HPLC was 30 kDa. Amino acid analysis showed that the amino acid composition of DNase gamma was similar to that of rat DNase I (molecular mass 32 kDa) but different with regard to alanine and lysine residues. The N-terminal amino acid sequence of DNase gamma was revealed to be not identical with that of rat DNase I. In accordance with previous studies, homogeneously purified DNase gamma requires both Ca2+ and Mg2+ for activity. This requirement could be partially supplied by Mn2+. Of the bivalent metal ions tested, Co2+, Ni2+, Cu2+ and Zn2+ inhibited DNase gamma activity. These bivalent cations also suppressed apoptotic DNA fragmentation in rat thymocytes irradiated by X-rays. The same order of inhibitory ability was observed for these bivalent metal ions in vivo (in intact cells) and in vitro, suggesting that the suppression of apoptotic DNA fragmentation at the cellular level is due to the inhibition of DNase gamma. DNase gamma activity was found to exist at high levels in spleen, lymph node, thymus, liver and kidney, but little was present in brain, heart or pancreas. On the basis of these findings, together with previous data, we conclude that DNase gamma is a novel DNase I-like endonuclease responsible for internucleosomal cleavage of chromatin during thymic apoptosis.
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PMID:Purification and properties of DNase gamma from apoptotic rat thymocytes. 930 16

An endonuclease named DNase gamma was purified to apparent homogeneity from rat splenocyte nuclei and its properties were characterized. We also determined the NH2-terminal and partial amino acid sequences of the proteolytic internal peptides. The molecular mass of gamma DNase was 33,000 daltons as determined by SDS-polyacrylamide gel electrophoresis. A native molecular mass of 30,000 was estimated by gel filtration. Purified DNase gamma is active in the presence of both Ca2+ and Mg2+ or Mn2+ alone and inhibited by Co2+, Ni2+, Cu2+, and especially Zn2+. Maximal activity was achieved at pH 7.2 in Mops-NaOH buffer. The sequence data on the NH2-terminal and seven internal peptides obtained by sequential digestions with Achromobacter protease I and endoproteinase Asp-N revealed that DNase gamma is a novel endonuclease that shows sequence homology with DNase I.
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PMID:Purification, characterization, and amino acid sequencing of DNase gamma from rat spleen. 932 79

A neutral Mg(2+)-dependent deoxyribonuclease from the Colorado potato beetle was isolated and characterized in physicochemical terms. An electrophoretically homogeneous preparation of the enzyme was obtained using salt fractionation, Sephadex G-100 gel filtration, and subsequent preparative isoelectrofocusing in an Ultrodex layer. The molecular weight of the purified DNase preparation (with a purification degree of 104) and its isoelectric point were 100 kD and 9.1, respectively. The enzyme activity was maximal at pH 7.2 and 46 degrees C in the presence of 10 mM Mg2+. The DNase of the Colorado beetle preferentially hydrolysed denatured DNA via the endonuclease pathway, degrading the substrate to oligonucleoside-3'-phosphates. As far as the physical and chemical properties are concerned, this Colorado beetle DNase seems different from previously investigated DNases of other insect species.
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PMID:Isolation, purification, and characterization of a neutral Mg(2+)-dependent deoxyribonuclease of the Colorado potato beetle Leptinotarsa decemlineata Say. 936 Mar 4

The endonuclease group of E colicins are a family of bacterial toxins whose cytotoxic activity in a producing host is inactivated by a specific immunity protein. The DNase of colicin E9 can be bound and inhibited by both cognate and noncognate immunity proteins, the dissociation constants for which span a range of 12-orders of magnitude. DNase binding specificity of the immunity proteins is governed primarily by helix II, the sequence of which is variable in this family of proteins. Heteronuclear NMR experiments have identified helix III along with helix II as the likely DNase binding site, although other regions of Im9 also showed perturbations on binding the E9 DNase. In the present work, we have used the NMR experiments as a guide for alanine scanning mutagenesis of Im9. Our data show that helices II and III of Im9 are indeed the DNase binding site and in addition quantitate the relative binding energy associated with each helix. We find that the conserved residues of helix III make the largest relative contribution toward E9 DNase binding. In conjunction with previous studies, the data suggest that specificity in the colicin-immunity system is governed by a dual recognition mechanism in which highly stabilizing interactions emanating from the conserved regions of an immunity protein act as the binding site anchor and these are modulated by interactions from neighboring, nonconserved amino acid residues. This modulation is likely to take the form of both favorable and unfavorable interactions, the balance of which define the specificity of the protein-protein interaction. The generality of such a dual recognition mechanism in other systems is also discussed.
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PMID:Specificity in protein-protein recognition: conserved Im9 residues are the major determinants of stability in the colicin E9 DNase-Im9 complex. 942 68

Two fundamental properties of monomeric actin were examined in this study, ie its interaction with DNase-I, and the inhibition of endonuclease activity consecutive to the association of the two molecules. In particular, the topological independence between catalytic site of DNase-I and interface with actin, structural changes in actin monomer and the absence of conformational changes in DNase-I were described. We demonstrated a loss of flexibility of antigenic structures in actin subdomain I (ie epitopes 18-28 and 95-105) as well as modification in the exposure of Cys10 and Cys374 after DNase-I binding. Furthermore, the conformational changes induced by DNase-I into the actin molecule weakened the interaction of CapZ to its binding site located in the C-terminal region of actin monomer. These structural changes were time-dependent. When actin was cleaved in the DNase-I binding loop (sequence 38-52) at position 42 by E coli A2 strain protease, a tight DNase-I binding to split actin and the conformational changes were still observed, whereas the DNase-I inhibition activity was completely abolished. Finally, when we substitute Ca2+ by Mg2+ (ATP-Mg2+ monomeric actin) which induces a tighter conformation of actin and partially restores the inhibitory ability of split actin, long-range conformational effects of DNase-I are prevented and the ternary complex DNase-I-actin-CapZ is obtained.
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PMID:Analysis of long-range structural effects induced by DNase-I interaction with actin monomeric form or complexed to CapZ. 945 49

The pseudorabies virus (PRV) DNase gene has previously been mapped within the PRV genome. To characterize further the enzymic properties of PRV DNase, this enzyme was expressed in Escherichia coli with the use of a pET expression vector. The protein was purified to homogeneity and assayed for nuclease activity in vitro. Recombinant PRV DNase exhibited an alkaline pH preference and an absolute requirement for Mg2+ ions that could not be replaced by Ca2+ and Na+ ions. Further studies showed that PRV DNase exhibited endonuclease, 5'-exonuclease and 3'-exonuclease activities in both single-stranded and double-stranded DNA. This activity occurred randomly and no significant base preference was demonstrated. The multiple biochemical activities of PRV DNase are similar to the activities of Neurospora crassa endo-exonuclease and E. coli RecBCD, two additional enzymes that are involved in recombination. Taken together, the similarity of action between N. crassa endo-exonuclease, E. coli RecBCD, and PRV DNase suggests that PRV DNase might have a role in the process of recombination that occurs during PRV infection.
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PMID:Recombinant pseudorabies virus DNase exhibits a RecBCD-like catalytic function. 946 90


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