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)

The stimulatory effect of Mg2+ and Mn2+ on the ribonuclease H (RNase H) functions of HIV-1 reverse transcriptase (RT) has been evaluated using a model 90-nt RNA template/36-nt DNA primer. Wild type enzyme exhibits similar endonuclease and directional processing activities in response to both cations, while RNase H activity (hydrolysis of double-stranded RNA) is only evident in the presence of Mn2+. Enzyme altered at the p66 residue Glu478 (Glu478-->Gln478), which participates in metal ion binding, is completely inactive in Mg2+. However, Mn2+ restores specifically its endoribonuclease activity. In the presence of Mn2+, mutant RT also catalyzes specific removal of the tRNA replication primer, eliminating the possibility of contaminating Escherichia coli RNase H in our recombinant enzyme. However, the efficiency with which mutant RT catalyzes transfer of nascent DNA between RNA templates (an event mandating RNase H activity) is severely reduced. These findings raise the possibility that directional processing activity is required to accelerate transfer of nascent DNA between templates during retroviral replication.
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PMID:Divalent cation modulation of the ribonuclease functions of human immunodeficiency virus reverse transcriptase. 754 83

The bacterial expression plasmids, pET3b and pET16b, that contain the integrase domain of the human foamy virus (HFV) reverse transcriptase were constructed and expressed in Escherichia coli. The histidine-tagged HFV IN protein was purified to near homogeneity by single-step Ni2+ chelate affinity chromatography. HFV-specific proteins of 39 and 120 kDa from virus-infected cells reacted with antisera raised against the recombinant IN protein. Purified recombinant HFV IN protein was active as an endonuclease specifically cleaving two nucleotides from a 20-bp oligodeoxynucleotide substrate that mimics the authentic 5' ends of HFV DNA. Substrates with mutations relatively close to the cleavage site were less efficiently cleaved or not cleaved at all compared with the HFV U5 DNA end. The purified recombinant protein was active as integrase with double-stranded oligodeoxynucleotide substrates. The reverse reaction of DNA strand transfer, the disintegration activity, was shown by efficient cleavage of an intermediate Y-shaped oligodeoxynucleotide. In the presence of Mn2+ as the preferred divalent cation, oligodeoxynucleotides were specifically and efficiently cleaved. In contrast, endonucleolytic cleavages in the presence of Mg2+ ions led to a broad range of reaction products with the His-tagged HFV IN protein. After further purification of the HFV IN by cation-exchange chromatography, the unspecific degradation of oligonucleotide substrate in the presence of Mg2+ was not detectable.
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PMID:Endonucleolytic cleavages and DNA-joining activities of the integration protein of human foamy virus. 768 24

A ribonuclease H activity from human placenta has been separated by ion exchange chromatography from the major RNase HI enzyme. Additional chromatographic steps allowed further purification, more than 3,000 fold compared to the crude extract in which it represents about 15% of the total RNase H activity. The enzyme requires Mg2+ ions for its activity, is strongly inhibited by the addition of Mn2+ ions or other divalent transition metal ions, and exhibits a pH optimum between 8.5 and 9. It shows a strong sensitivity to the SH-blocking agent N-ethylmaleimide. It has a strict specificity for double-stranded RNA-DNA duplexes and exhibits neither single-stranded nor double-stranded RNase (or DNase) activities. Therefore, this enzyme displays the characteristics of class II RNase H and is now termed RNase HII. Renaturation gel assays and gel filtration experiments proved a monomeric structure for the active enzyme with a native molecular weight of about 33 kDa. The human RNase HII acts as an endonuclease and releases oligoribonucleotides with 3'-OH and 5'-phosphate ends. It is therefore a candidate for the RNase H-mediated effect of antisense oligodeoxynucleotides.
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PMID:Purification and characterization of human ribonuclease HII. 781 13

We have studied the roles of metal cofactors Mg2+ and Mn2+ in modulating substrate specificities during the enzymatic cycle of TaqI endonuclease using steady state and single-turnover kinetics. In the presence of Mg2+, stringent discrimination of TaqI against single base-pair changes (star sites) is manifested by the loss of tight, specific binding in the early stage of the enzymatic cycle. In the presence of Mn2+, relaxed specificity for a star site sequence is attributed to formation of three distinct classes of the ternary complexes: the highly activated TaqI-cognate-Mn2+ complex; the partially activated TaqI-star-Mn2+ complex; and the ground state, inactive TaqI-nonspecific-Mn2+ complex. In addition to a high affinity for a TaqI-DNA complex, Mn2+ also binds to TaqI in a DNA-independent fashion. This may facilitate enzyme activation, which could account for the observed relaxation in substrate specificity. Thus, the TaqI-DNA-Mn2+ complex could be formed by either of two pathways: TaqI binding to DNA followed by the binding of Mn2+ or TaqI first binding to Mn2+ followed by the addition of DNA. The inactive, nonspecific TaqI-star-Mg2+ complex virtually prohibits transition state interactions, but a TaqI-star-Mn2+ complex attains a measurable single-turnover rate. In the late stages of the enzymatic cycle, high affinity of Mn2+ to a TaqI-DNA complex and to the TaqI enzyme may also account for a slower rate of product release.
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PMID:Stringent and relaxed specificities of TaqI endonuclease: interactions with metal cofactors and DNA sequences. 785 38

The repair of DNA requires the removal of abasic sites, which are constantly generated in vivo both spontaneously and by enzymatic removal of uracil, and of bases damaged by active oxygen species, alkylating agents and ionizing radiation. The major apurinic/apyrimidinic (AP) DNA-repair endonuclease in Escherichia coli is the multifunctional enzyme exonuclease III, which also exhibits 3'-repair diesterase, 3'-->5' exonuclease, 3'-phosphomonoesterase and ribonuclease activities. We report here the 1.7 A resolution crystal structure of exonuclease III which reveals a 2-fold symmetric, four-layered alpha beta fold with similarities to both deoxyribonuclease I and RNase H. In the ternary complex determined at 2.6 A resolution, Mn2+ and dCMP bind to exonuclease III at one end of the alpha beta-sandwich, in a region dominated by positive electrostatic potential. Residues conserved among AP endonucleases from bacteria to man cluster within this active site and appear to participate in phosphate-bond cleavage at AP sites through a nucleophilic attack facilitated by a single bound metal ion.
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PMID:Structure and function of the multifunctional DNA-repair enzyme exonuclease III. 788 81

The Escherichia coli RuvC protein is an endonuclease that resolves Holliday junctions. In vitro, the protein shows efficient structure-specific binding of Holliday junctions, yet the rate of junction resolution is remarkably low. We have mapped the sites of cleavage on a synthetic junction through which a crossover can branch migrate through 26 bp and find that > or = 90% of the junctions were cleaved at one site. This observation of sequence-specific cleavage suggests that inefficient resolution may be due to DNA binding events which occur away from the cleavage site and are therefore non-productive. Holliday junction resolution by RuvC protein can be stimulated by a number of factors including: (i) the presence of Mn2+ (rather than Mg2+) as the divalent metal cofactor, (ii) alkaline pH (< or = 10), and (iii) elevated temperature. These observations may indicate that other proteins are required for efficient RuvC-mediated resolution.
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PMID:Activation of RuvC Holliday junction resolvase in vitro. 804 10

We have purified a novel endonuclease from Escherichia coli that recognizes deoxyinosine, a deamination product of deoxyadenosine in DNA. This activity, which we named deoxyinosine 3' endonuclease, is different from the known hypoxanthine DNA N-glycosylases which have been partially characterized in E. coli and other organisms. The enzyme was purified 24,800-fold to apparent homogeneity. SDS- and activity PAGE analyses indicate that the enzyme has an apparent molecular mass of 25 kDa. Deoxyinosine 3' endonuclease recognized deoxyinosine in both single- and double-stranded DNA but exhibited a 4-fold preference for double stranded over single-stranded DNA. In addition to deoxyinosine, the enzyme recognized urea residues and AP sites. Deoxyinosine 3' endonuclease has an obligatory requirement for Mg2+, but other cations such as Co2+ and Mn2+ could partially replace Mg2+. The optimal pH for deoxyinosine 3' endonuclease was around 7.5. In contrast to most of the known repair enzymes, deoxyinosine 3' endonuclease makes an incision at the second phosphodiester bond 3' to a deoxyinosine or AP site, leaving behind the intact lesion on the nicked DNA. Therefore, deoxyinosine 3' endonuclease recognizes, but does not remove, the lesion from the DNA molecule. The biological significance of this novel activity is discussed with reference to other repair activities in E. coli.
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PMID:Purification and characterization of a novel deoxyinosine-specific enzyme, deoxyinosine 3' endonuclease, from Escherichia coli. 820 31

Terminases are enzymes common to all of the complex double-stranded DNA viruses and are required for viral assembly. These enzymes function to excise a single viral genome from a concatemeric DNA precursor and package it into a preformed protective protein shell or capsid. ATP hydrolysis by these enzymes has been described and appears to be critical to the packaging process. We have previously characterized the endonuclease activity of purified terminase from bacteriophage lambda [Tomka, M. A., & Catalano, C. E. (1993) J. Biol. Chem. 268, 3056-3065], and we describe here a kinetic characterization of the ATPase activity of the enzyme. lambda Terminase possesses a DNA-stimulated ATPase activity and hydrolyzes ATP to ADP and Pi. This activity requires divalent metal and is supported by all of the group IIa metals examined, as well as Mn2+. The reaction is also stimulated by NaCl, GTP, and dGTP. Of note is that neither of the guanosine nucleotides is hydrolyzed by the enzyme, while dATP is hydrolyzed at a rate comparable to that of ATP. Kinetic analysis of the ATPase activity revealed two apparent binding sites for ATP hydrolysis. The high-affinity site (Km = 5 microM) and low-affinity site (Km approximately 1.3 mM) hydrolyze ATP with kcat = 3 and 16 min-1, respectively. While the high-affinity site is unaffected by the presence of DNA, ATP hydrolysis at the low-affinity site is stimulated by DNA, which results from both a decrease in the Km and a concomitant increase in the kcat of the reaction.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Kinetic characterization of the ATPase activity of the DNA packaging enzyme from bacteriophage lambda. 821 75

Methods are reported for conjugating a cationic tris(methylpyridiniumyl)porphyrin-manganese(III) complex to oligonucleotides. The DNA cleaver motif used in the present work is related to Mn-TMPyP, a highly efficient artificial endonuclease able to hydroxylate C-H bonds of DNA sugars at nanomolar concentrations. A general coupling method has been developed by using porphyrin precursors having a linker attached at the para position of the meso phenyl group of the macrocycle and terminating with an activated ester of 1-hydroxybenzotriazole. Coupling reactions were monitored by HPLC. Purification of metalloporphyrin-oligonucleotide conjugates was performed by HPLC and desalting on Bio-Gel P-2. The purity of conjugates was always above 95%.
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PMID:Preparation of hybrid "DNA cleaver-oligonucleotide" molecules based on a metallotris(methylpyridiniumyl)porphyrin motif. 827 22

Cell death occurring by apoptosis has become widely recognized as an integral component of the life cycle of many cell types. Apoptosis can be induced in many tissues by a wide variety of endogenous signals, including glucocorticoids. However, even though there are glucocorticoid receptors present in almost all cells, only certain lymphoid cells are susceptible to glucocorticoid-induced apoptosis. The basis for this selective regulation of programmed cell death is unknown. Internucleosomal chromatin degradation is an integral characteristic of apoptosis, common to all cells undergoing this form of programmed cell death. Thus, we have developed an in vitro assay that recapitulates apoptotic DNA degradation in isolated nuclei and have identified a nuclease activity with internucleosomal specificity that is present in nuclear extracts of thymocytes undergoing glucocorticoid-induced apoptosis. We have now extended these studies to analyze the molecular properties of the crude enzyme and to further elucidate the mechanism of its regulation during the tissue-specific induction of apoptosis. In vitro, optimal internucleosomal cleavage activity was detected in the presence of millimolar concentrations of calcium and magnesium or manganese. Nuclease activity was inhibited by three agents previously shown to block apoptosis (zinc, aurintricarboxylic acid, and sodium), suggesting that the nuclease we detected in apoptotic thymocytes is responsible for the DNA degradation associated with apoptosis. Size-fractionation analysis of thymocyte nuclear extract under native conditions revealed a protein with an apparent molecular mass of 22.7 kilodaltons and a sedimentation coefficient of 3.5S. Interestingly, when extracts from control thymocytes, inactive in internucleosomal cleavage activity, were subjected to gel filtration or sucrose density gradient fractionation, internucleosomal cleavage activity was detected. The physical characteristics of this endonuclease activity were identical to those found in thymocyte extract from glucocorticoid-treated rats. Repressed or latent internucleosomal cleavage activity was also detected in hepatocyte nuclear extracts, but this activity was not activated by glucocorticoid treatment. These data suggest that glucocorticoid-induced apoptosis involves cell-specific activation of a latent endonuclease, rather than nuclease induction.
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PMID:Mechanism of tissue-specific induction of internucleosomal deoxyribonucleic acid cleavage activity and apoptosis by glucocorticoids. 839 69


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