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)

The bacteriophage lambda terminase is composed of two subunits, gpNu1 and gpA. In vitro, the holoenzyme is a site-specific endonuclease, helicase, ATPase, and can package lambda DNA into proheads. gpA possesses ATPase and helicase activities which are similar to those of the holoenzyme. Both terminase and gpA can hydrolyze a wide range of deoxyribo- and ribonucleoside triphosphates to inorganic phosphate and the corresponding diphosphate. Nucleoside diphosphates are not substrates for either protein. ATPase of both proteins is stimulated by double-stranded DNA. The ATPase of gpA is protein concentration-dependent, while that of terminase is not. Helicase activity of both proteins is not concentration-dependent, and requires a hydrolyzable triphosphate. ATP, dATP, and GTP supported helicase activity, while adenosine 5'-(beta, gamma-methylene)triphosphate, adenosine 5'-3-O-(thio)triphosphate, ADP, CTP, and UTP did not. The kinetic parameters of ATPase and helicase activities were similar for both proteins, but packaging with terminase was optimal only at a significantly higher level of ATP. Packaging was detectable at significant levels with CTP and UTP, but not with GTP. Packaging also differed from ATPase and helicase in the utilization of divalent metal cations and susceptibility to various inhibitors.
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PMID:The in vitro ATPases of bacteriophage lambda terminase and its large subunit, gene product A. The relationship with their DNA helicase and packaging activities. 817 94

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

The role of G protein mutations in the pathogenesis of adrenal cortex neoplasms is controversial. Two published studies disagree on the existence of a cysteine or histidine for arginine substitution at position 179 (R179C/H) of the GTP binding region of the alpha chain of an inhibitory G protein (Gi2alpha) in these tumors. Prior studies using detection by mutation-specific oligonucleotide hybridization showed either 3 of 11 or 0 of 56 tumors harbored mutations. To resolve this discrepancy and ascertain the importance of the R179C/H Gi2alpha mutation in the development of adrenal cortex tumors, we screened tumors from 29 patients (24 with adenoma, 5 with carcinoma) using a more sensitive assay employing polymerase chain reaction (PCR) and examination for restriction fragment length polymorphisms (RFLP). Detection of the potential R179C/H mutation by this technique was possible because the wild-type coding sequence includes the BSTU-1 restriction endonuclease recognition site CGCG, whereas the mutated gene does not. Results showed complete digestion of the amplified DNA samples from all 29 patients and the negative control DNA by BSTU-1, indicating that all tumor samples exhibited only the wild-type sequence. Direct sequencing of PCR product from four tumor samples confirmed the presence of only the wild-type sequence. The 0 of 29 rate of R179C/H mutations we found in Gi2alpha is different than the 3 of 11 positive rate (p < 0.05, Fishers' exact) previously reported but agrees with the report showing 0 of 56 mutations. We conclude a mutation at position 179 of Gi2alpha is not important in the pathogenesis of most adrenal cortical tumors.
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PMID:Gip-2 codon 179 oncogene mutations: absent in adrenal cortical tumors. 867 43

McrBC is a GTP-dependent restriction endonuclease of E. coli K12, selectively directed against DNA containing modified cytosine residues. McrB, one of its components, is responsible for the binding and, together with McrC, for the cleavage of DNAs containing two 5'-Pu(m)C sites separated by 40-80 base pairs. Gel retardation assays with wild-type and mutant McrB reveal that (i) single 5'-Pu(m)C sites in DNA can be sufficient to elicite binding by McrB. Binding to such substrates is, however, weak and strongly dependent on the sequence context of Pu(m)C sites. (ii) Strong DNA binding (K(ass) approximately 10(7)M[-1]) is dependent on the presence of at least two Pu(m)C sites, even if they are separated by less than 40 bp, and is modulated by the sequence context (-A(m)CCGGT- --> -A(m)CT(C/G)AGT- --> -AGG(m)CCT- --> -AAG(m)CTT-). (iii) DNA binding by McrB is accompanied by formation of distinct multiple complexes whose distribution is modulated by GTP. (iv) McrC, which cannot bind DNA by itself, moderately stimulates the DNA binding of McrB and converts McrB-DNA complexes to large aggregates. (v) Deletion of the C-terminal half of McrB, which harbors the three consensus sequences characteristic for guanine nucleotide binding proteins, leads to protein inactive in GTP binding and/or hydrolysis and in McrC-assisted DNA cleavage; the protein, however, remains fully competent in DNA binding. (vi) Mutations in McrB which lead to a reduction in GTP binding and/or hydrolysis can affect DNA binding, suggesting that the two activities are coupled in the full-length protein.
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PMID:The recognition of methylated DNA by the GTP-dependent restriction endonuclease McrBC resides in the N-terminal domain of McrB. 934 6

McrBC is a methylation-dependent endonuclease from Escherichia coli K-12. The enzyme recognizes DNA with modified cytosines preceded by a purine. McrBC restricts DNA that contains at least two methylated recognition sites separated by 40-80 bp. Two gene products, McrBL and McrBs, are produced from the mcrB gene and one, McrC, from the mcrC gene. DNA cleavage in vitro requires McrBL, McrC, GTP and Mg2+. We found that DNA cleavage was optimal at a ratio of 3-5 McrBL per molecule of McrC, suggesting that formation of a multisubunit complex with several molecules of McrBL is required for cleavage. To understand the role of McrBs, we have purified the protein and analyzed its role in vitro. At the optimal ratio of 3-5 McrBL per molecule of McrC, McrBs acted as an inhibitor of DNA cleavage. Inhibition was due to sequestration of McrC and required the presence of GTP, suggesting that the interaction is GTP dependent. If McrC was in excess, a condition resulting in suboptimal DNA cleavage, addition of McrBs enhanced DNA cleavage, presumably due to sequestration of excess McrC. We suggest that the role of McrBs is to modulate McrBC activity by binding to McrC.
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PMID:McrBs, a modulator peptide for McrBC activity. 973 25

The adeno-associated virus (AAV) nonstructural proteins Rep68 and Rep78 are site-specific DNA binding proteins, ATP-dependent site-specific endonucleases, helicases, and ATPases. These biochemical activities are required for viral DNA replication and control of viral gene expression. In this study, we characterized the biochemical properties of the helicase and ATPase activities of homogeneously pure Rep68. The enzyme exists as a monomer in solution at the concentrations used in this study (<380 nM), as judged by its mobility in sucrose density gradients. Using a primed single-stranded (ss) circular M13 substrate, the helicase activity had an optimum pH of 7 to 7.5, an optimum temperature of 45 degreesC, and an optimal divalent-cation concentration of 5 mM MgCl2. Several nucleoside triphosphates could serve as cofactors for Rep68 helicase activity, and the order of preference was ATP = GTP > CTP = dATP > UTP > dGTP. The Km values for ATP in both the DNA helicase reaction and the site-specific trs endonuclease reaction were essentially the same, approximately 180 microM. Both reactions were sigmoidal with respect to ATP concentration, suggesting that a dimer or higher-order multimer of Rep68 is necessary for both DNA helicase activity and terminal resolution site (trs) nicking activity. Furthermore, when the enzyme itself was titrated in the trs endonuclease and ATPase reactions, both activities were second order with respect to enzyme concentration. This suggests that a dimer of Rep68 is the active form for both the ATPase and nicking activities. In contrast, DNA helicase activity was linear with respect to enzyme concentration. When bound to ssDNA, the enzyme unwound the DNA in the 3'-to-5' direction. DNA unwinding occurred at a rate of approximately 345 bp per min per monomeric enzyme molecule. The ATP turnover rate was approximately 30 to 50 ATP molecules per min per enzyme molecule. Surprisingly, the presence of DNA was not required for ATPase activity. We estimated that Rep translocates processively for more than 1,300 bases before dissociating from its substrate in the absence of any accessory proteins. DNA helicase activity was not significantly stimulated by substrates that have the structure of a replication fork and contain either a 5' or 3' tail. Rep68 binds only to ssDNA, as judged by inhibition of the DNA helicase reaction with ss or double-stranded (ds) DNA. Consistent with this observation, no helicase activity was detected on blunt-ended ds oligonucleotide substrates unless they also contained an ss 3' tail. However, if a blunt-ended ds oligonucleotide contained the 22-bp Rep binding element sequence, Rep68 was capable of unwinding the substrate. This means that Rep68 can function both as a conventional helicase for strand displacement synthesis and as a terminal-repeat-unwinding protein which catalyzes the conversion of a duplex end to a hairpin primer. Thus, the properties of the Rep DNA helicase activity suggest that Rep is involved in all three of the key steps in AAV DNA replication: terminal resolution, reinitiation, and strand displacement.
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PMID:Biochemical characterization of adeno-associated virus rep68 DNA helicase and ATPase activities. 988 64

The methylation-dependent restriction endonuclease McrBC from Escherichia coli K12 cleaves DNA containing two R(m)C dinucleotides separated by about 40 to 2000 base-pairs. McrBC is unique in that cleavage is totally dependent on GTP hydrolysis. McrB is the GTP binding and hydrolyzing subunit, whereas MrC stimulates its GTP hydrolysis. The C-terminal part of McrB contains the sequences characteristic for GTP-binding proteins, consisting of the GxxxxGK(S/T) motif (position 201-208), followed by the DxxG motif (position 300-303). The third motif (NKxD) is present only in a non-canonical form (NTAD 333-336). Here we report a mutational analysis of the putative GTP-binding domain of McrB. Amino acid substitutions were initially performed in the three proposed GTP-binding motifs. Whereas substitutions in motif 1 (P203V) and 2 (D300N) show the expected, albeit modest effects, mutation in the motif 3 is at variance with the expectations. Unlike the corresponding EF-Tu and ras -p21 variants, the D336N mutation in McrB does not change the nucleotide specificity from GTP to XTP, but results in a lack of GTPase stimulation by McrC. The finding that McrB is not a typical G protein motivated us to perform a search for similar sequences in DNA databases. Eight microbial sequences were found, mainly from unfinished sequencing projects, with highly conserved sequence blocks within a presumptive GTP-binding domain. From the five sequences showing the highest homology, 17 invariant charged or polar residues outside the classical three GTP-binding motifs were identified and subsequently exchanged to alanine. Several mutations specifically affect GTP affinity and/or GTPase activity. Our data allow us to conclude that McrB is not a typical member of the superfamily of GTP-binding proteins, but defines a new subfamily within the superfamily of GTP-binding proteins, together with similar prokaryotic proteins of as yet unidentified function.
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PMID:The GTP-binding domain of McrB: more than just a variation on a common theme? 1049 20

The GTP-dependent restriction endonuclease McrBC of E. coli K12, which recognizes cytosine-methylated DNA, consists of two protein subunits, McrB and McrC. We have investigated the structural assignment and interdependence of the McrB subunit functions, namely (i) specific DNA recognition and (ii) GTP binding and hydrolysis. Extending earlier work, we have produced McrB variants comprising N- and C-terminal fragments. The variants McrB1-162 and McrB1-170 are still capable of specific DNA binding. McrB169-465 shows GTP binding and hydrolysis characteristics indistinguishable from full-length McrB as well as wild-type like interaction with McrC. Thus, DNA and GTP binding are spatially separated on the McrB molecule, and the respective domains function quite independently.
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PMID:Defining the location and function of domains of McrB by deletion mutagenesis. 1059 86

McrBC, a GTP-requiring, modification-dependent endonuclease of Escherichia coli K-12, specifically recognizes DNA sites of the form 5' R(m)C 3'. DNA cleavage normally requires translocation-mediated coordination between two such recognition elements at distinct sites. We have investigated assembly of the cleavage-competent complex with gel-shift and DNase I footprint analysis. In the gel-shift system, McrB(L) binding resulted in a fast-migrating specific shifted band, in a manner requiring both GTP and Mg(2+). The binding was specific for methylated DNA and responded to local sequence changes in the same way that cleavage does. Single-stranded DNA competed for McrB(L)-binding in a modification and sequence-specific fashion. A supershifted species was formed in the presence of McrC and GTPgammaS. DNase I footprint analysis showed modest cooperativity in binding to two sites, and a two-site substrate displayed protection in non-specific spacer DNA in addition to the recognition elements. The addition of McrC did not affect the footprint obtained. We propose that McrC effects a conformational change in the complex rather than a reorganization of the DNA:protein interface.
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PMID:Methyl-specific DNA binding by McrBC, a modification-dependent restriction enzyme. 1078 24

RNA splicing in archaea requires at least an endonuclease and a ligase, as is the case for the splicing of eukaryal nuclear tRNAs. Splicing endonucleases from archaea and eukarya are homologous, although they differ in subunit composition and substrate recognition properties. However, they all produce 2',3' cyclic phosphate and 5'-hydroxyl termini. An in vitro-transcribed, partial intron-deleted Haloferax volcanii elongator tRNA(Met) has been used to study splicing by H. volcanii cell extracts. Substrates and products were analyzed by nearest neighbor analyses using nuclease P1 and RNase T2, and fingerprinting analyses using acid-urea gels in the first dimension and gradient thin layer chromatography in the second dimension. The results suggest that 2',3' cyclic phosphate at the 3' end of the 5' exon is converted into the splice junction phosphate forming a 3',5'-phosphodiester linkage. This resembles the animal cell type systems where the junction phosphate preexists in the transcript, and differs from yeast type systems, where GTP is the source of junction phosphate.
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PMID:Junction phosphate is derived from the precursor in the tRNA spliced by the archaeon Haloferax volcanii cell extract. 1091 97


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