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)

Double-stranded, full-length linear DNA was synthesized in vitro by using single-stranded linear DNA as a self-priming template from the parvovirus Kilham rat virus and Escherichia coli DNA polymerase "large fragment" as the polymerizing enzyme. To ascertain the order of the synthesis of the cleavage fragments and to assess the accuracy of the in vitro synthesis, restriction endonuclease cleavage sites with known recognition sequences were mapped on the DNA. Comparing the cleavage pattern of the synthesized DNA with that of double-stranded viral DNA isolated from infected cells confirms that the in vitro synthesis produces a faithful copy of the viral single-stranded genome. Electron micrographs of the in vitro product reveal it to be a double-stranded linear molecule.
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PMID:In vitro synthesis of double-stranded DNA from the Kilham rat virus single-stranded DNA genome. 21 93

Purified preparations of the parvovirus, Kilham rat virus, have associated with them a protein with DNA polymerase activity. The enzyme has been separated from the other two or three viral proteins and purified 63-fold. The viral associated enzyme was found in a single peak of DNA polymerase activity after chromatography on DEAE-cellulose, DNA-cellulose, and phosphocellulose columns. It shares some properties in common with the host cellular DNA polymerases, described in the preceding paper (Salzman, L.A., and McKerlie, L. (1975) J. Biol. Chem. 250, 5589-5595), but also has some important distinguishing characteristics. The Kilham rat virus-associated DNA polymerase has increased enzyme activity in the presence of 0.02 M KCl and has a strong preference for a synthetic DNA polymer containing deoxyadenylate and deoxythymidylate. The enzyme has a molecular weight of approximately 75,000 plus or minus 3,000 and appears to contain endonuclease activity.
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PMID:Characterization of the deoxyribonucleic acid polymerase associated with Kilham rat virus. 23 24

During an electron-microscopic survey with the aim of identifying the parvovirus MVM transcription template, we observed previously unidentified structures of MVM DNA in lysates of virus-infected cells. These included double-stranded "lasso"-like structures and relaxed circles. Both structures were of unit length MVM DNA, indicating that they were not intermediates formed during replication; they each represented about 5% of the total nuclear MVM DNA. The proportion of these structures was unchanged after digestion with sodium dodecyl sulfate/Pronase and RNase and after mild denaturation treatment. Cleavage of the "lasso" structures with EcoRI restriction endonuclease indicated that the "noose" part of the "lasso" structure is located on the 5' side of the genomic single-stranded MVM DNA. A model is presented for the molecular nature of the circularization process of MVM DNA in which the "lasso" structures are identified as intermediates during circle formation. This model proposes a mechanism for circularization of linear DNAs.
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PMID:Mechanism for circularization of linear DNAs: circular parvovirus MVM DNA is formed by a "noose" sliding in a "lasso"-like DNA structure. 29 64

The double-stranded replicative form (RF) DNA of the autonomous parvovirus H-1 can be isolated from infected cells in a covalent complex with protein. The protein is present on most or all of the RF DNA, including actively replicating molecules, and is associated with the 5'-terminal endonuclease Hae III fragments of both the viral and complementary strands of RF. The size of the protein is estimated to be 60,000-70,000 daltons from its effect on buoyant density of DNA. DNA with covalently bound protein has not been found in H-1 virions.
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PMID:Covalent association of protein with replicative form DNA of parvovirus H-1. 29 59

The genome of the nondefective parvovirus minute virus of mice (MVM) is a linear DNA molecular weight 1.48 x 10(6), which is single stranded for approximately 94% of its length. In contrast to the genomes from defective parvoviruses MVM DNA does not contain a detectable inverted terminal redundancy. A combination of enzymatic and physical techniques has shown that the molecule contains a stable hairpin duplex of approximately 130 base pairs located at the 5' terminus of the genome. MVM DNA is efficiently utilized as a template-primer by a number of DNA polymerases, including reverse transcriptases. Polymerases lacking 5' to 3' exonuclease activity yield a duplex DNA product with a molecular weight 1.96 times that of the viral genome, in which the newly synthesized complementary strand is covalently attached to the template. This duplex contains an internal "nick" that can be sealed by DNA ligase to produce a self-complementary single-strand circle. The MVM DNA duplex is cleaved twice by EcoR-RI restriction endonuclease to yield three distinct fragments in molar amounts. These results suggest that the initiation of DNA synthesis in vitro occurs at a point within 100 bases of the 3' end of the genome, using the 3' terminus of viral DNA as a primer, and that the sequence of nucleotides in the genome is not permuted.
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PMID:DNA of minute virus of mice: self-priming, nonpermuted, single-stranded genome with a 5'-terminal hairpin duplex. 78 12

Partial denaturation mapping, restriction endonuclease digestion, and electron microscopy were used to determine which end of the linear duplex replicative-form (RF) DNA molecule contains the origin of RF replication for the parvovirus H-1. This origin was localized within approximately 300 base pairs of the arbitrarily designated right end of the RF DNA, in the EcoRI or HaeII-A fragment. Based on denaturation behavior in formamide, the right end was also found to have a relatively high guanine plus cytosine content, whereas the region adjacent to the left terminus of the RF DNA molecule was adenine plus thymine rich.
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PMID:Replication process of the parvovirus H-1. VIII. Partial denaturation mapping and localization of the replication origin of H-1 replicative-form DNA with electron microscopy. 83 45

The linear duplex replicative form (RF) DNA of the parvovirus H-1 has been characterized with respect to cleavage by the bacterial restriction endonuclease of Escherichia coli, EcoRI. RF DNA has a single cleavage site 0.22 genome length from the left end of the molecule. The molecular weight of H-1 RF DNA determined by gel electrophoresis is 3.26 X 10(6). H-1 RF DNA has been found to dimerize by hydrogen-bounded linkage at the molecular left end, and in some molecules the viral strand is covalently linked to the complementary strand. Some 10% of monomeric RF DNA also has a covalent linkage between the viral and complementary strands at the left end. The EcoRI-B fragment, containing the left end of the RF molecule, appears to be a replication terminus by its labeling characteristics for both RF and progeny DNA synthesis. These findings suggest that the left end of H-1 RF DNA has some type of "turn-around" structure and that this end is not an origin for DNA synthesis.
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PMID:Replication process of the parvovirus H-1. VI. Characterization of a replication terminus of H-1 replicative-form DNA. 85 76

The adeno-associated virus (AAV) Rep protein is required for both viral DNA replication and transactivation of the AAV promoters. Here we report the effects of mutations in the rep gene on transcription and replication in vivo and terminal repeat binding and terminal resolution site (trs) endonuclease activities in vitro. In all, we examined 10 in-frame deletions and 14 amino acid substitution mutations at eight positions. The point mutations were targeted to regions that are highly conserved among the parvovirus nonstructural proteins and include the extended ATPase domain of the AAV Rep protein. The mutations identify at least two noncontiguous regions of Rep which are essential for terminal repeat binding (amino acids 134 to 242 and amino acids 415 to 490). Mutations in either region render the protein inactive for both DNA replication and transactivation. In addition, mutations within a putative ATPase region also cause defects in replication and transactivation in vivo as well as in the ATP-dependent trs endonuclease activity in vitro. These results suggest that Rep transactivates via a novel mechanism which may require both DNA binding and an enzymatic activity, namely, ATPase or DNA helicase activity.
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PMID:Analysis of mutations in adeno-associated virus Rep protein in vivo and in vitro. 131 96

DNA amplification of the helper-dependent parvovirus AAV (adeno-associated virus) can be induced by a variety of genotoxic agents in the absence of coinfecting helper virus. Here we investigated whether the origin of AAV type 2 DNA replication cloned into a plasmid is sufficient to promote replication activity in cells treated by the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). A pUC19-based plasmid, designated pA2Y1, which contains the left terminal repeat sequences (TRs) representing the AAV origin of replication and the p5 and p19 promoter but lacks any functional parvoviral genes is shown to confer replication activity and to allow selective DNA amplification in carcinogen-treated cells. Following transfection of plasmid pA2Y1 or plasmid pUC19 as a control, density labeling by a bromodeoxyuridine and DpnI resistance assay suggested a semi-conservative mode of replication of the AAV origin-containing plasmid. Furthermore, the amount of DpnI-resistant full-length pA2Y1 DNA molecules was increased by MNNG treatment of cells in a dose-dependent manner. In addition, DNA synthesis of plasmid pA2Y1 was studied in vitro. Extracts derived from MNNG-treated CHO-9 and L1210 cells displayed greater synthesis of DpnI-resistant full-length pA2Y1 molecules than did nontreated controls. Experiments with specific enzyme inhibitors suggested that the reaction is largely dependent on DNA polymerase alpha, DNA primase, and DNA topoisomerase I. Furthermore, restriction endonuclease mapping analysis of the in vitro reaction products revealed the occurrence of specific initiation at the AAV origin of DNA replication. Though elongation was not very extensive, extracts from carcinogen-treated cells markedly amplified the AAV origin region. Our results, including electron microscopic examination, suggest that the AAV origin/terminal repeat structure is recognized by the cellular DNA replicative machinery induced or modulated by carcinogen treatment in the absence of parvoviral gene products.
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PMID:Origin of adeno-associated virus DNA replication is a target of carcinogen-inducible DNA amplification. 203 69

The genome DNAs of 12 strains of human parvovirus B19, isolated in Japan at two different times, 1981 and 1986 to 1987, were molecularly cloned in the plasmid pUC18. The cloned B19 DNAs were analysed by cleaving with restriction endonucleases, and were classified into several groups by genome type. The restriction endonuclease cutting patterns of B19 strains isolated during 1981 were similar to that of the group IV genome type, and the patterns of those isolated later were similar to that of group II, suggesting a correlation between the genome type and the prevalence. We conclude that the prevalences of B19 infection in Japan during 1981 and in 1986 to 1987 were caused by viruses differing in genome type, and that B19 viruses with similar genome types disseminated widely in Japan during each prevalence.
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PMID:The genome type of human parvovirus B19 strains isolated in Japan during 1981 differs from types detected in 1986 to 1987: a correlation between genome type and prevalence. 215 7


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