Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.30.2 (endonuclease)
 18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The cistron A protein induced by phage varphiX174 nicks (produces a single-strand break in) the viral strand of the superhelical varphiX duplex DNA, thereby forming a complex with the DNA. The protein, seen bound to the DNA in the electron microscope, was located in the restriction endonuclease fragment between nucleotides 4290 and 4330 on the varphiX map [Sanger, F., Air, G. M., Barrel, B. G., Brown, N. L., Coulson, A. R., Fiddes, J. C., Hutchison, C. A., III, Slocomb, P. M. Y. & Smith, M. (1977) Nature 265, 687-695]. Replication also was initiated at this point, thus identifying the site of cistron A protein nicking and binding as the origin of replication. The cisA-DNA complex (separated from free cistron A protein), upon the addition of Escherichia coli rep protein, ATP, and DNA binding protein, is unwound to generate a single-stranded linear [presumably the nicked (+) strand] and a circular [presumably the (-) strand] molecule. The cisA-DNA complex, upon the further addition of DNA polymerase III holoenzyme and deoxynucleoside triphosphates, supports replication to generate viral, single-stranded circles, as many as 15 circles per cisA-DNA complex. The replicating intermediates seen in the electron microscope are a novel form of "rolling circle" [Gilbert, W. & Dressler, D. H. (1969) Cold Spring Harbor Symp. Quant. Biol. 33, 473-485]. The 5' end (presumably with the cistron A protein bound to it) is locked in the replication fork and loops back to accompany the strand-separation and replication fork around the template [(-) strand] circle. Thus, the multiple functions of cistron A protein include: (i) nicking the viral strand at the origin of replication to initiate a round of replication, (ii) participating in a complex which supports fork movement in strand separation and replication, (iii) nicking again at the regenerated origin to produce a unit-length DNA, and (iv) ligating the newly generated 3'-OH end to the 5'-phosphate-complexed end to form a circular viral molecule.
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PMID:phiX174 cistron A protein is a multifunctional enzyme in DNA replication. 26 83

Among the mediators of interferon action are one enzyme that is activated by double-stranded RNA to convert ATP to (2'-5')An and a second enzyme, an endonuclease, that is activated by (2'-5')An to cleave single-stranded RNA. The binding of (2'-5')An to the endonuclease (partially purified from mouse Ehrlich ascites tumor cells) is revealed by its retention on nitrocellulose filters. This can serve as the basis for an assay of the enzyme. Activation of the enzyme is reversible and is lost upon removal of (2'-5')An:gel filtration of activated endonuclease on Sephacryl S-200 results in an inactive enzyme. The enzyme can be activated again, however, by addition of (2'-5')An. The elution volume of the nonactivated endonuclease from Sephadex G-200 indicates that its molecular weight is 185,000, unusually large for a nuclease. The elution volume of the maximally activated endonuclease from Sephadex G-200 equilibrated with (2'-5')An is not detectably different from that of enzyme that had not been previously activated that was passed through Sephadex G-200 not equilibrated with (2'-5')An. This indicates that the activation does not result in a large change in the size or conformation of the enzyme.
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PMID:Interferon, double-stranded RNA, and RNA degradation: activation of an endonuclease by (2'-5')An. 29 97

Photoalkylation of circular covalently closed DNA from phage PM2 with isopropyl alcohol by using a free radical photoinitiator and UV light of lambda greater than 305 nm led to the specific 8-substitution of purine moieties in the DNA, yielding 8-(2-hydroxy-2-propyl)adenine and 8-(2-hydroxy-2-propyl)guanine as the only detectable damage in the DNA. Using this specifically photoalkylated DNA as a substrate, we discovered in extracts of Micrococcus luteus an endonucleolytic activity that is directed towards 8-(2-hydroxy-2-propyl) purines in DNA. The activity is not a combination of a DNA-glycosylase and an apurinic site endonuclease. It is not inhibited by single-stranded DNA, by UV- or gamma-irradiated single-stranded DNA, or by normal or depurinated double-stranded DNA. however, gamma- or UV-(254 nm) irradiated double-stranded DNAs to inhibit the activity, hinting at the possibility of a common type of lesion in these damaged DNAs. Divalent cations are not required for the incising activity, and it is fully active in 1 mM EDTA, whereas caffeine and ATP cause inhibition. Extracts of mutant M. luteus lacking pyrimidine-dimer-directed endonucleases were found to contain the endonucleolytic activity in levels comparable to those present in the wild type. After the incision, we could demonstrate the specific excision of the 8-alkylated purines from the damaged DNA. The special conformational consequences of the 8-alkylation of purines, at the nucleotide level, namely their nonregular syn conformation, suggest that it is the distortion in the DNA that is recognized by the endonuclease.
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PMID:Endonucleolytic activity directed towards 8-(2-hydroxy-2-propyl) purines in double-stranded DNA. 29 58

The interaction of Escherichia coli DNA-dependent RNA polymerase (EC 2.7.7.6) with the replicative form of the DNA from the filamentous coliphage fd cleaved by the restriction endonuclease HindII has been studied by electron microscopy at low and high ionic strength. In the presence of ATP or GTP, and heparin, RNA polymerase binds to fd replicative-form DNA at a few specific sites which have been mapped. The map was oriented so that transcription is from right to left. Three main GTP initiator sites are found at 15%, 82% and 94% of the genome length. One main ATP initiator site is found which cannot be mapped with the same accuracy, and which is localized between 38% and 50%. In the absence of initiator triphosphates and heparin, the binding of the enzyme to fd DNA is much more heterogeneous and therefore the mapping is more difficult. Nevertheless it seems that the preferential binding regions correspond to the specific sites mapped in the presence of GTP or ATP. The mean number of polymerase molecules bound to DNA as a function of the molecular ratio enzyme to DNA present in the mixture has been determined. From these results a binding isotherm can be obtained. The apparent equilibrium constant (K approximately 10(9) M-1) which is derived certainly represents an under-estimated value, as discussed.
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PMID:Electron microscopy analysis of the interaction between Escherichia coli DNA-dependent RNA polymerase and the replicative form of phage fd DNA. 1. Mapping of the binding sites. 33 31

An in vitro complementation assay has been used for partial purification of uvrA+, uvrB+, and uvrC+ gene products from Escherichia coli. The uvrB+ and uvrC+ products cochromatograph on DEAE-cellulose and are completely resolved from the uvrA+ product, which has been further purified by phosphocellulose chromatography of the nonadsorbed protein fraction from the DEAE-cellulose. Neither the uvrB+/uvrC+ nor the uvrA+ product shows appreciable endonuclease activity on UV-irradiated DNA when tested separately. However, these factors complement each other to yield and ATP-dependent endonuclease activity specific for UV-irradiated DNA. Gel filtration experiments with the partially purified proteins indicate that the functional uvrA+ gene product has a molecular weight of 100,000. The uvrB+ gene product has an apparent molecular weight of 70,000, but it is presently unclear if this is the size of the uvrB+ product alone or the size of a complex of the uvrB+ and uvrC+ gene products.
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PMID:Reconstitution of an Escherichia coli repair endonuclease activity from the separated uvrA+ and uvrB+/uvrC+ gene products. 35 11

The restriction enzyme from a restriction and modification-deficient strain of Escherichia coli K mutated in the modification gene (hsdM) has been purified using an in vitro complementation assay with a mutant restriction enzyme from a strain lacking only restriction. The restriction enzyme from the hsdM mutant lacks all of the activities that are associated with the wild type enzyme: binding of unmodified DNA to filters, cleavage, or methylation of unmodified DNA and ATP hydrolysis. It is shown that the enzyme from this hsdM mutant cannot bind S-adenosylmethionine, an allosteric effector in the restriction reaction. In the absence of enzyme activation by S-adenosylmethionine, no binding to unmodified DNA takes place. A comparison with other mutant restriction enzymes allows us to outline the biochemical role of the subunits of the E. coli K restriction endonuclease.
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PMID:Characterization of a restriction enzyme from Escherichia coli K carrying a mutation in the modification subunit. 35 30

Extracts from interferon-treated, not virus infected EAT cells differ in several biochemical characteristics from extracts of untreated cells. Some of these differences are manifested only if the extracts are supplemented with ds RNA and ATP. Thus, in the extracts from interferon-treated cells these supplements activate a protein kinase and an endonuclease activity as well as an inhibitor of the translation of messenger RNA. The effect of the same supplements in extracts of untreated cells is much less pronounced. Other differences between the two types of extracts do not seem to depend on the addition of ds RNA and ATP. These include an impairment of mRNA cap methylation and an inhibition of peptide chain elongation that can be overcome by the addition of tRNA. The treatment of human (HeLa S3) cells with human interferon is manifested in the cell extract similarly to the treatment of EAT cells with mouse interferon. Studies are underway to isolate and characterize the ds RNA activated enzymes and the inhibitors and to establish how the presence of these in extracts from interferon-treated cells can account for the impairment of virus replication by interferon.
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PMID:Messenger RNA methylation, translation and degradation in extracts of interferon-treated cells. 35 50

The method developed for the total synthesis of a given DNA containing biologically specific sequences consists of the following. The DNA in the double-stranded form is carefully divided into short single-stranded segments with suitable overlaps in the complementary strands. All the segments are chemically synthesized starting with protected nucleosides and mononucleotides. The 5'-OH ends of the appropriate oligonucleotides are then phosphorylated with the use of [y-32P]ATP and polynucleotide kinase. A few to several neighboring oligonucleotides are then allowed to form bihelical complexes in aqueous solution, and the latter are joined end to end by polynucleotide ligase to form covalently linked duplexes. Subsequent heat-to-tail joining of the short duplexes leads to the total DNA. The methods are described for the construction of a biologically functional suppressor transfer RNA gene. The total work involved (i) the synthesis of a 126-nucleotide-long bihelical DNA corresponding to a known precursor to the tyrosine suppressor transfer RNA, (ii) the sequencing of the promoter region and the distal region adjoining the C-C-A end, which contained a signal for the processing of the RNA transcript, (iii) total synthesis of the 207 base-pair-long DNA, which included the control elements, as well as the Eco R1 restriction endonuclease specific sequences at the two ends, and (iv) full characterization by transcription in vitro and amber suppressor activity in vivo of the synthetic gene.
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PMID:Total synthesis of a gene. 36 49

The phi29 early mRNA's synthesized in infected Bacillus subtilis were studied by using sedimentation velocity analysis, polyacrylamide gel electrophoresis, and hybridization of phi29 DNA fragments generated by the restriction endonuclease Eco RI. Viral RNAs synthesized in vivo in the resence of chloramphenicol were found to hybridize to Eco RI-A, -C, and -D fragments, but not to Eco RI-B and -E fragments, of the viral genome. Major early mRNA sedimenting as 16S material in neutral sucrose gradients was examined in detail. Radioactive phi29 RNA, purified by sucrose gradient centrifugation, was hybridized to either the Eco RI-A or Eco RI-C DNA fragment. The RNA was eluted from the hybrids and then tested for complementary hybrid formation with Eco RI-A and -C fragments. RNA eluted from the Eco RI-A fragment annealed only to the Eco RI-A fragment and not to the Eco RI-C fragment. Similarly, RNA eluted from the Eco RI-C fragment hybridized to the Eco RI-C and -D fragments. Viral RNAs synthesized in vitro using B. subtilis RNA polymerase hybridized to both Eco RI-A and -C DNA fragments. Furthermore, RNA initiated with [gamma-(32)P]GTP also hybridized to both Eco RI-A and -C fragments. These results indicate that there are at least two efficient promotors for early transcription on the phi29 chromosome. In addition, a low-molecular-weight RNA initiated with [gamma-(32)P]ATP was found to hybridize exclusively with the Eco RI-A fragment. Kinetic studies of phi29 mRNA synthesis during the lytic cycle have shown that viral RNAs hybridizable to the Eco RI-A and -C fragments are synthesized immediately after phage infection. On the other hand, mRNA specific for the Eco RI-B fragment was not synthesized for several minutes after phage infection. Based on the results of the in vivo and in vitro transcription studies, a transcription map of the phi29 chromosome is proposed.
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PMID:Transcription of the genome of bacteriophage phi 29: isolation and mapping of the major early mRNA synthesized in vivo and in vitro. 40 15

Extracts of interferon-treated HeLa cells adsorbed to poly(I) . poly(C)-agarose have been used to synthesize 2'5'oligo(A). This oligonucleotide has been characterized by enzymatic digestion with alkaline phosphatase, snake venom phosphodiesterase, T2 ribonuclease and chromatography on DEAE, and PEI-cellulose. The oligonucleotide inhibits protein synthesis in vitro and activates an endonuclease present in extracts of control and interferon-treated cells. The metabolic stability of 2'5'oligo(A) has been investigated in these cell extracts. The oligonucleotide undergoes rapid degradation, particularly in the absence of ATP and of an energy regenerating system. Furthermore, the 2'5'oligo(A)-activated endonuclease reverts to an inactive state under these conditions, but can be reactivated upon further addition of 2'5'oligo(A). A possible role for the degradation of 2'5'oligo(A) in the mechanism of interferon action is discussed.
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PMID:Metabolic stability of 2' 5'oligo (A) and activity of 2' 5'oligo (A)-dependent endonuclease in extracts of interferon-treated and control HeLa cells. 42 14


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