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)

Relative abundances of early virus RNA species in the cytoplasm of cells infected with wild-type adenovirus type 5 (WT Ad5) and a temperature-sensitive "early" mutant, H5ts125 (ts125), were compared by hybridization kinetics using separated strands of HindIII restriction endonuclease fragments of Ad5 DNA. 1-beta-D-Arabinofuranosylcytosine (ara-C) was used to limit transcription to early virus genes in cells infected by WT virus. At 40.5 degrees C, a restrictive temperature for ts125, three to seven times as much virus RNA from all four early regions of the genome accumulated in the cytoplasm of cells infected by the mutant as accumulated in cells infected by WT. At 32 degrees C, no such difference in the relative abundances of cytoplasmic virus RNA was observed. The capacity to synthesize a 72,000-dalton (72K) virus polypeptide, presumably the single-stranded DNA-binding protein that is defective in ts125 at restrictive temperatures, was compared in cells infected at 40.5 degrees C in the presence of ara-C with the mutant or WT Ad5. The rate of 72K polypeptide synthesis, measured by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis of [35S]methionine-labeled polypeptides and autoradiography, was greater at 15 h after infection in ts125-infected cells than in cells infected by WT. A time course experiment showed that the rate of synthesis of the 72K polypeptide increased continuously in ts125-infected cells during the first 15 h of infection, relative to the rate in WT-infected cells. These data are consistent with the hypothesis that Ad5 early gene expression is modulated by the product of an early gene, the 72K DNA-binding protein.
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PMID:Possible role of the 72,000 dalton DNA-binding protein in regulation of adenovirus type 5 early gene expression. 20 22

The kinetics of accumulation of early virus RNA in the cytoplasm of KB cells infected at 40.5 degrees C by wild-type (WT) adenovirus type 5 and a temperature-sensitive "early" mutant, H5ts125 (ts125), were compared by hybridization of unlabeled RNA in solution to the (3)H-labeled l strand of Ad5 DNA HindIII restriction endonuclease fragment A. In the presence of 1-beta-d-arabinofuranosylcytosine, A(l) RNA accumulated in WT-infected cells for 9 h and then decreased in concentration to 6% of the 9-h concentration by 18 h. In ts125-infected cells, A(l) RNA accumulated for 12 h and then remained at the same concentration for at least 6 h thereafter. The concentrations of virus RNA from the four early transcription regions of the genome were measured at 15 h in cells infected at 40.5 degrees C in the presence of 1-beta-d-arabinofuranosylcytosine by: (i) ts125 and WT; (ii) two other ts early mutants, ts107 and ts149; and (iii) a revertant of ts125. The revertant and ts149, a mutant from a different complementation group than ts125, both accumulated all early virus cytoplasmic RNA species in amounts similar to, or less than, WT. However, both ts125 and ts107, independently isolated mutations in the 72,000-molecular-weight (72K) DNA-binding protein gene, accumulated cytoplasmic early RNA in excess of that found in WT infection. This pattern of RNA accumulation with the mutants and WT virus was the same in the nuclei as in the cytoplasm at 40.5 degrees C. At 32 degrees C, however, the abundance of nuclear virus RNA from all four early regions was the same in cells infected by either ts125 or WT. Differences in the relative abundance of nuclear RNA from the four early regions were observed in cells infected at 40.5 and 32 degrees C, but were not dependent upon the infecting virus genotype. These results are consistent with autoregulation of early gene expression by the 72K protein and support the hypothesis that the 72K protein either decreases the rate of early virus transcription or increases the rate of virus RNA degradation in the nucleus.
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PMID:Autoregulation of adenovirus type 5 early gene expression II. Effect of temperature-sensitive early mutations on virus RNA accumulation. 21 73

Replication in vitro of the replicative form (RF) I DNA of bacteriophage varphiX174 requires the phage-induced cistron A (cisA) protein, the host rep protein, DNA-binding protein, ATP, and DNA polymerase III plus replication factors. The rep protein is a single-stranded DNA-dependent ATPase. In this paper we show that varphiX174 RF I DNA cut by the cisA protein acts as a duplex DNA cofactor for the rep protein ATPase activity, provided that DNA-binding protein is present. In this latter reaction the duplex DNA is unwound by the rep protein with concomitant hydrolysis of ATP. The extents of ATP hydrolysis, DNA unwinding, and, where appropriate, DNA synthesis are proportional to the amounts of DNA-binding protein present. Two ATP molecules are hydrolyzed per base pair unwound. We propose that the obligatory requirement for the cisA protein in the unwinding of varphiX174 RF I DNA is not simply due to its endonuclease activity but rather is due to its provision of a site for the binding of the rep protein. The rep protein in the presence of DNA-binding protein, but in the absence of cisA protein, unwinds duplex DNA when one strand extends to generate a single-stranded leader region preceding the duplex. We show that rep protein translocates along the leader single strand in a 5'-to-3' direction only and then invades the duplex DNA. The rep protein shows a directional specificity for translocation and unwinding. A model is presented to explain the mechanism of DNA unwinding catalyzed by the rep protein.
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PMID:Enzyme-catalyzed DNA unwinding: studies on Escherichia coli rep protein. 22 1

A soluble extract prepared from T7-infected E. coli is able to initiate DNA synthesis on an exogenous T7 DNA template. We have developed a fractionation procedure to resolve and identify the proteins required for T7 DNA synthesis. By this method we have purified the following T7 replication-related proteins (each greater than 50% pure as judged by sodium dodecyl sulfate gel electrophoresis): T7 DNA-binding protein (27,000 daltons), T7 RNA polymerase (105,000 daltons), T7 DNA polymerase (gene 5-protein, 85,000 daltons, plus host-factor), T7 DNA ligase (40,000 daltons), and T7 DNA-priming protein (65,000 daltons). The T7 DNA-priming protein, synthesized between 7.5 and 15 min following infection, was not detectable if the infecting phage carried an amber mutation in gene 4. Using an in vitro complementation assay which specifically measures the stimulation of DNA synthesis in an extract prepared from T7 gene 4-mutant infected cells, we have purified the DNA-priming protein about 2,000-fold. The purified priming protein preparations are essentially free of endonuclease, exonuclease, DNA ligase and DNA polymerase activity, but they do contain measurable DNA-dependent RNA synthetic acitvity. The enzyme is rapidly inactivated by heating to 46 degrees C and by treatment with N-ethylmalemide. In the presence of T7 DNA-binding protein and all four ribonucleoside triphosphates, the DNA-priming protein enables T7 DNA polymerase to initiate DNA synthesis on intact duplex T7 DNA. Closer studies of its enzymatic function as well as of the possible roles of the other proteins in the T7 replication system will be presented in the accompanying paper.
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PMID:Studies on bacteriophage T7 DNA synthesis in vitro. I. Resolution of the T7 replication system into its components. 110 17

Lymphocytes from a common human leukemia, chronic lymphocytic leukemia, chronic lymphocytic leukemia, have a greatly enhanced capability of DNA repair and a concomitantly prolonged survival in vitro after damage to DNA. From these lymphocytes, we isolated and purified a DNA-binding protein with a molecular weight of 24,000. It binds tightly to both ultraviolet light (UV)-irradiated and single-stranded DNA. At 35 degrees it enhances the helix-coil transition of poly[d(A-T)] AND the UV-irradiated calf thymus DNA but is inefficient in ordinary native DNA. This protein also facilitates the rate of UV-endonuclease incision of UV DNA but does not induce any nicks by itself. This finding suggests that the protein may be involved in DNA repair by enhancing such activity, and also offers an explanation for our observation of increased DNA repair in chronic lymphocytic leukemia cells. When human metaphase chromosomes are exposed to the protein, it induces marked lengthening of chromatids suggesting that this protein may also act on complex chromosomes. By quantitative immunochemical determinations, such protein could not be found in lymphocyte extracts of three normal individuals.
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PMID:Some properties of a DNA-unwinding protein unique to lymphocytes from chronic lymphocytic leukemia. 111 55

A DNA-binding protein specific for ultraviolet irradiated DNA has been purified extensively from human placenta. The binding preparation is free of exonuclease, polymerase, endonuclease, and N-glycosidase activity. The binding activity is salt dependent and is specific for double-stranded irradiated DNA. DNA from which the pyrimidine dimers have been monomerized by the action of photolyase (photoreactivating enzyme) remains an effective substrate for the binding protein, suggesting that the protein recognizes photoproducts other than pyrimidine dimers. This is supported by the finding that DNA irradiated under conditions which introduce only pyrimidine dimers is not a substrate for the binding protein. Examination of three of the xeroderma pigmentosum complementation groups has revealed no deficiency in this binding activity.
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PMID:A DNA binding protein from human placenta specific for ultraviolet damaged DNA. 127 48

The genome structure of a spontaneously generated HSV-1 strain 17 variant, 1720, has been determined by restriction endonuclease and Southern blot analysis. The short segment of 1720 is unaltered compared to the parental strain 17 genome, whereas the long segment is extensively rearranged. Almost all of TRL (approximately 9.2 kb) has been deleted and consequently IRL is converted into unique sequence. Sequences from approximately 9200 nucleotide position (np) to 97,000 np are present in inverted orientation, covalently bound to sequences in the prototype orientation from approximately 94,000 np to the L/S junction at 126,372 np. Thus, sequences from 94,000 np to 97,000 np are now diploid, with one copy in the normal orientation and location, and the other at the long terminus as an inverted repeat; no inversion of the intervening unique sequences occurs about this novel inverted repeat. In contrast, normal inversions of the long and short segments occur to give four equimolar genomic isomers, indicating that the novel long terminus has gained an "a" sequence. The duplication of sequences between 94,000 np and 97,000 np results in a genome containing two copies of UL43 and one complete and one partial copy each of genes UL42 and UL44 encoding the 65 kD DNA-binding protein and glycoprotein C, respectively. The variant has been shown to grow normally in vitro following high multiplicity infection.
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PMID:A HSV-1 variant (1720) generates four equimolar isomers despite a 9200-bp deletion from TRL and sequences between 9200 np and 97,000 np in inverted orientation being covalently bound to sequences 94,000-126,372 np. 132 42

We have developed a simple procedure for rapid determination of a DNA sequence recognized by a DNA binding protein based on immobilization of the protein on nitrocellulose filters. The procedure consists of the following steps: A recombinant protein with a functional DNA binding domain is expressed in E. coli. The protein is purified to homogeneity, immobilized on nitrocellulose paper, and exposed to a pool of double stranded oligonucleotides carrying in the central part a 20 bp random sequence, which is flanked by conserved sequences with restriction endonuclease recognition sites for analytical and subcloning purposes and sequences complementary to polymerase chain reaction primers. Oligonucleotides retained by the DNA-binding protein are liberated by increasing the ionic strength and used in a new binding process after amplification by the polymerase chain reaction technique. Finally the amplified product is cloned for determination of the DNA sequence selected by the DNA-binding protein. Murine Zn-finger and basic helix-loop-helix DNA binding proteins were used to demonstrate the efficiency of the method. We show that the yield of oligonucleotides binding to the protein was increased by several consecutive rounds of filter binding and amplification, and that the protein extracted a specific sequence from the pool of random oligonucleotides.
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PMID:Determination of recognition-sequences for DNA-binding proteins by a polymerase chain reaction assisted binding site selection method (BSS) using nitrocellulose immobilized DNA binding protein. 147 93

Two independent pathways of transcriptional regulation that show functional homology have been identified in yeast. It has been demonstrated previously that SWI5 encodes a zinc finger DNA-binding protein whose transcription and cellular localization both are cell cycle regulated. We show that ACE2, whose zinc finger region is nearly identical to that of SWI5, shows patterns of cell cycle-regulated transcription and nuclear localization similar to those seen previously for SWI5. Despite their similarities, SWI5 and ACE2 function in separate pathways of transcriptional regulation. SWI5 is a transcriptional activator of the HO endonuclease gene, whereas ACE2 is not. In contrast, ACE2 is a transcriptional activator of the CTS1 gene (which encodes chitinase), whereas SWI5 is not. An additional parallel between the SWI5/HO pathway and the ACE2/CTS1 pathway is that HO and CTS1 both are cell cycle regulated in the same way, and HO and CTS1 both require the SWI4 and SWI6 transcriptional activators. Overproduction of either SWI5 or ACE2 permits transcriptional activation of the target gene from the other pathway, suggesting that the DNA-binding proteins are capable of binding in vivo to promoters that they do not usually activate. Chimeric SWI5/ACE2 protein fusion experiments suggest that promoter specificity resides in a domain distinct from the zinc finger domain.
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PMID:Parallel pathways of gene regulation: homologous regulators SWI5 and ACE2 differentially control transcription of HO and chitinase. 173 Apr 13

The major apurinic (AP) DNA-binding protein was purified from a HeLa cell line and from the SV40-transformed cell line AT5BIVA derived from a patient with the repair deficiency syndrome ataxia telangiectasia (AT). This protein appears to be identical with the major cellular apurinic/apyrimidinic endonuclease. The two endonucleases differ in their molecular weight (HeLa, 37,600; AT, 38,900) and their dissociation equilibrium constant for AP sites (HeLa, 7.8 X 10(-11) M; AT, 28.3 X 10(-11) M). These variances might be the consequence of a different post-translational modification. Evidence for this interpretation stems from the observation that the AP DNA binding activity of AP endonuclease, as measured in a glass-fiber filter binding assay, is inactivated upon incubation with snake venom phosphodiesterase and that the AP endonuclease from AT cells in 5-10-fold more sensitive than the HeLa enzyme. For both enzymes, the diesterase treatment leads to the formation of a protein of Mr 35,500 which might be the unmodified precursor of AP endonuclease. The loss of AP DNA binding does not reduce but rather increases the catalytic activity of AP endonuclease when measured at excess substrate concentration.
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PMID:Comparison of apurinic DNA-binding protein from an ataxia telangiectasia and a HeLa cell line. Evidence for an altered processing of apurinic/apyrimidinic endonuclease. 241 10


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