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 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

Differential accessibility to DNA in tumor cell chromatin is important to growth, differentiation apoptosis, and the targeting of DNA modifying drugs. We now show that endonuclease accessibility to DNA in the nuclei of A431 human carcinoma cells is increased within 90 min by nontoxic nanomolar levels of okadaic acid, known to inhibit protein phosphatase 2A. This genomic hypersensitivity was partly enhanced by joint treatment with epidermal growth factor and okadaic acid but did not appear without the latter. Nuclei with greater DNA susceptibility showed a decrease in M(r) 80,000 DNA binding protein doublet specific for dAT-rich sequences concurrent with the "apparent" hyperphosphorylation of a M(r) 70,000 nuclear matrix protein. We propose that some of the tumor-promoting effects of okadaic acid may be partly associated with its ability to promote genomic susceptibility.
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PMID:Accessibility to DNA in carcinoma chromatin is promoted by nanomolar okadaic acid: effect on AT-rich DNA binding proteins. 133 Feb 92

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

As a means of determining how elongating RNA polymerase responds to a protein in its path, transcription has been carried out in vitro with the purified Escherichia coli enzyme on templates associated with a sequence-specific DNA binding protein. The major RNA species generated is the length expected from RNA polymerase which has transcribed to the position of the bound protein and is unable to elongate further. The binding proteins used are two mutants of the EcoRI endonuclease which are defective in cleavage function but retain high affinity for the wild-type recognition sequence (Wright, D. J., King, K., and Modrich, P. (1989) J. Biol. Chem. 264, 11816-11821). Blockage of RNA polymerase occurs on linear and circular templates and, although efficient with both proteins, is more effective for the EcoRI derivative with the slower dissociation rate. The protein-blocked transcription complexes are stable over time and remain in an active form, resuming elongation when the blocking protein is displaced by an increase in ionic strength. These paused ternary complexes, if treated with the termination factor rho, undergo release. The 3' ends of the blocked-length RNAs from DNAs of distinct sequences reveal that the ternary complexes are positioned at a constant distance from the protein block, 14 nucleotides upstream of the EcoRI recognition sequence. This information is combined with exonuclease III footprinting data to position the 3' end of the nascent RNA chain in the ternary complex quite near (approximately 7 nucleotides) the leading edge of RNA polymerase.
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PMID:Elongation by Escherichia coli RNA polymerase is blocked in vitro by a site-specific DNA binding protein. 169 18

We have used enzymic and chemical probes to search for altered DNA conformations in the 5' flanking region of the gene for a high mobility group protein (HMG-T) from trout. This search was conducted in order to identify potential genetic elements that might be involved in the transcriptional control of the HMG-T gene. We identified, in supercoiled plasmid DNA molecules containing a 900 base pair insert of the 5' region of the gene, an S1-sensitive site situated within an (AT)12 sequence approximately 120 base pairs upstream from the start of the HMG-T gene. Chemical modification of supercoiled DNA with the single-strand-selective reagent bromoacetaldehyde was limited to a region coincident with the S1 nuclease site. T7 endonuclease I, a probe highly specific for four-way helical junctions, cleaved predominantly at the boundaries of the (AT)12 stretch. These data are most consistent with the interpretation that the (AT)12 sequence adopts a cruciform structure when torsionally stressed by negative supercoiling. DNase I footprinting analyses demonstrated that HMG-T protects two regions almost equidistant from the center of the (AT)12 sequence, indicating that HMG-T is a sequence-specific DNA binding protein.
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PMID:Induction by torsional stress of an altered DNA conformation 5' upstream of the gene for a high mobility group protein from trout and specific binding to flanking sequences by the gene product HMG-T. 283 70

The location of nucleotide sequences within the bovid herpesvirus 1 (BHV-2) genome homologous to herpes simplex virus 1 (HSV-1) DNA were investigated. BHV-2 DNA was digested with restriction endonucleases and blotted to nitrocellulose paper. The blots were then probed with plasmids containing HSV-1 genes for thymidine kinase (TK), the major DNA binding protein (ICP8), the major capsid protein (VP5) and genes for HSV-1 glycoproteins gB, gD, and gC. Except for HSV-1 gC, each HSV-1 gene tested hybridized to BHV-2 nucleotide sequences that were located either on both sides of a restriction endonuclease cleavage site, within a small restriction endonuclease fragment, or to an area common to two overlapping restriction fragments. Thus, we were able to localize BHV-2 nucleotide sequences homologous to the HSV-1 ICP8 gene between 0.38 and 0.41 map units (m.u.), and BHV-2 nucleotide sequences homologous to the HSV-1 VP5 gene between 0.24 and 0.27 m.u. In addition, BHV-2 nucleotide sequences homologous to HSV-1 genes for TK, gB and gD were found to lie on both sides of restriction endonuclease cleavage sites at 0.30, 0.35, and 0.94 m.u., respectively.
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PMID:Genomic location of bovid herpesvirus type 2 nucleotide sequences homologous to five herpes simplex virus type 1 genes. 284 79

DNA binding proteins present in the cytoplasm and nuclei of term placenta were isolated by DNA-cellulose chromatography and analysed by electrophoresis in high resolution polyacrylamide gradient gels. A denatured DNA specific protein of approximate molecular weight 34 000 daltons was the predominant DNA binding protein of the cytoplasm; this protein consisted of over 65% of the total DNA binding proteins of the 0.15 M NaCl eluate of the cytoplasm. The cytoplasmic extracts contained two additional DNA binding proteins of molecular weight 24 000 and 18 000 daltons and these proteins bound preferentially to ds DNA. All the three DNA binding proteins were also present in the nuclei and electrophoresis of histones in adjacent lanes indicated that they are not histones. The 34 000-dalton DNA binding protein has been purified by ammonium sulphate fractionation followed by phosphocellulose (PC) chromatography. The DBP eluted from the PC column between 0.125-0.15 M potassium phosphate. PC fractions containing electrophoretically pure 34 KD DBP showed an endonuclease activity capable of converting plasmid pBR 322 DNA to the linear form. Maximum endonucleolytic activity was observed in the presence of 3-5 mM Mg2+ and the enzyme activity was completely inhibited by 3 mM ethylenediamine tetraacetate.
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PMID:DNA-binding proteins of human placenta: purification and characterization of an endonuclease. 609 9

We have introduced the DNA binding protein (DBP) gene of human adenovirus type 5 (Ad5) into high molecular weight DNA of permissive human cells by cotransformation of tk- cells with the cloned DBP and HSV-1 thymidine kinase genes. 110 tk+ cell lines were isolated after selection in HAT medium. The amount and arrangement of adenovirus sequences in the tk+ cell lines were analyzed by restriction endonuclease digestion and filter hybridization. Twelve of the 110 lines carry at least a segment of the DBP gene while only three of these contain the entire DBP gene at approximately one copy per cell. Cytoplasmic, polyadenylated DBP mRNA is made in all three cell lines though the amount is very low compared to that present in infected HeLa cells. The cell line U13-2 which contains approximately 1/30 the steady-state level of DBP mRNA found in infected HeLa cells produces a few percent of the amount of DBP made during the peak period of DBP synthesis in infected cells. The other two lines contain lower levels of DBP mRNA and do not synthesize detectable levels of the protein. When these DBP-tk+ cell lines are infected with adenovirus mutants containing temperature-sensitive (ts) mutations in the DBP gene, only U13-2 permits some viral DNA replication (and hence late gene expression) at the nonpermissive temperature, indicating that sufficient quantities of DBP from the integrated gene are produced to allow complementation of the ts mutation in this cell line. However, growth of these ts mutants (as measured by virus production) is only partially complemented in U13-2 at the nonpermissive temperature.
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PMID:Construction of human cell lines which contain and express the adenovirus DNA binding protein gene by cotransformation with the HSV-1 tk gene. 609 56

Single-strand DNA binding protein (SSB) from Escherichia coli abolishes transfection of E.coli by viral M13mp2 DNA at levels that inhibit transfection by M13mp2 replicative form (RF) DNA by approx. 25%. Synthesis of M13mp2 RF DNA (SS leads to DS) has been carried out using DNA polymerase I (Klenow fragment) and a unique 15-nucleotide primer. A time course for in vitro synthesis showed that the increase in transfection in the presence of SSB paralleled DNA synthesis after an initial lag period for transfection. Digestion of replication products with restriction endonucleases and S1 endonuclease indicates that only those molecules that are fully or almost fully duplex transfect competent cells in the presence of SSB.
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PMID:Preferential transfection with M13mp2 RF DNA synthesized in vitro. 630 39

Extracts of the yeast Saccharomyces cerevisiae support DNA replication on exogenous yeast 2-microns plasmid DNA templates. A crude extract from a S. cerevisiae cell division cycle mutant, cdc8-1, expressed the temperature-sensitive phenotype since it could be inactivated at 42 degrees C in vitro. This heat-inactivated extract was fully complemented by the addition of either wild-type or cdc8-1 single-stranded DNA binding protein (SSB). restoration by SSB of the activity of the mutant cell extract allowed replication like that of a wild-type crude extract, as shown by bidirectional DNA synthesis from the in vivo origin. The DNA binding protein specifically stimulates the reaction catalyzed by yeast DNA polymerase I, a true DNA replicase, using the hybrid of phi X174 single-stranded DNA and a restriction endonuclease fragment as a template. It also increases processivity of DNA polymerase I at least 10-fold. Escherichia coli SSB, but not T4 gene 32 protein, can substitute for yeast SSB. Both restoration of DNA synthesis in the heated mutant cell extract and stimulation of the DNA polymerase I reaction by SSB from cdc8-1 cells are inactivated at nonpermissive temperatures, suggesting that yeast SSB is the CDC8 gene product.
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PMID:Yeast 2-microns plasmid DNA replication in vitro: purification of the CDC8 gene product by complementation assay. 633 94


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