Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.27.1 (RNase)
 16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The presence of a nuclear DNA polymerase in mouse sperm from adult testes has been confirmed and the properties of this enzyme further investigated. This activity was shown to be greatly enhanced by treating the spermatozoa with methanol or ethanol before incubation in the reaction medium or by their addition in small amounts to this medium. It was protected against degradation by nuclear proteases by adding soybean trypsin inhibitor and was stimulated by ATP. It was found to be Mg2+ dependent (optimum concentration: 7.5 mM), DNA dependent, and all four deoxynucleoside triphosphates were needed for optimal reaction. The radioactive acid-precipitable product of polymerization was not eliminated by organic solvents, nor by pronase, ribonuclease or by nuclease S1; however, it was converted to a large extent to acid-soluble products by pancreatic deoxyribonuclease. Since it was only partially solubilized by Triton X-100, it therefore did not appear to be preferentially associated with the nuclear membranes. The activity recovered after incubation depended also on the pH (optimum at pH 8.3) and did not work well in a medium for DNA polymerase alpha. The temperature for maximum incorporation of nucleotides was found to be 32 degrees C and, under our conditions, the reaction was linear for 30 min. The DNA polymerase activity was inhibited by low and high concentrations of KCl. It was not lowered by N-ethylmaleimide or p-hydroxymercuribenzoate; urea slightly stimulated the reaction and this stimulation was reversed by subsequent treatment with N-ethylmaleimide. Actinomycin D (40 mug/ml), ethidium bromide (25--50 muM), netropsin (5--50 mug/ml), and spermidine (0.5--2.5 mM) lowered the polymerization of DNA precursors. The nuclear enzyme could shift from the endogenous template to activated exogenous calf thymus DNA, the resulting nuclear radioactivity being reduced. The endogenous DNP template ability was not increased by deoxyribonuclease activation according to the method of Aposhian and Kornberg (J. Biol. Chem. (1962) 237, 519--525) suggesting that the amount of DNA polymerase associated with chromatin was probably limiting the reaction. The DNA polymerase activity detected in mouse sperm nuclei has numerous properties of low molecular weight DNA polymerases (DNA polymerase beta) reported in several eukaryotic organisms.
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PMID:Further characterization of a DNA polymerase activity in mouse sperm nuclei. 1 3

Phosphorothioate oligodeoxycytidine (S-dCn) was used as a model compound to examine the impact of the number of phosphorothioate linkages and their position on the inhibition of human DNA polymerases and RNase H in vitro. S-dCn with a chain length longer than 15 could inhibit human DNA polymerases and RNase H activities, in a linkage number-dependent manner. Longer oligomers were more potent inhibitors than shorter ones. Kinetic studies indicated that S-dC28 was a competitive inhibitor of DNA polymerase alpha and beta with respect to the DNA template, whereas it was a noncompetitive inhibitor of polymerases gamma and delta. S-dC28 was also a competitive inhibitor of RNase H1 and H2 with respect to RNA-DNA duplex. Susceptibility of these enzymes to inhibition by S-dC28 was in the order of delta approximately gamma greater than alpha greater than beta and RNase H1 greater than RNase H2. Structural-activity relationships were explored with a group of S-dC28 analogs that have phosphorothioate internucleotide linkages at various positions. The inhibitory effect depended on the total number of thioate linkages, rather than the position of the linkages within the oligomer or the chain length itself. No sequence specificity was found. In the presence of the complementary RNA, antisense phosphorothioates (S-oligos) exerted a biphasic effect on RNase H activity. At low concentrations S-oligos could enhance the cleavage of the RNA portion of S-oligo-RNA duplex, whereas at high concentrations (in excess of the complementary RNA) S-oligos could inhibit RNase H and protect the complementary RNA from degradation. Together, these results suggest that the non-sequence-specific inhibitory effect of S-oligos should be taken into consideration in designing antisense inhibitors. This inhibitory activity could be avoided by decreasing the number of phosphorothioate linkages at the backbone, and S-oligos of 15-20 residues are preferable in antisense molecule design.
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PMID:Phosphorothioate oligonucleotides are inhibitors of human DNA polymerases and RNase H: implications for antisense technology. 137 82

Human immunodeficiency virus (HIV) reverse transcriptase (RT) uses host tRNA(Lys) partially annealed to the primer binding site (PBS) as primer for the initiation of cDNA synthesis. When assaying cDNA synthesis with a template-primer complex formed by an RNA fragment carrying the PBS site and bovine tRNA(Lys) we noticed that an excess of primer tRNA inhibited strongly the DNA polymerase activity of a recombinant HIV RT (p66-p51 heterodimeric form) produced in transformed yeast cells. The same inhibitory effect was observed with animal DNA polymerase alpha, while avian retrovirus RT was neither affected by tRNA(Lys) nor by its specific primer tRNA(Trp). Although the strongest inhibition was observed with tRNA(Lys), other tRNas like tRNA(Phe) and tRNA(Trp) inhibited also the HIV RT, whereas tRNAs specific for valine, proline and glycine had no effect on enzyme activity. Digestion of tRNA(Lys) with pancreatic RNase abolished the inhibition; on the other hand T1 RNase digestion had no effect on the inhibition suggesting a role of the anticodon region in this effect. The 12- and 14-mers corresponding to the anticodon regions of the three bovine tRNA(Lys) isoacceptors inhibited RT activity, indicating that at least an important part of the inhibitory effect could be ascribed to this tRNA region. A strong stimulation of DNA polymerase activity was observed when the effect of tRNA(Lys) was assayed on a recombinant HIV reverse transcriptase produced in a protease deficient yeast strain, which leads to the production of an active p66 enzyme. The same tRNAs that inhibited strongly the heterodimeric form stimulated the p66 form of HIV reverse transcriptase. The results suggest that although both enzymatic forms are able to interact with tRNA(Lys) the topography, as well as the functional implications of the interaction between the precursor and the mature form of HIV reverse transcriptase with the tRNA(Lys) primer, are different.
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PMID:Inhibition of the p66/p51 form of human immunodeficiency virus reverse transcriptase by tRNA(Lys). 168 23

Wheat DNA polymerase A has been purified from wheat germ. The previous purification procedure (Castroviejo, M. et al. (1979) Biochem. J. 181, 183-191; Tarrago-Litvak, L. et al. (1975) FEBS Lett. 59, 125-130), has been improved leading to a higher degree of purity. Several biochemical properties of the enzyme are described. Interestingly, wheat DNA polymerase A is able to copy natural poly(A)+ mRNA into cDNA, in a way that is similar to that of the human immunodeficiency virus reverse transcriptase (HIV-RT). All four dXTP and the oligo dT primer were required for cDNA synthesis. The cDNA product was completely digested in the presence of DNase I and predigestion of the mRNA template with RNase decreased dramatically the cDNA synthesis. The animal DNA polymerase gamma can not copy natural mRNA. Substances, known to alter the enzymatic activities have been used to compare enzymes properties. In the presence of glycerol, ethidium bromide or spermine, wheat DNA polymerase A, HIV-RT and DNA polymerase gamma behave similar and they differ from animal DNA polymerase alpha. Nevertheless, DNA polymerase A is more resistant than HIV-RT and DNA polymerase gamma to the chain terminator ddTTP, while the wheat enzyme is more inhibited than DNA polymerase gamma but more resistant than HIV-RT in the presence of N3-TTP.
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PMID:Wheat embryo DNA polymerase A reverse transcribes natural and synthetic RNA templates. Biochemical characterization and comparison with animal DNA polymerase gamma and retroviral reverse transcriptase. 169 Oct 20

We have developed a nuclear lysate system from infected, synchronized cells capable of synthesizing unit-length parvoviral DNA in vitro. It was necessary to supplement the nuclear lysates with the polyamines, spermidine and spermine, to prevent degradation of template and product DNAs. In this system RF, RI, and single-stranded progeny DNAs were synthesized. Label incorporated in viral RF DNA in vivo appeared first in RI DNA and then in single-stranded DNA during incubation in vitro. By sedimentation the product DNAs were identical to those found in infected cells. Their viral identity was confirmed by hybridization. The addition of ribonucleotides, RNase, or alpha-amanitin did not affect parvoviral DNA synthesis in this system. The results with the specific inhibitors of mammalian DNA polymerases, aphidicolin, N-ethylmaleimide, and 2',3'-dideoxythymidine 5'-triphosphate indicated that DNA polymerase alpha was required for synthesis of parvoviral DNA in the nuclear lysates. This requirement was confirmed by experiments with antibody to bovine DNA polymerase alpha.
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PMID:Replication of parvoviral DNA. I. Characterization of a nuclear lysate system. 626 Sep 87

The factor(s) derived from fibrosarcoma-induced suppressor T cells was sensitive to pronase and neuraminidase, but not to trypsin, beta-galactosidase, DNase, or RNase. Protein and RNA, but not DNA, synthesis were required to mediate suppression. Suppressor T cell-derived factor(s) could be precipitated by a 50% saturated ammonium sulfate (SAS) solution. The 50% SAS fraction inhibited both in vitro and in vivo spleen cell blastogenesis, whereas the 80% and unprecipitated fractions had no inhibitory activity. Using Sephadex G-200 chromatography, the 2nd protein fraction (fraction II) contained an inhibitor of both DNA polymerases (IDP) and DNA synthesis (IDS) activity, which possessed no cytotoxic activity. In vitro DNA polymerase alpha activity was suppressed by fraction II, whereas DNA polymerase beta and gamma activities remained unchanged. Molecular weight of IDP/IDS, as determined by Sephadex G-200 gel filtration chromatography, was approximately 14,500. Attempts to separate IDP/IDS activities found in fraction II by anion-exchange chromatography and slab gel electrophoresis were not successful, which suggested that the 2 activities were the same or very similar molecules.
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PMID:Suppressor cell activity in tumor-bearing mice. III. Co-purification of a factor inhibiting cellular DNA synthesis and DNA polymerase activity. 645 73

A diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) binding subunit has been resolved from a high molecular weight (640,000) multiprotein form of DNA polymerase alpha [deoxynucleoside triphosphate:DNA nucleotidyltransferase (DNA-directed), EC 2.7.7.7] from HeLa cells [DNA polymerase alpha 2 of Lamothe, P., Baril, B., Chi, A., Lee, L. & Baril, E. (1981) Proc. Natl. Acad. Sci. USA 78, 4723-4727]. The Ap4A binding activity copurifies with the DNA polymerizing activity during the course of purification. Hydrophobic chromatography on butylagarose resolves the Ap4A binding activity from the DNA polymerase. The Ap4A binding activity is protein in nature since the binding of Ap4A is abolished by treatment of the isolated binding activity with proteinase K but is insensitive to treatment with DNase or RNase. The molecular weight of the Ap4A binding protein, as determined by polyacrylamide gel electrophoresis under nondenaturing conditions or by NaDodSO4/polyacrylamide gel electrophoresis after photoaffinity labeling of the protein with [32P]Ap4A is 92,000 or 47,000. The binding activity of this protein is highly specific for Ap4A.
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PMID:Resolution of the diadenosine 5',5"'-P1,P4-tetraphosphate binding subunit from a multiprotein form of HeLa cell DNA polymerase alpha. 657 66

Using a synthetic telomere DNA template and whole cell extracts, we have identified proteins capable of synthesizing the telomere complementary strand. Synthesis of the complementary strand required a DNA template consisting of 10 repeats of the human telomeric sequence d(TTAGGG) and deoxy- and ribonucleosidetriphosphates and was inhibited by neutralizing antibodies to DNA polymerase alpha. No evidence for RNA-independent synthesis of the lagging strand was observed, suggesting that a stable DNA secondary structure capable of priming the lagging strand is unlikely. Purified DNA polymerase alpha/primase was capable of catalyzing synthesis of the lagging strand with the same requirements as those observed in crude cell extracts. A ladder of products was observed with an interval of six bases, suggesting a unique RNA priming site and site-specific pausing or dissociation of polymerase alpha on the d(TTAGGG)10 template. Removal of the RNA primers was observed upon the addition of purified RNase HI. By varying the input rNTP, the RNA priming site was determined to be opposite the 3' thymidine nucleotide generating a five-base RNA primer with the sequence 5'-AACCC. The addition of UTP did not increase the efficiency of priming and extension, suggesting that the five-base RNA primer is sufficient for extension with dNTPs by DNA polymerase alpha. This represents the first experimental evidence for RNA priming and DNA extension as the mechanism of mammalian telomeric lagging strand replication.
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PMID:Synthesis of the mammalian telomere lagging strand in vitro. 911 15

Flap endonuclease-1 (FEN1) is proposed to participate in removal of the initiator RNA of mammalian Okazaki fragments by two pathways. In one pathway, RNase HI removes most of the RNA, leaving a single ribonucleotide adjacent to the DNA. FEN1 removes this ribonucleotide exonucleolytically. In the other pathway, FEN1 removes the entire primer endonucleolytically after displacement of the 5'-end region of the Okazaki fragment. Cleavage would occur beyond the RNA, a short distance into the DNA. The initiator RNA and an adjacent short region of DNA are synthesized by DNA polymerase alpha/primase. Because the fidelity of DNA polymerase alpha is lower than that of the DNA polymerases that complete DNA extension, mismatches occur relatively frequently near the 5'-ends of Okazaki fragments. We have examined the ability of FEN1 to repair such errors. Results show that mismatched bases up to 15 nucleotides from the 5'-end of an annealed DNA strand change the pattern of FEN1 cleavage. Instead of removing terminal nucleotides sequentially, FEN1 appears to cleave a portion of the mismatched strand endonucleolytically. We propose that a mismatch destabilizes the helical structure over a nearby area. This allows FEN1 to cleave more efficiently, facilitating removal of the mismatch. If mismatches were not introduced during synthesis of the Okazaki fragment, helical disruption would not occur, nor would unnecessary degradation of the 5'-end of the fragment.
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PMID:Cleavage of substrates with mismatched nucleotides by Flap endonuclease-1. Implications for mammalian Okazaki fragment processing. 1032 52