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The Journal of Biological Chemistry Apr 1986The V1 nuclease from cobra venom preferentially hydrolyzes double helical RNA and has been used extensively for detecting RNA secondary structure. To increase the...
The V1 nuclease from cobra venom preferentially hydrolyzes double helical RNA and has been used extensively for detecting RNA secondary structure. To increase the utility of this enzyme as an RNA structure probe, we have investigated its properties and substrate specificity, using assays for polynucleotide hydrolysis based on fluorescent polynucleotide derivatives. Enzymatic activity requires both Na+ and Mg2+, with optima at 100 and 0.3 mM, respectively. From the sharp decrease in enzyme activity above 100 mM Na+ we estimate that 3-4 ionic interactions between the protein and polynucleotide phosphates take place. Analysis of products remaining after extensive V1 digestion also shows that the minimum size substrate is 4-6 nucleotides long. Helical RNAs and DNAs have Michaelis constants a factor of 3-10 times lower than most single-stranded RNAs. However, poly(epsilon A) has a Michaelis constant equal to the best synthetic double helices tested and is hydrolyzed at a rate comparable to helical RNA. The major V1 cutting sites in yeast tRNAPhe have Michaelis constants lower than any synthetic polymers. These data suggest that V1 nuclease recognizes any 4-6-nucleotide segment of polynucleotide backbone with an approximately helical conformation, but does not require that the bases be paired in a helix. A few single-stranded V1 cleavage sites are known in tRNA and rRNA, and their structures are consistent with the suggested V1 recognition site.
Topics: Base Sequence; DNA; Endoribonucleases; Kinetics; Magnesium; Nucleic Acid Conformation; Polynucleotides; RNA; RNA, Transfer, Amino Acyl; Sodium; Spectrometry, Fluorescence; Substrate Specificity
PubMed: 2420800
DOI: No ID Found -
The Journal of Biological Chemistry Oct 1992
Review
Topics: Biological Evolution; DNA; DNA Transposable Elements; DNA-Binding Proteins; Polynucleotides; RNA; Recombination, Genetic
PubMed: 1383220
DOI: No ID Found -
Journal of Virology Feb 2011Members of the human APOBEC3 family of editing enzymes can inhibit various mobile genetic elements. APOBEC3A (A3A) can block the retrotransposon LINE-1 and the...
Members of the human APOBEC3 family of editing enzymes can inhibit various mobile genetic elements. APOBEC3A (A3A) can block the retrotransposon LINE-1 and the parvovirus adeno-associated virus type 2 (AAV-2) but does not inhibit retroviruses. In contrast, APOBEC3G (A3G) can block retroviruses but has only limited effects on AAV-2 or LINE-1. What dictates this differential target specificity remains largely undefined. Here, we modeled the structure of A3A based on its homology with the C-terminal domain of A3G and further compared the sequence of human A3A to those of 11 nonhuman primate orthologues. We then used these data to perform a mutational analysis of A3A, examining its ability to restrict LINE-1, AAV-2, and foreign plasmid DNA and to edit a single-stranded DNA substrate. The results revealed an essential functional role for the predicted single-stranded DNA-docking groove located around the A3A catalytic site. Within this region, amino acid differences between A3A and A3G are predicted to affect the shape of the polynucleotide-binding groove. Correspondingly, transferring some of these A3A residues to A3G endows the latter protein with the ability to block LINE-1 and AAV-2. These results suggest that the target specificity of APOBEC3 family members is partly defined by structural features influencing their interaction with polynucleotide substrates.
Topics: APOBEC-3G Deaminase; Animals; Base Sequence; Cytidine Deaminase; DNA, Single-Stranded; Dependovirus; HEK293 Cells; Haplorhini; HeLa Cells; Humans; Long Interspersed Nucleotide Elements; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Plasmids; Polynucleotides; Proteins; Retroelements; Sequence Alignment; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Structure-Activity Relationship; Substrate Specificity
PubMed: 21123384
DOI: 10.1128/JVI.01651-10 -
The Journal of Biological Chemistry Aug 1981Escherichia coli recA protein catalyzes a specific proteolytic cleavage of repressors in vitro when it is activated by interaction with a single-stranded polynucleotide...
Escherichia coli recA protein catalyzes a specific proteolytic cleavage of repressors in vitro when it is activated by interaction with a single-stranded polynucleotide and nucleoside triphosphate. The ATP analogue adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S) satisfies the NTP requirement. We show here that despite its activity in repressor cleavage, ATP gamma S is hydrolyzed at a negligible rate by the recA protein DNA-dependent nucleoside triphosphatase activity. In the presence of DNA, ATP gamma S binds tightly to recA protein in a complex that can be detected because it is trapped by a nitrocellulose filter. One ATP gamma S molecule is bound per recA monomer. These results suggest that a ternary complex of recA protein, DNA, and nucleoside triphosphate is the species active in repressor cleavage. The activation of recA protein by small, defined oligonucleotides in place of DNA is described and characterized.
Topics: Adenosine Triphosphate; Affinity Labels; Bacterial Proteins; Bacteriophage lambda; DNA, Single-Stranded; DNA-Binding Proteins; Escherichia coli; Kinetics; Oligonucleotides; Polynucleotides; Rec A Recombinases; Repressor Proteins; Ribonucleotides; Transcription Factors; Viral Proteins; Viral Regulatory and Accessory Proteins
PubMed: 6455420
DOI: No ID Found -
Nucleic Acids Research Aug 19794', 6-Diamidine-2-phenylindole forms fluorescent complexes with synthetic DNA duplexes containing AT, AU and IC base pairs; no fluorescent complexes were observed with...
4', 6-Diamidine-2-phenylindole forms fluorescent complexes with synthetic DNA duplexes containing AT, AU and IC base pairs; no fluorescent complexes were observed with duplexes containing GC base pairs or with duplexes containing a single AT base pair sandwiched between GC pairs. The binding site size is one molecule of dye per 3 base pairs. The intrinsic binding constants are higher for alternating sequence duplexes than for the corresponding homopolymer pairs. With the exception of the four-stranded helical poly rI which exhibits considerable fluorescence enhancement upon binding of the ligand, none of the single- or multi- stranded polyribonucleotides and ribo-deoxyribonucleotide hybrid structures form fluorescent complexes with the dye. Poly rI is the only RNA which forms a DNA B-like structure (Arnott et al. (1974) Biochem. J. 141, 537). The B conformation of the helix and the absence of guanine appear to be the major determinants of the specificity of the fluorescent binding mode of the dye. Nonfluorescent interactions of the dye with polynucleotides are nonspecific; UV absorption and circular dichroic spectra demonstrate binding to synthetic single- and double-stranded DNA and RNA analogs, including those containing GC base pairs.
Topics: Amidines; Chemical Phenomena; Chemistry; Circular Dichroism; Fluorescent Dyes; Indoles; Kinetics; Nucleic Acid Conformation; Polydeoxyribonucleotides; Polyribonucleotides; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet
PubMed: 493113
DOI: 10.1093/nar/6.11.3519 -
The Journal of General Physiology Jul 1966The anatomy of DNA molecules isolated from mature bacteriophage is reviewed. These molecules are linear, duplex DNA consisting mainly of uninterrupted polynucleotide...
The anatomy of DNA molecules isolated from mature bacteriophage is reviewed. These molecules are linear, duplex DNA consisting mainly of uninterrupted polynucleotide chains. Certain phage (T5 and PB) contain four specifically located interruptions. While the nucleotide sequence of most of these molecules is unique (T5, T3, T7, lambda), some are circular permutations of each other (T2, T4, P22). Partial degradation of these DNA molecules by exonuclease III predisposes some of them to form circles upon annealing, but indicating they are terminally redundant.
Topics: Bacteriophages; Centrifugation, Density Gradient; DNA, Viral; Microscopy, Electron; Models, Theoretical; Molecular Weight; Nucleotidases; Nucleotides; Polynucleotides
PubMed: 5967428
DOI: 10.1085/jgp.49.6.143 -
Journal of Bacteriology Jul 1973Polynucleotide sequence relatedness studies were carried out to determine the extent of divergence present in members of the tribe Salmonelleae and between salmonellae...
Polynucleotide sequence relatedness studies were carried out to determine the extent of divergence present in members of the tribe Salmonelleae and between salmonellae and other enteric bacteria. Typical Salmonella were 85 to 100% related. Two groups of biochemically atypical Salmonella showed somewhat lower binding to typical salmonellae and to each other. Arizona were 70 to 80% related to salmonellae. Two groups of Arizona were detected. These groups correlated with the presence of monophasic or diphasic flagellar antigens. Salmonella and Arizona were no more related to Citrobacter than to Escherichia coli (45-55%). Relatedness of Salmonella and Arizona to other enterobacteria ranged from 20 to 40% with klebsiellae and shigellae, to 20 to 25% with erwiniae, and to less than 20% with edwardsiellae and Proteus mirabilis.
Topics: Base Sequence; Biological Evolution; Chromatography; DNA, Bacterial; DNA, Single-Stranded; Enterobacteriaceae; Escherichia; Hot Temperature; Hydroxyapatites; Molecular Weight; Nucleic Acid Denaturation; Nucleic Acid Hybridization; Phosphorus Isotopes; Polynucleotides; Salmonella; Species Specificity; Spectrophotometry
PubMed: 4717519
DOI: 10.1128/jb.115.1.307-315.1973 -
The Biochemical Journal Sep 1976Echinomycin is a peptide antibiotic which binds strongly to double-helical DNA up to a limit of approximately one molecule per five base-pairs. There is no detectable...
Echinomycin is a peptide antibiotic which binds strongly to double-helical DNA up to a limit of approximately one molecule per five base-pairs. There is no detectable interaction with rRNA and only extremely feeble non-specific interaction with poly(rA)-poly(rU). Heat denaturation of DNA greatly decreases the binding, and similarly limited interaction is observed with naturally occurring single-stranded DNA. Association constants for binding to nine double-helical DNA species from different sources are presented; they vary by a factor of approximately 10, but are not simply related to the gross base composition. The interaction with DNA is ionic-strength-dependent, the binding constant falling by a factor of 4 when the ionic strength is raised from 0.01 to 0.10mol/litre. From the effect of temperature on the association constant for calf thymus DNA, the enthalpy of interaction is calculated to be about -13kJ/mol (-3kcal/mol). Binding of echinomycin persists in CsCl gradients and the buoyant density of nicked bacteriophage PM2 DNA is decreased by 25 mg/ml. Echinomycin interacts strongly with certain synthetic poly-deoxynucleotides, the binding constant decreasing in the order poly(dG)-poly(dC) greater than poly(dG-dC) greater than poly(dA-dT). For the latter two polymers the number of base-pairs occluded per bound antibiotic molecule is calculated to be three, whereas for poly(dG)-poly(dC) it is estimated to be four to five. Poly(dA)-poly(dT) and poly(dI)-poly(dC) interact only very weakly with the antibiotic. Poly(dI-dC) interacts to a slightly greater extent, but the binding curve is quite unlike that seen with the three strongly binding synthetic polynucleotides. Echinomycin affects the supercoiling of closed circular duplex bacteriophage PM2 DNA in the characteristic fashion of intercalating drugs. At low ionic strength the unwinding angle is almost twice that of ethidium. Likewise the extension of the helix, determined from changes in the viscosity of rod-like sonicated DNA fragments, is nearly double that expected for a simple (monofunctional) intercalation process. On this basis the interaction process is characterized as bifunctional intercalation. At higher ionic strength the unwinding angle relative to that of ethidium and the helix extension per bound echinomycin molecule fall, indicating a smooth progression towards more nearly monofunctional intercalation. Two simpler compounds which act as analogues of the quinoxaline chromophores of echinomycin, quinoxaline-2-carboxamide and the trypanocidal drug Bayer 7602, interact with DNA very much more weakly than does echinomycin, showing that the peptide portion of the antibiotic plays an essential role in determining the strength and specificity of the interaction.
Topics: Binding Sites; DNA; DNA, Bacterial; DNA, Circular; Echinomycin; Osmolar Concentration; Poly C; Poly G; Poly I-C; Poly U; Poly dA-dT; Quinoxalines; Temperature; Ultracentrifugation; Viscosity
PubMed: 985413
DOI: 10.1042/bj1570721 -
Journal of Virology Oct 1979The DNAs of different members of the Papillomavirus genus of papovaviruses were analyzed for nucleotide sequence homology. Under standard hybridization conditions (Tm -... (Comparative Study)
Comparative Study
The DNAs of different members of the Papillomavirus genus of papovaviruses were analyzed for nucleotide sequence homology. Under standard hybridization conditions (Tm - 28 degrees C), no homology was detectable among the genomes of human papillomavirus type 1 (HPV-1), bovine papillomavirus type 2 (BPV-2), or cottontail rabbit (Shope) papillomavirus (CRPV). However, under less stringent conditions (i.e., Tm - 43 degrees C), stable hybrids were formed between radiolabeled DNAs of CRPV, BPV-1, or BPV-2 and the HindIII-HpaI A, B, and C fragments of HPV-1. Under these same conditions, radiolabeled CRPV and HPV-1 DNAs formed stable hybrids with HincII B and C fragments of BPV-2 DNA. These results indicate that there are regions of homology with as much as 70% base match among all these papillomavirus genomes. Furthermore, unlabeled HPV-1 DNA competitively inhibited the specific hybridization of radiolabeled CRPV DNA to bpv-2 DNA fragments, indicating that the homologous DNA segments are common among these remotely related papillomavirus genomes. These conserved sequences are specific for the Papillomavirus genus of papovaviruses as evidenced by the lack of hybridization between HPV-1 DNA and either simian virus 40 or human papovavirus BK DNA under identical conditions. These results indicate a close evolutionary relationship among the papillomaviruses and further establish the papillomaviruses and polyoma viruses as distinct genera.
Topics: Base Sequence; Bovine papillomavirus 1; Cottontail rabbit papillomavirus; DNA, Viral; Genes, Viral; Nucleic Acid Conformation; Nucleic Acid Hybridization; Papillomaviridae; Polynucleotides
PubMed: 232171
DOI: 10.1128/JVI.32.1.199-207.1979 -
Proceedings of the National Academy of... Dec 1963
Topics: DNA; DNA, Bacterial; Escherichia coli; Genetics; Hydrolases; Phosphates; Phosphorus Isotopes; Polydeoxyribonucleotides; Polynucleotides; Polyribonucleotides; RNA; RNA, Bacterial; Research
PubMed: 14096186
DOI: 10.1073/pnas.50.6.1116