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Environmental Health Perspectives Aug 1981In this paper, we consider the effects of metal ions on the accuracy of catalysis by DNA polymerases. Certain activating and nonactivating metal ions have been shown to...
In this paper, we consider the effects of metal ions on the accuracy of catalysis by DNA polymerases. Certain activating and nonactivating metal ions have been shown to diminish the fidelity of DNA synthesis in vitro with a variety of DNA polymerases. There is a significant correlation between the metals that decrease fidelity and those that have been reported to be mutagenic and carcinogenic. Thus, metal carcinogens are no exception to the general postulate that carcinogens can be identified by their interactions with DNA.
Topics: Animals; DNA Replication; DNA-Directed DNA Polymerase; Enzyme Activation; Humans; Metals; Polynucleotides
PubMed: 7274184
DOI: 10.1289/ehp.8140197 -
Nucleic Acids Research May 1988We have developed general methods for joining together, via cleavable disulfide bonds, either two unprotected polynucleotides or a polynucleotide and a peptide or...
We have developed general methods for joining together, via cleavable disulfide bonds, either two unprotected polynucleotides or a polynucleotide and a peptide or protein. To join two oligonucleotides, each is first converted to an adduct in which cystamine is joined to the 5'-terminal phosphate of the oligonucleotide by a phosphoramidate bond. The adducts are mixed and reduced with dithiothreitol. The dithiothreitol is then removed by dialysis. Oxidation by atmospheric oxygen occurs to yield the required dimer. To join an oligonucleotide to a cysteine-containing peptide or protein, the 5'-cystamine oligomer is first converted to a 2'-pyridyldisulfide adduct and then reacted with an excess of the peptide or protein. If the peptide does not contain a free cysteine residue, it is first treated with iminothiolane to introduce one or more sulfhydryl groups. We have used these procedures to join a 16 mer deoxynucleotide probe and MDV-1 RNA, a substrate of Q beta RNA polymerase. This adduct hybridizes with a complementary target DNA. We have also joined a 16mer probe to peroxidase and MDV-1 RNA to human IgG. The probe-peroxidase adduct maintains enzymatic activity and the MDV-1 RNA-IgG adduct binds to a complementary anti-IgG.
Topics: Chemical Phenomena; Chemistry; Disulfides; Immunoglobulin G; Oligodeoxyribonucleotides; Phosphorus Radioisotopes; Proteins; RNA, Viral
PubMed: 3375070
DOI: 10.1093/nar/16.9.3671 -
The Journal of Biological Chemistry Dec 1994Construction of chimaeric DNA molecules in vitro relies traditionally on two enzymatic steps catalyzed by separate protein components. Site-specific restriction...
Construction of chimaeric DNA molecules in vitro relies traditionally on two enzymatic steps catalyzed by separate protein components. Site-specific restriction endonucleases are used to generate linear DNAs with defined termini that can then be joined covalently at their ends via the action of DNA ligase. A novel approach to the synthesis of recombinant DNAs exploits the ability of a single enzyme, vaccinia DNA topoisomerase, to both cleave and rejoin DNA strands with extreme specificity at each step. Placement of the CCCTT cleavage motif for vaccinia topoisomerase near the end of a duplex DNA permits efficient generation of a stable, highly recombinogenic protein-DNA adduct that can religate only to acceptor DNAs that contain complementary single-strand extensions. Linear DNAs containing CCCTT cleavage sites at both ends (bivalent substrates) can be activated by topoisomerase and inserted into a plasmid vector in a simple and rapid in vitro procedure that is especially well suited to the molecular cloning of polymerase chain reaction-amplified DNAs. Activation of polyvalent (e.g. branched) DNA substrates by topoisomerase offers a potentially powerful method for the synthesis of two- and three-dimensional polynucleotide networks.
Topics: Base Sequence; Cloning, Molecular; DNA Topoisomerases, Type I; DNA, Recombinant; Molecular Sequence Data; Polynucleotides; Vaccinia virus
PubMed: 7798275
DOI: No ID Found -
Journal of Bacteriology Mar 1972Polynucleotide sequence similarity tests were carried out to determine the extent of divergence present in a number of Escherichia coli strains, obtained from diverse...
Polynucleotide sequence similarity tests were carried out to determine the extent of divergence present in a number of Escherichia coli strains, obtained from diverse human, animal, and laboratory sources, and closely related strains of Shigella, Salmonella, and the Alkalescens-Dispar group. At 60 C, relative reassociation of deoxyribonucleic acid (DNA) from the various strains with E. coli K-12 DNA ranged from 100 to 36%, with the highest level of reassociation found for three strains derived from K-12, and the lowest levels for two "atypical" E. coli strains and S. typhimurium. The change in thermal elution midpoint, which indicates the stability of DNA duplexes, ranged from 0.1 to 14.5 C, with thermal stability closely following the reassociation data. Reassociation experiments performed at 75 C, at which temperature only the more closely related DNA species form stable duplexes, gave similar indications of relatedness. At both temperatures, Alkalescens-Dispar strains showed close relatedness to E. coli, supporting the idea that they should be included in the genus Escherichia. Reciprocal binding experiments with E. coli BB, 02A, and K-12 yielded different reassociation values, suggesting that the genomes of these strains are of different size. The BB genome was calculated to be 9% larger than that of K-12, and that of 02A 9% larger than that of BB. Calculation of genome size for a series of E. coli strains yielded values ranging from 2.29 x 10(9) to 2.97 x 10(9) daltons. E. coli strains and closely related organisms were compared by Adansonian analysis for their relatedness to a hypothetical median strain. E. coli 0128a was the most closely related to this median organism. In general, these data compared well with the data from reassociation experiments among E. coli strains. However, anomalous results were obtained in the cases of Shigella flexneri, S. typhimurium, and "atypical" E. coli strains.
Topics: Base Sequence; Chromosomes, Bacterial; Cytosine; DNA, Bacterial; Enterobacteriaceae; Escherichia coli; Guanine; Hot Temperature; Hydroxyapatites; Molecular Weight; Nucleic Acid Denaturation; Phosphates; Phosphorus Isotopes; Polynucleotides; Salmonella typhimurium; Shigella; Spectrophotometry
PubMed: 4551757
DOI: 10.1128/jb.109.3.953-965.1972 -
Proceedings of the National Academy of... Aug 1987A polynucleotide helical structure containing two strands of poly(A) and one of poly(U) is reported. As shown by spectroscopic observations, the complex only forms when...
A polynucleotide helical structure containing two strands of poly(A) and one of poly(U) is reported. As shown by spectroscopic observations, the complex only forms when the poly(A) strands are of Mr between 9000 and 50,000 (degree of polymerization congruent to 28-150), whereas the size of the poly(U) strand has no effect. This limitation may explain why poly(A.A.U) was not seen in previous investigations. The potential of the poly(A) tails of mRNA for formation of this triple helix and of A.A.U or/and A.A.T triplet formation to contribute to the binding of specific RNA strands to gene-encoding nucleic acid double helices are noted.
Topics: Circular Dichroism; Macromolecular Substances; Molecular Weight; Nucleic Acid Conformation; Nucleic Acid Denaturation; Poly A; Poly U
PubMed: 3474644
DOI: 10.1073/pnas.84.15.5120 -
FEBS Letters Feb 2011APE-independent base excision repair (BER) pathway plays an important role in the regulation of DNA repair mechanisms. In this study it has been found that recently...
APE-independent base excision repair (BER) pathway plays an important role in the regulation of DNA repair mechanisms. In this study it has been found that recently discovered tyrosyl-DNA phosphodiesterase 1 (Tdp1) catalyzes the AP site cleavage reaction to generate breaks with the 3'- and 5'-phosphate termini. The removal of the 3'-phosphate is performed by polynucleotide kinase phosphatase (PNKP). Tdp1 is known to interact stably with BER proteins: DNA polymerase beta (Pol β), XRCC1, PARP1 and DNA ligase III. The data suggest a role of Tdp1 in the new APE-independent BER pathway in mammals.
Topics: Base Sequence; DNA Glycosylases; DNA Repair; DNA Repair Enzymes; DNA-(Apurinic or Apyrimidinic Site) Lyase; Humans; Phosphoric Diester Hydrolases; Phosphotransferases (Alcohol Group Acceptor); Polynucleotides; Recombinant Proteins; Substrate Specificity
PubMed: 21276450
DOI: 10.1016/j.febslet.2011.01.032 -
The Journal of Biological Chemistry May 1977A core-associated enzyme, designated as polynucleotide 5'-triphosphatase, has been purified from vaccinia. Fractionation on ADP-agarose of the soluble extract from...
A core-associated enzyme, designated as polynucleotide 5'-triphosphatase, has been purified from vaccinia. Fractionation on ADP-agarose of the soluble extract from detergent-disrupted cores followed by chromatography on poly(U)-agarose produced an 80-fold purification of the enzyme. The enzyme has an approximate molecular weight of 113,000 and is composed of two polypeptides with approximate molecular weights of 90,000 and 26,000. Divalent metal ions are necessary for enzymatic activity, which occurs optimally at pH 8.4. The enzyme acts upon 5'-ATP- and 5'-GTP-terminated RNA and also on 5'-ATP-terminated poly(A), catalyzing the hydrolysis of only the gamma-phosphate. The presumed biological role of the enzyme based upon this specificity is the participation in the initial step in the sequence of reactions through which the primary 5' termini of vaccinia messenger RNA are capped with the groups m7G(5')ppp(5')NmpN-.
Topics: Chemical Phenomena; Chemistry; Enzyme Activation; Kinetics; Molecular Weight; Nucleotidases; Peptides; Poly A; Polynucleotides; RNA; RNA, Messenger; RNA, Viral; Structure-Activity Relationship; Vaccinia virus
PubMed: 856813
DOI: No ID Found -
Journal of Virology Jan 1975The DNA of the BK virus (BKV) human papovavirus was found to be heterogeneous, consisting of at least four discrete species of DNA. Only the largest of these four... (Comparative Study)
Comparative Study
The DNA of the BK virus (BKV) human papovavirus was found to be heterogeneous, consisting of at least four discrete species of DNA. Only the largest of these four species, BKV DNA (i), which has a molecular weight calculated to be 96% that of simian virus 40 (SV40) DNA, was infectious. Homogeneous preparations of BKV DNA were obtained, however, from virions purified after low multiplicity infections of human embryonic kidney cells. BKV DNA (i) was shown to contain a single R-Eco RI and four R-Hind cleavage sites. The R-Eco RI site was localized in the largest R-Hind cleavage fragment. Radiolabeled BKV DNA reassociated slightly faster than SV40 DNA; 20 to 30% polynucleotide sequence homology was demonstrated between the genomes of BKV and SV40 when the reaction was monitored by chromatography on hydroxyapatite.
Topics: Animals; Base Sequence; Cell Line; DNA, Viral; Endonucleases; Haplorhini; Humans; Kidney; Molecular Weight; Nucleic Acid Conformation; Phosphates; Phosphorus Radioisotopes; Polynucleotides; Polyomavirus; Simian virus 40
PubMed: 163342
DOI: 10.1128/JVI.15.1.173-181.1975 -
Proceedings of the National Academy of... Feb 2000A variety of different DNA polymers were electrophoretically driven through the nanopore of an alpha-hemolysin channel in a lipid bilayer. Single-channel recording of... (Comparative Study)
Comparative Study
A variety of different DNA polymers were electrophoretically driven through the nanopore of an alpha-hemolysin channel in a lipid bilayer. Single-channel recording of the translocation duration and current flow during traversal of individual polynucleotides yielded a unique pattern of events for each of the several polymers tested. Statistical data derived from this pattern of events demonstrate that in several cases a nanopore can distinguish between polynucleotides of similar length and composition that differ only in sequence. Studies of temperature effects on the translocation process show that translocation duration scales as approximately T(-2). A strong correlation exists between the temperature dependence of the event characteristics and the tendency of some polymers to form secondary structure. Because nanopores can rapidly discriminate and characterize unlabeled DNA molecules at low copy number, refinements of the experimental approach demonstrated here could eventually provide a low-cost high-throughput method of analyzing DNA polynucleotides.
Topics: Bacterial Toxins; Base Composition; Biochemistry; DNA, Single-Stranded; Hemolysin Proteins; Lipid Bilayers; Molecular Weight; Nucleic Acid Conformation; Phosphatidylcholines; Polydeoxyribonucleotides; Temperature
PubMed: 10655487
DOI: 10.1073/pnas.97.3.1079 -
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