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At least 721 records · Page 40Linked to original sources
PubMed1984

Characterization of the Escherichia coli SSB-113 mutant single-stranded DNA-binding protein. Cloning of the gene, DNA and protein sequence analysis, high pressure liquid chromatography peptide mapping, and DNA-binding studies.

The ssb-113 (formerly lexC113) gene encoding a mutant single-stranded DNA binding protein (SSB) has been cloned into plasmid pSC101 resulting in 5- to 10-fold more mutant protein than strains carrying only one (chromosomal) copy of the gene. Analysis of tryptic and chymotryptic peptides of the mutant protein by high pressure liquid chromatography and solid phase protein sequencing has shown that the ssb-113 mutation results in the substitution of serine for proline at residue 176 of SSB. This change could only occur in one step by a C leads to T transition in the DNA sequence. Physicochemical studies of the homogeneous mutant protein have shown that it binds as well as wild type SSB to single-stranded DNA and that it is a slightly better helix-destabilizing protein than wild type SSB as measured by its ability to lower the thermal melting transition of poly[d(A-T)]. In vivo studies of ssb-113 strains carrying the cloned ssb-113 gene in pSC101 have shown that overproduction of the mutant protein does not complement the temperature-sensitive conditional lethality caused by the ssb-113 mutation when present in single gene copy in contrast to effects recently observed in ssb-1 strains overproducing the ssb-1 encoded protein (Chase, J. W., Murphy, J. B., Whittier, R. F., Lorensen, E., and Sninsky, J. J. (1983) J. Mol. Biol. 164, 193-211). Also noted in this report are two corrections to the DNA sequence of wild type SSB, one of which places glycine (codon GGC) at residue 133 rather than serine as previously reported (Sancar, A., Williams, K. R., Chase, J. W., and Rupp, W. D. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 4274-4278). The second correction to the DNA sequence is in the serine 39 codon, previously reported to be TCA and now correctly shown to be TCC.

Amino Acid Sequence↗
PubMed1984

Characterization of the structural and functional defect in the Escherichia coli single-stranded DNA binding protein encoded by the ssb-1 mutant gene. Expression of the ssb-1 gene under lambda pL regulation.

The ssb-1 gene encoding a mutant single-stranded DNA binding protein (SSB-1) has been cloned into a vector placing its expression under lambda pL regulation. This construction results in more than 100-fold increased expression of the mutant protein following temperature induction. Tryptic peptide analysis of the mutant protein by high-pressure liquid chromatography and solid-phase protein sequencing has shown that the ssb-1 mutation results in these substitution of tyrosine for histidine at residue 55 of SSB. This change could only occur in one step by a C----T transition in the DNA sequence which has been confirmed. Physicochemical studies of the homogeneous mutant protein have shown that in contrast to that of the wild-type SSB, the tetrameric structure of SSB-1 is unstable and gradually dissociates to monomer as the protein concentration is decreased from about 10 microM to less than 0.5 microM. The SSB-1 tetramer appears to be stable to elevated temperature (45 degrees C) but the monomer is not. We estimate the normal cellular concentration of SSB-1 (single chromosomal gene) to be 0.5-1 microM. Thus, there is a plausible physical explanation for our previous finding that increased expression of ssb-1 reverses the effects of a single gene (chromosomal) copy amount of SSB-1 (Chase, J.W., Murphy, J.B., Whittier, R.F., Lorensen, E., and Sninsky, J.J. (1983) J. Mol. Biol. 164, 193-211). However, even though the in vivo effects of ssb-1 and most of the in vitro defects of SSB-1 protein are reversed simply by increasing SSB-1 protein concentration, the mutant protein is not as effective a helix-destabilizing protein as wild-type SSB as measured by its ability to lower the thermal melting transition of poly[d-(A-T)].

Chromatography, Affinity↗
PubMed1983

Inhibition of DNA-dependent RNA polymerase from Escherichia coli by pyran copolymer.

Pyran copolymer, a potent inhibitor of DNA-dependent RNA polymerase from Escherichia coli, prevented polyribonucleotide synthesis by blocking both the initiation and elongation steps. The inhibition was noncompetitive with respect to template and nucleotide triphosphate substrates. Template binding and the stability of the nascent RNA chain were not affected by the inhibitor.

DNA-Directed RNA Polymerases↗
PubMed1983

Investigation of the nature and specificity of antinuclear antibody in dogs.

A liquid phase radioimmunoassay was used to test sera from 34 dogs in 3 categories, determined by presence of antinuclear antibody (ANA) and disease, for antibodies against native canine DNA and dAdT, a synthetic double-stranded DNA analog. Antibodies to dAdT were absent in healthy dogs which, as a group, had levels of DNA antibodies consistent with those reported for healthy persons. Canine patients with a variety of illnesses, but which remained ANA negative, had slightly increased levels of binding of DNA and dAdT. As a group, ANA-positive dogs had significantly increased binding of dAdT and native DNA, which was shown to be mainly, but not entirely, double stranded. In the ANA-positive group, no correlation was found among ANA titer, % DNA binding, and % dAdT binding, indicating that these 3 procedures detect antibodies with differing specificities. In dogs, ANA are heterogeneous in antigenic specificity. Antibodies to double-stranded nucleic acid in dogs do not appear to be as specific for systemic lupus erythematosus as they are reported to be in persons with autoimmune disease.

Animals↗
PubMed1977

Characterization of a new reverse transcriptase of possibly cellular origin in the chicken system.

The properties of an RNA-dependent DNA polymerase, which occurs ubiquitously in the allantoic fluid of uninfected, leukosis-virus-free eggs, are described. It is shown that the enzyme can synthesize faithful transcripts from natural RNA (globin mRNA). With biochemical and immunological methods, the enzyme can be clearly distinguished from the reverse transcriptases of the known chicken RNA tumour viruses and therefore seems to be a member of a so far unknown class of chicken polymerases. Our data show that in the chicken system reverse transcriptase can occur without connection to the replication of RNA tumour viruses and without relationship to the induction of malignancy.

Animals↗
PubMedDate not supplied

[Interactions between distamycin A analogs bound to DNA].

The experimental binding isotherms of the distamycin A analog to 8 natural and synthetic DNAs were analyzed. The shapes of binding isotherms suggest that the bound ligand molecule induces transitions of DNA (B-form) into two perturbated conformation states. These transitions are responsible for the existence of positive and negative cooperative effects on binding of distamycin analogs to DNA. At low levels of binding positive cooperative effects play a dominating role whereas at high levels of binding negative cooperative effects are observed. These cooperative effects can be described by the aid of a potential of pairwise interactions between nearest neighbour bound antibiotic molecules. A detailed analysis of experimental binding isotherms shows that characteristic distances over which these interactions are extended depend on the AT content of DNA. The energetical and structural parameters characterising the allosteric transitions of DNA to the perturbated states are obtained.

Allosteric Regulation↗
PubMed1984

Different ability of novobiocin and coumermycin A1 to interact with nucleic acids.

The possibility of two structurally related antibiotics, Coumermycin A1 and Novobiocin, to interact with nucleic acids was investigated. Only Coumermycin A1 was able to form complexes with DNA showing an apparent affinity constant comparable to that of the interaction with ribosomal RNA. A binding specificity for A + T complementary and repeating sequences was also exhibited by Coumermycin A1. In view of the different behaviour of the two compounds some considerations are made on their mode of action; although they are acting on the same target enzymes in Escherichia coli, they may affect the functions of eukaryotic cells through a different mechanism not equally specific and probably distinct for each of the two antibiotics.

Aminocoumarins↗
PubMed1983

Stimulation of calf thymus DNA alpha-polymerase by ATP.

ATP stimulates the activity of the A and C forms of calf thymus DNA alpha-polymerase on several natural and synthetic primer-templates. Stimulations ranging from 1.5- to 8-fold were observed on gapped bacteriophage fd replicative form DNA, poly(dA) x oligo(dT)10, and poly(dT) x oligo(A)10, at ATP concentrations of 1-5 mM. CTP, dATP, and dCTP can substitute for ATP but are less effective. The nonhydrolyzable ATP analogs, adenyl-5'-yl imidodiphosphate and adenosine 5'-O-(thiotriphosphate), and other deoxy- and ribonucleoside triphosphates are essentially inactive for stimulation. Stimulation does not result from polymerase-associated DNA primase activity. The size of products synthesized processively by each enzyme on the two homopolymer templates was determined by gel filtration of primers extended under conditions where the enzyme did not react with a given 3'-OH terminus more than once. The size of products synthesized by the A and D forms on poly(dA) x oligo(dT)10 increased by a factor of 3-6 in the presence of ATP. This suggests a direct effect of ATP on the primer elongation reaction. Finally, presynthesis incubation of enzyme plus template at 37 degrees C in the presence or absence of ATP demonstrates that ATP stabilizes the enzyme against breakdown.

Adenosine Triphosphate↗
PubMed1977

On the fidelity of DNA replication. Effect of metal activators during synthesis with avian myeloblastosis virus DNA polymerase.

The effect of metal activators on the fidelity of DNA synthesis has been examined. Using the DNA polymerase from avian myeloblastosis virus, the accuracy of Co2+-, M2+-, and Ni2+-activated DNA synthesis was determined with different polynucleotide templates. With poly[d(A-T)] as the template, the error frequency for dCMP incorporation was 1:1400, 1:1100, and 1:600 for Mg2+, Co2+, and Mn2+, respectively, at maximally activating concentrations. The error frequency was invariant with respect to [Mg2+] but increased with greater than activating concentrations of Co2+ and Mn2+. This increase resulted from differential rates of complementary and noncomplementary nucleotide incorporation. The enhanced error frequency was nonspecific as it occurred with all polynucleotide templates and with all noncomplementary deoxy- and ribonucleotides which were tested. Nearest neighbor analyses of the reaction products indicated that the noncomplementary deoxynucleotides were incorporated as single base substitutions. The fidelity of Ni2+-activated DNA synthesis was invariant with respect to [Ni2+] and was similar to that obtained using Mg2+. During DNA synthesis with Mg2+, the addition of Co2+, Mn2+, or Ni2+ resulted in a decrease in the fidelity of DNA synthesis. The relationship between decreases in the fidelity of DNA synthesis and metal mutagenesis, or carcinogenesis, or both, is considered.

Avian Myeloblastosis Virus↗
PubMed1987

O-Alkyl deoxythymidines are recognized by DNA polymerase I as deoxythymidine or deoxycytidine.

The O2- and O4-methyldeoxythymidine triphosphates (O-alkyl dTTP) can be used to substitute for dTTP in Escherichia coli DNA polymerase I (Pol I)-catalysed synthesis of poly[deoxyadenosine-deoxythymidine] (dA-dT). When incorporated into the polynucleotide, no detectable perturbation of structure occurred with even 20% O-methyldeoxythymidine in place of dT. However, on replication of such polymers with Pol I, significant amounts of deoxyguanosine triphosphate (dGTP) were incorporated, as well as high levels of deoxyadenosine triphosphate (dATP), indicating tautomer-like behaviour. Higher homologues, such as O4-ethyl (e4) dTTP or O4-isopropyl (ip4) dTTP, could also replace dTTP, but with lower efficiency. Nevertheless, their presence, like O4-methyl (m4) dT substitutions, caused transitions as well as inhibiting enzyme digestion with a variety of 3' nucleases, particularly to the 3'----5' exonuclease activity (proofreading) of polymerases. Further proof of mutagenicity comes from site-directed experiments placing m4dT or e4dT in place of dT at position 587 in am3 of phi X174, in which all revertants sequenced had A----G transitions. This implies that, since m4dT and e4dT are poorly repaired in eukaryotes, it is likely that they will remain in the DNA and lead to effects on enzyme activity, as well as mutations which contribute to the carcinogenicity of N-nitroso compounds.

Alkylation↗
PubMed1987

Catalytic activities of synthetic octadeoxyribonucleotides as coenzymes of poly(ADP-ribose) polymerase and the identification of a new enzyme inhibitory site.

The catalytic activity of highly purified poly(ADP-ribose) polymerase was determined at constant NAD+ concentration and varying concentrations of sDNA or synthetic octadeoxyribonucleotides of differing composition. The coenzymic activities of deoxyribonucleotides were compared in two ways: graphic presentation of the activation of poly(ADP-ribose) polymerase in the presence of a large concentration range of deoxyribonucleotides and by calculating kD values for the deoxyribonucleotides. As determined by method i, auto-mono-ADP-ribosylation of the enzyme protein at 25 nM NAD+ was maximally activated at 1:1 octamer/enzyme molar ratios by the octadeoxyribonucleotide derived from the regulatory region of SV40 DNA (duplex C). At a 0.4:1 sDNA/enzyme ratio, sDNA was the most active coenzyme for mono-ADP-ribosylation. At 200 microM NAD+, resulting in polymer synthesis and with histones as secondary polymer acceptors, duplex C was the most active coenzyme, and the octamer containing the steroid hormone receptor binding consensus sequence of DNA was a close second, whereas sDNA exhibited an anomalous biphasic kinetics. sDNA was effective on mono-ADP-ribosylation at a concentration 150-200 -times lower than on polymer formation. When comparison of deoxyribonucleotides was based on method ii (kD values), by far the most efficiently binding coenzyme for both mono and polymer synthesis was sDNA, followed by duplex C, with (dA-dT)8 exhibiting the weakest binding. The synthetic molecule 6-amino-1,2-benzopyrone (6-aminocoumarin) competitively inhibited the coenzymic function of synthetic octadeoxyribonucleotides at constant concentration of NAD+, identifying a new inhibitory site of poly(ADP-ribose) polymerase.

ADP Ribose Transferases↗
PubMed1990

Molecular recognition in the minor groove of the DNA helix. Studies on the synthesis of oligonucleotides and polynucleotides containing 3-deaza-2'-deoxyadenosine. Interaction of the oligonucleotides with the restriction endonuclease EcoRV.

An improved procedure for the preparation of 3-deaza-2'-deoxyadenosine (d3CA) is described which is suitable for the synthesis of gram quantities of this analogue. Using phosphoramidite chemistry d3CA has been incorporated into the Eco RV restiction endonuclease recognition sequence (underlined) present in the self-complementary dodecamer d(GACGATATCGTC). The modified oligonucleotides have been thoroughly characterised by nucleoside composition analysis, circular dichroism and thermal melting studies. Studies with Eco RV show that incorporation of d3CA into either the central or outer dA-dT base-pair results in a substantial reduction in the rate of cleavage. The two-step conversion of d3CA to 3-deaza-2'-deoxyadenosine-5'-O-triphosphate (d3CATP) via the 5'-O-tosylate is also described. d3CATP is not a substrate in the poly[d(AT)].poly[d(AT)] primed polymerisation for either E. coli DNA polymerase I or Micrococcus luteus DNA polymerase. In a more detailed kinetic analysis d3CATP was shown to be a competitive inhibitor of E. coli DNA polymerase I with respect to dATP.

Adenosine Triphosphate↗