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At least 55 records · Page 3Linked to original sources
PubMed1992

In vitro DNA synthesis by DNA polymerase I and DNA polymerase alpha on single-stranded DNA containing either purine or pyrimidine monoadducts.

The capacity of the large fragment of DNA polymerase I from Escherichia coli and of DNA polymerase alpha from Drosophila embryo to replicate single-stranded M13mp10 DNA containing either purine or pyrimidine monoadducts was compared. The monoadducts were respectively induced by cisplatinum and by furocoumarin photoaddition. For both types of lesions, it is observed that the eukaryotic enzyme is more inhibited than the prokaryotic one. By mapping the arrest sites produced by furocoumarin monoadducts on the synthesis catalysed by DNA polymerase alpha, we show that, in contrast with the photoreaction observed with double-stranded DNA, these compounds do not show a strong sequence specificity in reacting with single-stranded DNA.

Animals↗
PubMed1978

DNA modifying enzymes of Agrobacterium tumefaciens: effect of DNA topoisomerase, restriction endonuclease, and unique DNA endonuclease on plasmid and plant DNA.

Extracts from Agrobacterium tumefaciens strain ID135 contain three enzymes that have been characterized and partially purified. The first enzyme, a DNA topoisomerase, appeared to relax only negatively twisted DNA. The second enzyme, Atu I, a type II restriction endonuclease, generated the identical DNA digestion pattern as EcoRII when several DNAs were used. The third enzyme, endonuclease A, showed a preference for superhelical DNAs as substrates. When plasmid pCK135DNA, obtained from the virulent strain IDI135 of A. tumefaciens, or plant DNA was exposed to the three enzymes, changes in DNA patterns were observed due to either conformational changes or digestion of the DNAs. These enzymes may function in vivo in the processing and incorporation of bacterial DNA in plant cells.

Animals↗
PubMed2001

Indirect readout of DNA sequence at the primary-kink site in the CAP-DNA complex: alteration of DNA binding specificity through alteration of DNA kinking.

The catabolite activator protein (CAP) sharply bends DNA in the CAP-DNA complex, introducing a DNA kink, with a roll angle of approximately 40 degrees and a twist angle of approximately 20 degrees, between positions 6 and 7 of the DNA half-site, 5'-A(1)A(2)A(3)T(4)G(5)T(6)G(7)A(8)T(9)C(10)T(11)-3' ("primary kink"). CAP recognizes the base-pair immediately 5' to the primary-kink site, T:A(6), through an "indirect-readout" mechanism involving sequence effects on the energetics of primary-kink formation. CAP recognizes the base-pair immediately 3' to the primary-kink site, G:C(7), through a "direct-readout" mechanism involving formation of a hydrogen bond between Glu181 of CAP and G:C(7). Here, we report that substitution of the carboxylate side-chain of Glu181 of CAP by the one-methylene-group-shorter carboxylate side-chain of Asp changes DNA binding specificity at position 6 of the DNA half site, changing specificity for T:A(6) to specificity for C:G(6), and we report a crystallographic analysis defining the structural basis of the change in specificity. The Glu181-->Asp substitution eliminates the primary kink and thus eliminates indirect-readout-based specificity for T:A(6). The Glu181-->Asp substitution does not eliminate hydrogen-bond formation with G:C(7), and thus does not eliminate direct-readout-based specificity for G:C(7).

Base Sequence↗
PubMed1994

Interaction of DNA polymerase and DNA helicase within the bacteriophage T4 DNA replication complex. Leading strand synthesis by the T4 DNA polymerase mutant A737V (tsL141) requires the T4 gene 59 helicase assembly protein.

The bacteriophage T4 tsL141 (A737V) mutant in T4 DNA polymerase is temperature-sensitive for DNA replication and an antimutator for some types of mutations. In the accompanying paper (Spacciapoli, P., and Nossal, N. G. (1993) J. Biol. Chem. 269, 438-446), we show that the purified A737V T4 DNA polymerase is less processive than the wild type enzyme as a polymerase, but is more processive as an exonuclease. The bacteriophage T4 multienzyme replication complex reconstituted with the A737V mutant polymerase is defective in both lagging and leading strand synthesis. On lagging strand templates, the A737V polymerase is stimulated by the gene 44/62 and 45 polymerase accessory proteins and the gene 32 DNA binding protein, but is still arrested at pause sites much more frequently than the wild type. In contrast to wild type T4 DNA polymerase, the A737V polymerase does not catalyze leading strand synthesis on a forked duplex template with the polymerase accessory proteins, 32 protein, and the gene 41 protein helicase. The A737V polymerase requires the T4 gene 59 helicase assembly protein, as well as the other proteins, to carry out this reaction. Each of these defects is suppressed by the intragenic L771F mutation that suppresses the antimutator phenotype of the A737V, polymerase in vivo (Reha-Krantz, L. J., Stocki, S., Nonay, R., and Maughan, C. (1989) J. Cell. Biochem. 13D, 140).

Bacteriophage T4↗
PubMed1992

[The content of DNA, RNA and protein and the fresh weight:DNA, protein:DNA and RNA:DNA ratios in tissue of male calves, bulls, short-scrotum bulls and oxen during the growth period].

In male calves, in bulls, in short scrotum bulls and in oxen of different age the content of DNA, RNA and protein in various tissues (cerebrum, cerebellum, spinal cord, ventricles, lung, liver, cortex of the kidney, spleen, testicles, M. semimembranosus) was analysed. The fresh weight: DNA-, the protein: DNA- and the RNA: DNA-ratios were calculated. In the age of 78 weeks the short scrotum bulls had a body weight of 577 +/- 44, the bulls of 550 +/- 21 and the oxen of 462 +/- 41 kg. The testicles of the short scrotum bulls were smaller than that of the bulls. The differences in the content of DNA, RNA and protein per g tissue between male calves, bulls, short scrotum bulls and oxen of the same age were in part significant. The total content of DNA, of RNA and of protein for the 78 weeks old as well as the number of nuclei of the lung, the liver, the spleen and the testicles for the 6 and 78 weeks old animals were calculated. The biochemical parameters can be used for the characterization of the inhibition of growth of the different organs of calves and cattle by malnutrition and diseases.

Animals↗
PubMed1982

Design and characterization of N2-arylaminopurines which selectively inhibit replicative DNA synthesis and replication-specific DNA polymerases: guanine derivatives active on mammalian DNA polymerase alpha and bacterial DNA polymerase III.

The 2-amino substituted derivatives of guanine, N2-(p-n-butylphenyl)guanine (BuPG) and N2-(3',4'-trimethylenephenyl) guanine (TMPG), were synthesized and found to selectively inhibit, respectively, HeLa cell DNA polymerase alpha (po1 alpha) and B. subtilis DNA polymerase III (po1 III). Both purines, like their corresponding uracil analogs, BuAu and TMAU (2,9), were specifically competitive with dGTP in their inhibitory action on their target polymerases. BuPG, the pol alpha-specific purine, was also toxic for HeLa cells in vivo, selectively inhibiting DNA synthesis. These N2-substituted purines, in contrast to the 6-substituted uracils, provide a structural basis for the synthesis of nucleosides and nucleotides with considerable potential as probes for the analysis of the structure of specific replicative DNA polymerases and their function in cellular DNA metabolism.

Bacillus subtilis↗
PubMed2002

DNA topology: topological transformation of DNA by reaction of cisplatin-DNA-core histone complexes with human DNA topoisomerase.

Cis-diamminedichloroplatinium(II), an active antitumor agent binds to core-histone -SV40 DNA complexes prepared by reaction of DNA with core-histone, and alters the fiber-like structure into the loosened structure. In order to model the cisplatin-modified chromatin complexes in cell, the complexes were reacted with human DNA topo II. We found the generations of unique topologically isomers such as trefoil knot (and catenane) and pseudo catenane (and pseudo knot) by reaction of cis-DDP--linearDNA--core-histone complexes with DNA topo I. The results are discussed in relation with a possible recombinational role of topo II (or topo I) on the reaction with cis-DDP--DNA--core-histone complexes.

Cisplatin↗
PubMedDate not supplied

DNA-DNA interstrand cross-linking by 2,5-bis(1-aziridinyl)-3,6-bis(carbethoxyamino)-1,4-benzoquinone: covalent structure of the dG-to-dG cross-links in calf thymus DNA and a synthetic DNA duplex.

The products of the alkylation reaction of reduced 2,5-bis(1-aziridinyl)-3,6-bis(carbethoxyamino)-1,4-benzoquinone (AZQ, 1a) with duplex DNA were studied using calf thymus DNA and a synthetic oligodeoxynucleotide. Reaction of calf thymus DNA with a mixture of AZQ and ascorbic acid followed by enzymatic digestion of the sugar phosphate backbone afforded numerous AZQ-derived products including substances identified as monoadducts of AZQ with both dG and dA (with the former in greater abundance) and two diadducts, as would be expected for intrastrand or interstrand cross-links, with one containing two dG residues per AZQ and the other one each of dG and dA (with the former adduct in greater abundance). The nucleotide connectivity and covalent structure of the dG-to-dG interstrand cross-links were studied in greater detail using a synthetic DNA duplex containing the nucleotide sequence 5'-d(GGGCCC), where it appeared that the predominant interstrand cross-links bridged dG residues on opposite strands and were separated by two intervening base pairs [5'-d(GNNC)]. The covalent structure of this lesion was tentatively identified as 2b, in which the N7 atoms of two dG residues have been alkylated by the aziridine functions of AZQ, based upon the results of piperidine fragmentation and characterization of the enzymatic and acidic hydrolysates of the cross-linked DNA.

Alkylation↗
PubMed1980

Deletions and DNA rearrangements within the transposable DNA element IS2. A model for the creation of palindromic DNA by DNA repair synthesis.

Three derivatives of mutant ga10P-308::IS2-I of Escherichia coli were characterized by DNA sequence analysis. Deletions and DNA sequence rearrangements were observed which apparently were initiated at short A-T rich inverted repeats within IS2. Two of the mutants carried newly synthesized DNA sequences which were inverted copies of already existing IS2 sequences. Thus long stretches with twofold symmetry were formed. It is discussed whether these inverted repeats were formed by DNA repair synthesis which was initiated at the A-T rich palindromes of IS2.

Base Sequence↗
PubMed1993

[The content of DNA, RNA and protein and the wet weight:DNA, the protein:DNA and the RNA:DNA ratio in 19 different tissues of bovine fetuses of different body weight].

In 3 groups of fetuses (n = 6 each) of cattle with a body mass of 4.18 +/- 2.1 (about 160 days old), of 9.72 +/- 0.97 (about 200 days old) and of 17.12 +/- 2.61 kg (about 230 days old) the content of DNA, RNA and protein in 19 different tissues was analysed. The wet weight:DNA-, the protein:DNA- and the RNA:DNA-ratios were calculated. The growth of the different tissues in the mentioned period of the development of the fetuses by hyperplasia and hypertrophy is discussed.

Animals↗
PubMed1988

RglB facilitated cloning of highly methylated eukaryotic DNA: the human L1 transposon, plant DNA, and DNA methylated in vitro with human DNA methyltransferase.

In vitro methylation of Bluescribe plasmid DNA (pBS) with human placental DNA methyltransferase to 6% 5-methylcytosine (mC) reduced transformation efficiencies in rglB+ host strains C600 and DS410 by almost 2 orders of magnitude. By contrast, the rglB- derivative of DS410 showed no reduction in transformation efficiency with methylation while the rglB- derivative of C600 was partially tolerant to methylation. Further, we show that the 1.8 kilobase (kb) and 1.2 kb KpnI fragments derived from the human L1 repeat have respectively 18.3% and 2.3% mC in vivo. Using these hyper- and hypo-methylated genomic segments ligated into the pBS plasmid, transformants with the highly methylated 1.8 kb L1 insert were recovered at 17 to 40 fold higher frequency with the rglB- host strains than with the rglB+ hosts. In addition, recombinant phage (lambda 2001) containing inserts of plant genomic DNA with 26.7% mC (from Petunia hybrida) when plated on rglB- hosts gave titres up to 222 times higher than on the rglB+ strains.

Cell Line↗
PubMed1997

DNA sequencing and comparative sequence analysis reveal that the Escherichia coli genomic DNA may replace the target DNA during molecular cloning: evidence for the erroneous assembly of E. coli DNA into database sequences.

DNA sequencing and similarity search of databases provide experimental evidence that portions of the host Escherichia coli genome may get ligated into the cloning vector, resulting in clones containing nontargeted inserts. Several lines of evidence suggest that this non-targeted ligation, as observed by us while subcloning troponin I cDNA, is presumably due to a recombination-mediated mechanism by which host DNA replaces the target DNA in the cloning vector. The E. coli genome mapping to 64-65 min and 92.8-00.1 min, the latter containing insertion sequences, appears to be the hotspot regions involved in this process. We examined the possibility that some sequences reported in the databases may also contain genomic sequences of E. coli. A search of current databases revealed that a rat hepatic glutathione transporter cDNA contains a 2.2-kb-long portion of the E. coli genome that has been wrongly assembled into its 5' untranslated and coding regions. In addition, about 30 sequences in databases, including a Yersinia pestis toxin gene, showed relatively high sequence identity with those portions of the E. coli genome that were present in the nonauthentic clones.

Animals↗
PubMed2000

Interactions of the DNA ligase IV-XRCC4 complex with DNA ends and the DNA-dependent protein kinase.

The DNA-dependent protein kinase (DNA-PK), consisting of Ku and the DNA-PK catalytic subunit (DNA-PKcs), and the DNA ligase IV-XRCC4 complex function together in the repair of DNA double-strand breaks by non-homologous end joining. These protein complexes are also required for the completion of V(D)J recombination events in immune cells. Here we demonstrate that the DNA ligase IV-XRCC4 complex binds specifically to the ends of duplex DNA molecules and can act as a bridging factor, linking together duplex DNA molecules with complementary but non-ligatable ends. Although the DNA end-binding protein Ku inhibited DNA joining by DNA ligase IV-XRCC4, it did not prevent this complex from binding to DNA. Instead, DNA ligase IV-XRCC4 and Ku bound simultaneously to the ends of duplex DNA molecules. DNA ligase IV-XRCC4 and DNA-PKcs also formed complexes at the ends of DNA molecules, but DNA-PKcs did not inhibit ligation. Interestingly, DNA-PKcs stimulated intermolecular ligation by DNA ligase IV-XRCC4. In the presence of DNA-PK, the majority of the joining events catalyzed by DNA ligase IV-XRCC4 were intermolecular because Ku inhibited intramolecular ligation, but DNA-PKcs still stimulated intramolecular ligation. We suggest that DNA-PKcs-containing complexes formed at DNA ends enhance the association of DNA ends via protein-protein interactions, thereby stimulating intermolecular ligation.

Animals↗
PubMed1984

Hepatitis B viral DNA-RNA hybrid molecules in particles from infected liver are converted to viral DNA molecules during an endogenous DNA polymerase reaction.

Particles purified from the liver of hepatitis B virus-infected patients were previously shown by us to incorporate 32P-deoxynucleotides into viral DNA and DNA-RNA hybrid molecules when incubated in a DNA polymerase reaction mixture. In this investigation, similar particles from duck and ground squirrel livers infected with viruses closely related to HBV were also shown to incorporate 32P-deoxynucleotides into viral-specific DNA and DNA-RNA hybrid molecules when incubated in vitro in a DNA polymerase reaction mixture. The particles from duck hepatitis B virus-infected liver contained a heterogeneous population of hybrid molecules, while those from ground squirrel hepatitis virus-infected liver contained hybrid molecules with densities similar to those found in HBV particles including a distinct population of molecules with an average density of 1.57 g/cm3. Brief endogenous DNA polymerase reactions with particles from all three livers, resulted in incorporation of 32P-deoxynucleotides into viral DNA of DNA-RNA hybrid as well as viral DNA molecules. When the reaction was continued in the presence of a 1000-fold molar excess of unlabeled deoxynucleotides, a decrease in [32P]DNA in the DNA-RNA hybrid region of the Cs2SO4 density gradient and a proportional increase in [32P]DNA in the DNA region of the gradient was observed. This effect was seen most dramatically with particles isolated from freshly obtained ground squirrel hepatitis virus-infected livers in which 90% of the pulse labeled DNA in the hybrid species at the buoyant density of 1.57 g/cm3 appeared to be converted to a form with the buoyant density of pure DNA (1.42 g/cm3). Storage of virus particles at 4 degrees, or prior freezing of infected ground squirrel liver almost completely abolished the ability of the endogenous DNA polymerase activity to incorporate 32P-deoxynucleotides into hybrid molecules, while incorporation into DNA molecules was apparently unaffected. These results suggest that different enzymatic activities catalyze synthesis of the viral DNA in DNA-RNA hybrids and in molecules with buoyant density of pure DNA. Thus particles from infected liver synthesize DNA of DNA-RNA hybrid molecules which can be converted in the particles into molecules with the buoyant density of pure DNA. This indicates that DNA-RNA hybrids may be intermediates in viral DNA replication and that the mechanism of hepatitis B virus (and closely related viruses of ground squirrels and ducks) DNA replication differs from that known for other DNA viruses.

Animals↗
PubMed2002

The DNA-dependent protein kinase interacts with DNA to form a protein-DNA complex that is disrupted by phosphorylation.

DNA double-strand breaks are a serious threat to genome stability and cell viability. One of the major pathways for the repair of DNA double-strand breaks in human cells is nonhomologous end-joining. Biochemical and genetic studies have shown that the DNA-dependent protein kinase (DNA-PK), XRCC4, DNA ligase IV, and Artemis are essential components of the nonhomologous end-joining pathway. DNA-PK is composed of a large catalytic subunit, DNA-PKcs, and a heterodimer of Ku70 and Ku80 subunits. Current models predict that the Ku heterodimer binds to ends of double-stranded DNA, then recruits DNA-PKcs to form the active protein kinase complex. XRCC4 and DNA ligase IV are subsequently required for ligation of the DNA ends. Magnesium-ATP and the protein kinase activity of DNA-PKcs are essential for DNA double-strand break repair. However, little is known about the physiological targets of DNA-PK. We have previously shown that DNA-PKcs and Ku undergo autophosphorylation, and that this correlates with loss of protein kinase activity. Here we show, using electron spectroscopic imaging, that DNA-PKcs and Ku interact with multiple DNA molecules to form large protein-DNA complexes that converge at the base of multiple DNA loops. The number of large protein complexes and the amount of DNA associated with them were dramatically reduced under conditions that promote phosphorylation of DNA-PK. Moreover, treatment of autophosphorylated DNA-PK with the protein phosphatase 1 catalytic subunit restored complex formation. We propose that autophosphorylation of DNA-PK plays an important regulatory role in DNA double-strand break repair by regulating the assembly and disassembly of the DNA-PK-DNA complex.

Adenosine Triphosphate↗
PubMed1998

DNA synthesis on discontinuous templates by human DNA polymerases: implications for non-homologous DNA recombination.

DNA polymerases catalyze the synthesis of DNA using a continuous uninterrupted template strand. However, it has been shown that a 3'-->5' exonuclease-deficient form of the Klenow fragment of Escherichia coli DNA polymerase I as well as DNA polymerase of Thermus aquaticus can synthesize DNA across two unlinked DNA templates. In this study, we used an oligonucleotide-based assay to show that discontinuous DNA synthesis was present in HeLa cell extracts. DNA synthesis inhibitor studies as well as fractionation of the extracts revealed that most of the discontinuous DNA synthesis was attributable to DNA polymerase alpha. Additionally, discontinuous DNA synthesis could be eliminated by incubation with an antibody that specifically neutralized DNA polymerase alpha activity. To test the relative efficiency of each nuclear DNA polymerase for discontinuous synthesis, equal amounts (as measured by DNA polymerase activity) of DNA polymerases alpha, beta, delta (+/- PCNA) and straightepsilon (+/- PCNA) were used in the discontinuous DNA synthesis assay. DNA polymerase alpha showed the most discontinuous DNA synthesis activity, although small but detectable levels were seen for DNA polymerases delta (+PCNA) and straightepsilon (- PCNA). Klenow fragment and DNA polymerase beta showed no discontinuous DNA synthesis, although at much higher amounts of each enzyme, discontinuous synthesis was seen for both. Discontinuous DNA synthesis by DNA polymerase alpha was seen with substrates containing 3 and 4 bp single-strand stretches of complementarity; however, little synthesis was seen with blunt substrates or with 1 bp stretches. The products formed from these experiments are structurally similar to that seen in vivo for non-homologous end joining in eukaryotic cells. These data suggest that DNA polymerase alpha may be able to rejoin double-strand breaks in vivo during replication.

Animals↗