Showing papers on "DNA clamp published in 1983"

Two DNA methyltransferases from murine erythroleukemia cells: purification, sequence specificity, and mode of interaction with DNA.

Abstract: Dye-ligand chromatography on Cibacron blue F3GA-agarose has been used to resolve two species of DNA (cytosine-5-)-methyltransferase from nuclear extracts of uninduced Friend murine erythroleukemia cells. Each species has been highly purified; the activities in the first and second peaks were associated with polypeptides of Mr 150,000 and 175,000, respectively. Analysis of substrate specificity with synthetic DNAs and restriction fragments of phi X174 replicative form DNA and pBR322 DNA showed that neither enzyme had dependence on the sequence context of CpG dinucleotides; poly(dG-dC) had the greatest methyl-accepting activity of any unmethylated DNA substrate tested. De novo methylation by both enzymes was inefficient relative to methylation of hemimethylated sites. Methyl-accepting activity was strongly dependent on DNA chain length. This observation suggests that binding to DNA, followed by one-dimensional diffusion of enzyme along the DNA molecule, is important in the mechanism by which DNA methyltransferase locates its recognition sites.

Infidelity of DNA synthesis associated with bypass of apurinic sites

Abstract: The mutagenic potential of apurinic sites in vivo has been studied by transfection of depurinated phi X174 DNA containing amber mutations into SOS-induced Escherichia coli spheroplasts. Mutagenicity is abolished by treatment of the depurinated DNA with an apurinic endonuclease from Hela cells, establishing the apurinic site as the mutagenic lesion. The frequency of copying apurinic sites in vitro was analyzed by measuring the extent of DNA synthesis using E. coli DNA polymerase I and avian myeloblastosis DNA polymerase. The inhibition of DNA synthesis by apurinic sites was less with avian myeloblastosis DNA polymerase, suggesting that this error-prone enzyme copies apurinic sites with greater frequency. Consistent with this conclusion is the observation that, upon transfection into (normal) spheroplasts, the reversion frequency of depurinated phi X174 am3 DNA copied with avian myeloblastosis virus DNA polymerase is much greater than that of the same DNA copied with E. coli DNA polymerase I. Sequence analysis of the DNA of 33 revertant phage produced by depurination indicates a preference for incorporation of deoxyadenosine opposite putative apurinic sites. The combined results indicate that mutagenesis resulting from apurinic sites is associated with bypass of these noncoding lesions during DNA synthesis.

Specific binding of a cellular DNA replication protein to the origin of replication of adenovirus DNA.

Abstract: Nuclear factor I, a 47-kilodalton protein, purified from nuclear extracts of uninfected HeLa cells, is involved in the initiation and possibly the elongation of replicating adenovirus (Ad) DNA in vitro. The binding of nuclear factor I to DNA has been monitored by a filter binding assay of nuclear factor I to DNA has been monitored by a filter binding assay using plasmid pLA1 DNA, which contains a 3,290 base-pair fragment derived from the left-hand terminus (coordinates, 0-9.4 map units) of Ad serotype 5 DNA. Nuclear factor I binds selectively to a double-stranded fragment spanning nucleotides 0-451 to the Ad genome. The retention of the 451-base-pair DNA fragment-nuclear factor I complex on nitrocellulose filters does not require Mg2+ or ATP and is resistant to high ionic strength. DNase I protection experiments revealed that nuclear factor I binds to a nucleotide sequence located at position 17-48, close to the terminus of Ad DNA. This 32-nucleotide sequence contains four "consensus" sequences present in various serotypes of Ad DNA and is capable of forming higher ordered structures. The role of nuclear factor I and this DNA sequence in the generation of Ad preterminal protein-dCMP initiation complex is discussed.

Identification of the epsilon-subunit of Escherichia coli DNA polymerase III holoenzyme as the dnaQ gene product: a fidelity subunit for DNA replication.

Abstract: Based on extensive genetic and biochemical studies, the multisubunit DNA polymerase III holoenzyme is considered responsible for the chain-elongation stage in replication of the genome of Escherichia coli and is thus expected to be the major determinant of fidelity as well Previous experiments have shown that two mutations conferring a very high mutation rate on E coli, mutD5 and dnaQ49, decrease severely the 3' leads to 5' exonucleolytic editing activity of the polymerase III holoenzyme To identify more precisely the nature of these mutations, we have carried out genetic mapping and complementation experiments From these studies and experiments by others, we conclude that the most potent general mutator mutations in E coli occur in a single gene, dnaQ To define further the role of the dnaQ gene, we have used two-dimensional gel electrophoresis to compare the labeled dnaQ gene product with purified polymerase III holoenzyme The dnaQ product comigrates with the epsilon-subunit, a 25-kilodalton protein of the polymerase III "core" enzyme We conclude that the epsilon-subunit of polymerase III holoenzyme has a special role in defining the accuracy of DNA replication, probably through control of the 3' leads to 5' exonuclease activity

Construction of a plasmid that overproduces the large proteolytic fragment (Klenow fragment) of DNA polymerase I of Escherichia coli.

Abstract: Using currently available gene fusion techniques, we have constructed plasmids that direct the overproduction of the carboxyl-terminal two-thirds of DNA polymerase I, corresponding to the proteolytically derived "Klenow fragment." We have obtained overproduction amounting to several percent of the cellular protein using constructs in which expression is directed either from the lac promoter or from the leftward promoter of phage lambda. The polymerase fragment has been purified to homogeneity from such overproducing strains by a rapid three-stage purification procedure, yielding material capable of carrying out the same reactions (polymerization, 3' labeling, DNA sequence analysis) as the proteolytically derived fragment. The availability of such overproducing strains should greatly facilitate structural and mechanistic studies of DNA polymerase I. Moreover, the techniques we have described for the cloning and expression of a gene fragment should be generally applicable for the study of protein structure and function in other systems.

Mutator strains of Escherichia coli, mutD and dnaQ, with defective exonucleolytic editing by DNA polymerase III holoenzyme

Abstract: The closely linked mutD and dnaQ mutations confer a vastly increased mutation rate on Escherichia coli and thus might define a gene with a central role in the fidelity of DNA replication. To look for the biochemical function of the mutD gene product, we have measured the 3' leads to 5' exonucleolytic editing activity of polymerase III holoenzyme from mutD5 and dnaQ49 mutants. The editing activities of the mutant enzymes are defective compared to wild type, as judged by two assays: (i) decreased excision of a terminal mispaired base from a copolymer substrate and (ii) turnover of dTTP to dTMP during replication with a phage G4 DNA template. Thus, the mutD (dnaQ) gene product is likely to control the editing (proofreading) capacity of polymerase III holoenzyme.

Cross-linking of DNA induced by chloroethylnitrosourea is presented by O6-methylguanine-DNA methyltransferase.

Abstract: The DNA repair enzyme O6-methylguanine-DNA methyltransferase has been used as a reagent to analyse the initial reaction sites of alkylating agents such as chloroethylnitrosourea that cross-link DNA. The transferase can be employed for this purpose because it removes substituted ethyl groups from DNA, as shown by its ability to act on O6-hydroxyethylguanine residues in DNA. The enzyme counteracts the formation of interstrand cross-links induced by bis-chloroethylnitrosourea, but not those induced by nitrogen mustard. Once formed, chloroethylnitrosourea-induced cross-links are not broken by the enzyme. In agreement with deductions from experiments with living cells, it is concluded that chloroethylnitrosourea act by forming reactive monoadducts at the O6 position of guanine and/or the O4 position of thymine, which subsequently generate -CH2CH2- bridges to the complementary DNA strand. A new method for quantitating interstrand cross-links in DNA has been employed.

Adenovirus DNA replication in vitro: synthesis of full-length DNA with purified proteins.

Abstract: A protein required for the elongation of replicating intermediates of adenovirus (Ad) DNA to full length has been isolated and characterized. This factor, isolated from nuclear extracts of uninfected HeLa cells, has been designated nuclear factor II. In the presence of Ad DNA with proteins at each 5' end (Ad DNA-protein) and three proteins coded for by the Ad genome [the preterminal protein (pTP), the DNA polymerase (Ad Pol), and the DNA binding protein (Ad DBP)], nuclear factor II complementing activity is detected only in the presence of host nuclear factor I. Highly purified preparations of nuclear factor II that are free of detectable DNA polymerase alpha, beta, and gamma activities contain a DNA topoisomerase activity. Furthermore, type I DNA topoisomerases purified from HeLa cells and calf thymus substitute for nuclear factor II complementing activity in the in vitro Ad DNA replication system. These results indicate that a protein that is involved in higher order DNA structure is required for Ad replication. This protein plus the purified proteins described above carry out the initiation and synthesis of full-length 36,000-base-pair Ad DNA.

Transformasomes: specialized membranous structures that protect DNA during Haemophilus transformation.

Abstract: The mechanism by which Haemophilus protects donor DNA from cellular restriction and degradative enzymes during transformation is unclear. In this report, we demonstrate that donor DNA enters Haemophilus influenzae through specialized membranous extensions, which we have termed "transformasomes." DNA within transformasomes is in a protected state--resistant to external DNase and cellular restriction enzymes, although remaining unmodified and double-stranded. The ability of donor DNA to exit from transformasomes is dependent on its topological conformation. Circular DNA remains intact within transformasomes, while linear DNA rapidly exits and undergoes homologous recombination. Protected donor DNA can be preferentially removed from the surface of competent cells by extraction with organic solvents. Structurally intact transformasomes containing donor DNA could be partitioned into the organic layer and can be further purified by density centrifugation.

High-efficiency ligation and recombination of DNA fragments by vertebrate cells

Abstract: DNA-mediated gene transfer (transfection) is used to introduce specific genes into vertebrate cells. Events soon after transfection were quantitatively analyzed by determining the infectivity of the DNA from an avian retrovirus and of mixtures of subgenomic fragments of this DNA. The limiting step of transfection with two DNA molecules is the uptake by a single cell of both DNA's in a biologically active state. Transfected cells mediate ligation and recombination of physically unlinked DNA's at nearly 100 percent efficiency.

Protein covalently bound to minus-strand DNA intermediates of duck hepatitis B virus.

Abstract: Analysis of duck hepatitis B viral DNA by gel electrophoresis, Southern blotting, and binding to benzoylated naphthoylated DEAE-cellulose showed that a protein is bound to the minus-strand virion DNA as well as to the full-length single strand, minus-strand species, and minus-strand DNA intermediates isolated from replicating complexes present in infected duck liver. By utilizing a modified dideoxynucleotidyl sequencing method, it was shown that the protein is covalently bound to the smallest detectable growing strands (ca. 30 bases) and that minus-strand synthesis begins at a unique site. These results support the notion that the protein may function as a primer for synthesis of the minus-strand DNA.

Isolation of an intact DNA polymerase-primase from embryos of Drosophila melanogaster.

Abstract: A procedure has been devised for the purification of intact DNA polymerase alpha from early embryos of Drosophila melanogaster The purified enzyme consists of at least three polypeptides with Mrs of 182,000, 60,000, and 50,000 These are related antigenically to the alpha (Mr 148,000), beta (Mr 58,000), and gamma (Mr 46,000) subunits, respectively, of the DNA polymerase described previously [Banks, G R, Boezi, J A & Lehman, I R (1979) J Biol Chem 254, 9886-9892] The alpha subunit (Mr 182,000) has a molecular weight indistinguishable from that observed in extracts of freshly harvested embryos and presumably present in vivo As in the previous preparation, the alpha subunit is required for DNA polymerase activity and is very likely the catalytic subunit of the enzyme The ratio of primase to polymerase remains constant throughout the purification Thus, the primase is very likely an integral component of the Drosophila DNA polymerase alpha The purified DNA polymerase-primase contains no detectable endo- or exodeoxyribonuclease and has pH, MgCl2, (NH4)2SO4, and NaCl optima identical to those reported previously In contrast, the Km for dTTP is 37 microM as compared with 175 microM for the previous enzyme Sensitivities to aphidicolin and N-ethylmaleiimide and resistance to dideoxy TTP are unchanged

Terminal transferase: use of the tailing of DNA and for in vitro mutagenesis

Abstract: Publisher Summary Terminal deoxynucleotidyltransferase catalyzes the addition of deoxynucleotides to the 3´ termini of DNA. In 1962, this enzyme was recognized as being different from DNA polymerase, and the name was changed to terminal transferase. By use of a tailing method, which adds homopolymer deoxynucleotide tails to denatured DNA, any double-stranded DNA fragment can be joined to a cloning vehicle. Because each DNA fragment carries the same type of tail, it cannot hybridize with another molecule of the same species. Thus, after cloning, each transformant should represent the desired recombinant DNA. These advantages make this technique particularly useful for the construction of recombinant DNA molecules for cloning. This chapter presents an improved procedure for the efficient addition of homopolymer tails to DNA, and also describes a new procedure for the addition of a single nucleotide to the 3´ end of a DNA as a method for in vitro mutagenesis, which can prove to be useful in investigating the control and expression of genes.

Incorporation of 5-Aza-2'-deoxycytidine-5'-triphosphate into DNA. Interactions with mammalian DNA polymerase alpha and DNA methylase.

Abstract: In order to understand further the molecular mode of action of 5-Aza-2'-deoxycytidine (5-AZA-dCyd), a potent antileukemic agent, we prepared enzymatically 5-Aza-2'-deoxycytidine 5'-triphosphate (5-AZA-dCTP) and performed studies with purified DNA polymerase alpha and DNA methylase from mammalian cells. DNA polymerase alpha catalyzed the incorporation of 5-AZA-dCTP into DNA. The apparent Km value for 5-AZA-dCTP was estimated to be 3.0 microM; the Km of dCTP was 2.0 microM. The apparent Vmax of 5-AZA-dCTP was slightly lower than that for dCTP. 5-AZA-dCTP was a weak competitive inhibitor (Ki 4.3 microM) with respect to dCTP. Template studies with 5-AZA-dCTP showed that this nucleotide analogue was incorporated into poly(dIC), but not into poly(dAT), suggesting that the incorporation follows the rules of Watson-Crick base pairing. Incorporation of 5-AZA-dCTP into hemimethylated DNA produced a significant inhibition of DNA methylase. These results show that 5-AZA-dCTP is a very good substrate for DNA polymerase alpha and that its incorporation into DNA inhibits DNA methylation.

Poly(ADP-ribose) polymerase auto-modification and interaction with DNA: electron microscopic visualization.

Abstract: The interaction between purified calf thymus poly(ADP-ribose) polymerase and its activating co-purified DNA (sDNA) was investigated by electron microscopy. We have shown that the enzyme-DNA complex possesses a nucleosome-like structure. The enzyme-bound DNA (sDNA) was found to be enriched in single-stranded regions and branched structures, presumed to be replication forks. The auto-ribosylated polymerase as well as the branched poly(ADP-ribose) formed were visualized by dark field electron microscopy during the auto-ADP-ribosylation reaction and the possible mechanism of this phenomenon is discussed.

4 – Exonuclease III: Use for DNA Sequence Analysis and in Specific Deletions of Nucleotides

Abstract: Publisher Summary Exonuclease III of Escherichia coli catalyzes the sequential hydrolysis of mononucleotides from the 3´ termini of duplex DNA molecules. Using a high ratio of exonuclease III to DNA ends and moderate concentration of salt (90 mM KC1), digestion of DNA is relatively synchronous, removing approximately 10 nucleotides per minute from each 3´ terminus at room temperature. Authors have developed an improved enzymic method for DNA sequence analysis based on the partial digestion of duplex DNA with exonuclease III to produce DNA molecules with 3' ends shortened to varying lengths. The major steps underlying genetic engineering technology include isolation and specific cleavage of DNA, ligation of DNA fragments to a cloning vector, transformation and selection of the desired clone, confirming the cloned gene by physical mapping and DNA sequencing, and expression of the cloned gene. Usually several vectors are needed to serve these functions. However, it is most convenient if a single vector can serve all these functions.

The major herpes simplex virus DNA-binding protein holds single-stranded DNA in an extended configuration.

Abstract: Properties of the major DNA-binding protein found in herpes simplex virus-infected cells were investigated by using a filter binding assay and electron microscopy. Filter binding indicated that the stoichiometry of binding of the protein with single-stranded DNA is approximately 40 nucleotides per protein molecule at saturation. Strong clustering of the protein in DNA-protein complexes, indicative of cooperative binding, was seen with the electron microscope. Measurements of single-stranded fd DNA molecules saturated with protein and spread for electron microscopy by using both the aqueous and formamide spreading techniques indicated that the DNA is held in an extended configuration with a base spacing of approximately 0.13 nm per base.