Luteus DNA Research Articles

Preferential binding of DNA methyltransferase and increased de novo methylation of deoxyinosine containing DNA

Mammalian DNA-cytosine 5-methyltransferases methylate cytosines in deoxyinosine containing DNA polymers more rapidly than in other synthetic or naturally occurring DNAs. The initial methylation rate of poly(dI-dC)·poly(dI-dC) is about 10-times higher than that of poly-(dG-dC)·poly(dG-dC) or of the native Micrococcus luteus DNA. In competitive binding experiments, DNA methyltransferase has about 10-fold higher affinity for the dI-containing alternating DNA polymer than for poly(dG-dC) · poly(dG-dC). The observed high methyl accepting capacity of poly(dI-dC) · poly(dI-dC) may be a useful methodological advance to determine de novo DNA methyltransferase activity in extracts of mammalian cells.

Mouse ascites DNA methylase: characterisation of size, proteolytic breakdown and nucleotide recognition

We have purified the DNA methylase from mouse ascites tumour cells to a specific activity of 11 500 units per mg protein using denatured Micrococcus luteus DNA as methyl acceptor. Methyl groups are transferred to cytosines almost exclusively in CpG dinucleotides. The purified enzyme contains two polypeptides of molecular mass 185 and 160 kDa, and an antiserum raised in a rabbit to the purified enzyme specifically reacts with these two proteins in crude extracts. The two proteins can be partially separated by affinity chromatography when activity is associated with the 185 kDa protein which can be proteolytically degraded to give polypeptides of 170 and later 100 and 50 kDa. Only the 185 kDa methylase is lost when cells are treated with azadeoxycytidine and this is the predominant form firmly bound in the nucleus of dividing cells. Antibody bound to the 185 kDa band in protein blots will itself bind native DNA methylase, which can be detected by its binding 14C-labelled, azacytosine-containing DNA.

Inhibition of Micrococcus luteus DNA gyrase by norfloxacin and 10 other quinolone carboxylic acids.

The ability of norfloxacin, amifloxacin, cinoxacin, ciprofloxacin, flumequine, nalidixic acid, ofloxacin (OFL), oxolinic acid, perfloxacin, pipemidic acid, and rosoxacin to inhibit the in vitro supercoiling activity of Micrococcus luteus DNA gyrase was compared with the ability of each drug to inhibit the growth of the M. luteus strain from which the gyrase was purified. The potency of the quinolones as DNA gyrase inhibitors did not always correlate with antimicrobial potency. For example, OFL was a less potent inhibitor of gyrase than rosoxacin, yet the MIC of OFL was 16-fold lower than that of rosoxacin. Similarly, the MICs of norfloxacin and ciprofloxacin (the most potent of the antibiotics tested in these assays) were several hundredfold lower than the MIC of nalidixic acid (the least potent of these antibiotics), but the inhibition of purified gyrase by these two quinolones was only 8- to 16-fold lower than that of nalidixic acid. These results suggest that factors in addition to inhibition of gyrase supercoiling activity are important in determining the potency of these drugs. Further studies indicated that the uptake of norfloxacin, OFL, and amifloxacin by M. luteus cells may not account for the large differences in MICs observed for these drugs (MICs of 0.8, 2.0, and 128 micrograms/ml, respectively).

Methylated DNA-binding protein from human placenta recognizes specific methylated sites on several prokaryotic DNAs.

Methylated DNA-binding protein (MDBP) from human placenta recognizes specific DNA sequences containing 5-methylcytosine (m5C) residues. Comparisons of binding of various prokaryotic DNAs to MDBP indicate that m5CpG is present in the recognition sites for this protein but is only part of the recognition sequence. Specific binding to MDBP was observed for bacteriophage XP12 DNA, which naturally contains approximately 1/3 of its residues as m5C, and for Micrococcus luteus DNA, M13mp8 replicative form (RF) DNA, and pBR322 when these three DNAs were methylated at CpG sites by human DNA methyltransferase. Five DNA regions binding to MDBP have been localized by DNase I footprinting or restriction mapping in methylated pBR322 and M13mp8 RF DNAs. A comparison of their sequences reveals a common 5'-m5CGRm5CG-3' element or closely related sequence in which one of the m5C residues may be replaced by a T. In addition to this motif, one upstream and one downstream m5CpG as well as other common residues over an approximately 20-bp long region may be recognized by MDBP.

Mechanism of rat liver DNA methyltransferase interaction with anti-benzo[a]pyrenediol epoxide modified DNA templates.

We investigated the methylation reaction catalyzed by 1500-fold purified rat liver DNA methyltransferase (DMase) on native Micrococcal luteus DNA (ML-DNA) and poly(dC-dG) templates containing covalently bound (+)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE), the strongly carcinogenic, principal metabolite of benzo[a]pyrene. Since eukaryotic DNA methyltransferases recognize the dinucleotide 5'd[CG] in DNA as a substrate for methylation, the model polynucleotide poly(dC-dG) was used to study in more detail the mode of interaction and effect on incorporation. With either of these BPDE-modified templates, a progressive inhibition of methylation was correlated with increasing amount of BPDE substitution. The effect of BPDE-dG adducts did not alter the apparent km with respect to the concentration of d[CG] in either unmodified or BPDE-modified poly(dC-dG) (km = 10 microM) but lowered the relative apparent Vmax. In assays in which perturbation by salt of preformed enzyme-DNA complex is measured, no change in the relative stability to either unsubstituted or the carcinogen-modified template was noted, thus, excluding any change in the ionic component of this interaction. However, in competition-type experiments, BPDE-DNA is an inhibitor of the methylation reaction on native DNA. When BPDE-DNA is allowed to interact with the enzyme before the addition of native competitor DNA, the methylation rate is not stimulated, suggesting very tight hydrophobic binding of the enzyme to BPDE-DNA and an inhibition in the dissociation of DMase from the template following a methylation event.(ABSTRACT TRUNCATED AT 250 WORDS)

Human placental DNA methyltransferase: DNA substrate and DNA binding specificity.

We have partially purified a DNA methyltransferase from human placenta using a novel substrate for a highly sensitive assay of methylation of hemimethylated DNA. This substrate was prepared by extensive nick translation of bacteriophage XP12 DNA, which normally has virtually all of its cytosine residues replaced by 5-methylcytosine (m5C). Micrococcus luteus DNA was just as good a substrate if it was first similarly nick translated with m5dCTP instead of dCTP in the polymerization mixture. At different stages in purification and under various conditions (including in the presence or absence of high mobility group proteins), the methylation of m5C-deficient DNA and that of hemimethylated DNA were compared. Although hemimethylated , m5C-rich DNAs were much better substrates than were m5C-deficient DNAs and normal XP12 DNA could not be methylated, all of these DNAs were bound equally well by the enzyme. In contrast, from the same placental extract, a DNA-binding protein of unknown function was isolated which binds to m5C-rich DNA in preference to the analogous m5C-poor DNA.

Chlorophyll Photosensitized Electron Transfer across Lipid Bilayer Membranes

Figure 1. Electrophoretic mobility of single DNA topoisomers containing different amount of UV photoproducts. Left: electrophoretic pattern on 1% agarose gel of plasmid pAT 153. The population of single topoisomers was obtained by exposure of naturally supercoiled DNA to Micrococcus luteus DNA topoisomerase I. Right: relative mobility of each visible band as a function of pyrimidine dimer content. RFI: fully supercoiled DNA; RFII: circular DNA containing at least one-single strand nick.

Inhibition of Micrococcus luteus DNA topoisomerase I by UV photoproducts.

The activity of Micrococcus luteus DNA topoisomerase I on UV-irradiated supercoiled DNA was studied under either processive or distributive reaction conditions. Changes in DNA structure caused by UV irradiation reduce the rate of DNA relaxation at very low concentration of photoproducts. Under processive conditions the inhibition of the topoisomerase I by photoproducts can be quantitated by measuring the amount of substrate left in the replicative form I band. The mode of action of DNA topoisomerase I was affected by the presence of photoproducts in the DNA substrate, although the ability of the enzyme to form a covalent complex with UV-irradiated supercoiled DNA was not changed. The inhibition of topoisomerase I by UV photoproducts has been compared to the effects of single-stranded DNA and UV-irradiated duplex linear DNA on the enzyme, and the results suggest that the inhibition by photoproducts is caused by changes in the conformation of the supercoil. Our findings indicate the possibility that DNA topoisomerase I plays a role in repair.

The effect of netropsin on the electrochemical oxidation of DNA at a graphite electrode

The interaction of netropsin with calf thymus, Bacillus cereus and Micrococcus luteus DNAs and with the RNA of phage f2 was studied by means of differential pulse voltammetry at a paraffin-wax-impregnated spectroscopic graphite electrode. It was found that the oxidation voltammetric peaks of double-helical calf thymus and Bacillus cereus DNAs were lowered when netropsin was added to the DNA solution. The peak corresponding to electro-oxidation of adenine residues was lowered more than that corresponding to electro-oxidation of guanine residues. Under the same experimental conditions the oxidation peaks of double-helical Micrococcus luteus DNA, double-helical RNA and thermally denatured samples of all DNAs used were almost uninfluenced by the addition of netropsin. The results observed were explained by ( i) decreased flexibility of the segments to which netropsin was bound, and ( ii) the specific binding of netropsin to the segments of double-helical DNA rich in adenine.thymine pairs.

459—The effect of netropsin on the electrochemical oxidation of DNA at a graphite electrode

The interaction of netropsin with calf thymus, Bacillus cereus and Micrococcus luteus DNAs and with the RNA of phage f2 was studied by means of differential pulse voltammetry at a paraffin-wax-impregnated spectroscopic graphite electrode. It was found that the oxidation voltammetric peaks of double-helical calf thymus and Bacillus cereus DNAs were lowered when netropsin was added to the DNA solution. The peak corresponding to electro-oxidation of adenine residues was lowered more than that corresponding to electro-oxidation of guanine residues. Under the same experimental conditions the oxidation peaks of double-helical Micrococcus luteus DNA, double-helical RNA and thermally denatured samples of all DNAs used were almost uninfluenced by the addition of netropsin. The results observed were explained by ( i) decreased flexibility of the segments to which netropsin was bound, and ( ii) the specific binding of netropsin to the segments of double-helical DNA rich in adenine thymine pairs.

Sequence specificity of DNA adenine methylase in the protozoan Tetrahymena thermophila.

The sequence specificity of the Tetrahymena DNA-adenine methylase was determined by nearest-neighbor analyses of in vivo and in vitro methylated DNA. In vivo all four common bases were found to the 5' side of N6-methyladenine, but only thymidine was 3'. Homologous DNA already methylated in vivo and heterologous Micrococcus luteus DNA were methylated in vitro by a partially purified DNA-adenine methylase activity isolated from Tetrahymena macronuclei. The in vitro-methylated sequence differed from the in vivo sequence in that both thymidine and cytosine were 3' nearest neighbors of N6-methyladenine.

Organization and nucleotide sequence of nuclear 5S rRNA genes in yellow lupin (Lupinus luteus).

Genomic blots of yellow lupin (Lupinus luteus) DNA digested with restriction nucleases and probed with 32P-labelled Lupinus 5S RNA reveal that 5S DNA is organized as tandemly repeated sequences of one size class, 342 bp. The DNA is extensively methylated. Two cloned BamHI ribosomal repeats were sequenced, revealing sequence divergence within both the coding and spacer regions.

Differential activity of DNA methyltransferase in the life cycle of Chlamydomonas reinhardi.

Two molecular weight forms of DNA (cytosine-5-)-methyltransferase [S-adenosyl-L-methionine:DNA (cytosine-5-)- methyltransferase, EC 2.1.1.37], both active in assays in vitro, were isolated from the green alga Chlamydomonas reinhardi at various stages of the life cycle. The enzyme with Mr 60,000 was found in vegetative cells and gametes of both male (mt-) and female (mt+) mating types. The enzyme with Mr 200,000 was specific to gametic cells and zygotes, which are the only stages at which methylation of chloroplast DNA occurs in vivo. Chloroplast DNA from gametes was shown to be methylated on both strands at most if not all methylation sites and the Mr 200,000 enzyme was shown to methylate both unmethylated and hemimethylated sites, the latter at an elevated rate. Micrococcus luteus DNA showed the same nearest-neighbor frequencies of methylation after methylation by each molecular weight component. The data suggest strongly that the Mr 200,000 enzyme is the active multimeric form of the Mr 60,000 enzyme and that it acts as both initiation and maintenance methylase. It is proposed that methylation of chloroplast DNA in female gametes and zygotes is regulated by assembly of the multimeric Mr 200,000 active enzyme, which in turm determines the maternal inheritance of chloroplast DNA.

Competitive binding studies of compounds that interact with DNA utilizing fluorescence polarization

The increase in the fluorescence polarization of acridine orange upon binding to DNA molecules is used as the basis of a competitive method to study the interaction of a variety of fluorescent and non-fluorescent compounds with DNA. Test compounds that interact with DNA inhibit both the binding of acridine orange to DNA and the accompanying increase in fluorescence polarization. Actinomycin D exhibits a dose-dependent inhibition of acridine orange-DNA binding with Micrococcus luteus DNA, calf thymus DNA, and poly(dG-dC); no detectable inhibition of acridine orange intercalation into poly(dA-dT) is observed. In contrast, proflavine shows similar acridine orange inhibition for poly(dA-dT), calf thymus DNA and M. luteus DNA.

E. coli and M. luteus DNA topoisomerase I can catalyze catenation or decatenation of double-stranded DNA rings

Escherichia coli and Micrococcus luteus DNA topoisomerase I are found to promote catenation of double-stranded DNA rings. At low DNA concentration dimeric catenanes are the major catenated products; at high DNA concentration or when spermidine is present, catenanes containing more than two rings are formed. There is no requirement of extensive sequence homology between the component rings forming a catenane; dimeric catenanes between Pseudomonas phage PM2 DNA and E. coli plasmid pBR322 are readily formed. The formation of a dimeric catenane by these type I topoisomerases, however, requires the presence of at least one preexisting single-chain scission in one of the two component rings. This is in contrast to the cases with the type II DNA topoisomerases which can form catenanes made of covalently closed rings only. The catenanes formed by the type I enzymes can be unlinked by the same enzymes, or by DNA gyrase, a type II enzyme, upon dilution of the isolated catenanes. The catenation and decatenation of duplex DNA rings adds a fourth type of reaction promoted by these type I DNA topoisomerases to the three reported previously: relaxation of superhelical DNA, interconversion between single-stranded DNA rings with and without knots and the intertwining of single-stranded DNA rings of complementary sequences into a covalently closed duplex ring with a high linking number. All four topoisomerization reactions involve the crossing of one DNA strand through a transient break of another DNA strand. The new reaction reported here suggests that such a crossover event might not require pairing of complementary nucleotide sequences.

Methylation of viral DNA in Vivo and in Vitro

Methylation of herpesvirus saimiri DNA and adenovirus type 5 DNA was examined by labelling with [6- 3H]uridine and [2- 3H]adenosine and separating the bases by thin layer chromatography. The number of methyl groups was less than one per genome in both DNAs, indicating that these viral DNAs are not substrates for methylation in vivo. In a long-term in vitro incubation with purified rat liver DNA methylase adenovirus type 5 DNA and herpes simplex virus type I DNA were methylated to about the same degree as M. luteus DNA (1 methyl group per 42 bases). In a short-term incubation the viral DNAs accepted considerably less methyl groups than M. luteus DNA. This suggests that viral DNAs have lower affinity for DNA methylases than bacterial DNAs. Rat liver DNA was a competitive inhibitor for the in vitro methylation of adenovirus type 5 DNA by rat liver DNA methylase. The higher affinity of the enzyme for cellular DNA may explain why viral DNA is not methylated in the cell.

Construction and mapping of recombinant plasmids used for the preparation of DNA fragments containing the Escherichia coli lactose operator and promoter.

Three DNA restriction fragments of established sequence containing the Escherichia coli lac genetic controlling regions were cloned. In each case a recombinant plasmid was constructed which was suitable for the subsequent large scale purification of the lac fragment. A 789-base pair HindII fragment, containing the lac operator, promoter, and cyclic AMP receptor protein binding site, was ligated into the single HindII site of the amplifiable plasmid minicolicin E1 DNA (pVH51). A 203-base pair Hae III fragment containing the same genetic sites was ligated into the single Eco RI site of pVH51 which had been "filled in" by the Micrococcus luteus DNA polymerase. Thus, the lac fragment was inserted between two Eco RI sites. Plasmids containing multiple copies of this Eco RI fragment were then constructed. A 95-base pair Alu I fragment containing the lac promoter and operator was cloned similarly. Also, the 203-base pair fragment was cloned into the Eco RI site of pVH51 using a 300-base pair linker fragment (isolated by RPC-5 column chromatography) which permitted retention of its Hae III ends. Mapping studies on pVH51 DNA with a number of DNA restriction endonucleases, including Alu I, Taq I, and Hpa II, are described.

Conformation and circular dichroism of DNA

AbstractCD spectra of calf thymus, C. perfringens, E. coli, and M. luteus DNA have been measured in the vacuum‐uv region to about 168 nm for the A‐, B‐, and C‐forms. The positive band at about 187 nm and the negative band at about 170 nm found for each type and form of DNA are sensitive to the source of the DNA and the base–base interactions of the double‐stranded helix. The A‐form spectra confirm that these bands are indeed sensitive to secondary structure. In the near‐uv, the CD of B‐form DNA is well analyzed as a linear combination of 27% A‐form and 78% C‐form. However, an analysis of the extended spectrum demonstrates that the near‐uv analysis is not correct. The extended analysis shows that the base–base interactions are similar for B‐ and C‐forms in solution, which implies that these two forms have nearly the same number of base pairs per turn. Various types of CD difference spectra are also discussed.

Comparison of the G4 and φX174 phage genomes by electron microscopy

The order of G4 Haemophilus influenzae Rd (H indII) DNA fragments was mapped by electron microscopy. G4 H indII DNA fragments were annealed to φX174 viral singles-tranded DNA circles and found to partially hybridize. Eight to eighteen percent of the φX174 DNA circles had a G4 DNA fragment attached, depending upon the fragment and the time of annealing. The G4 H indII restriction enzyme cleavage map was aligned with the φX174 restriction enzyme cleavage map by analyzing under the electron microscope partial heteroduplex molecules consisting of φX174 single-stranded viral DNA circles annealed with φX174 Arthrobacter luteus (AluI) DNA fragments as markers and G4 HindII DNA fragments. These results indicate that the gene order of the bacteriophages G4 and φX174 is the same. Despite the high frequency of φX174/G4 heteroduplex DNA molecules observed under the electron microscope, heterologous marker rescue experiments with G4 DNA fragments and φX174 mutant viral DNA strand circles were not successful, except in the case of a φX174 amber mutation in gene E ( am3).

Core nucleosomes by digestion of reconstructed histone-DNA complexes

Reconstructed complexes of the inner histones (H2A, H2B, H3, H4) and a variety of DNAs were digested with micrococcal nuclease to yield very homogeneous populations of core nucleosomes (nu 1). Nucleosomes containing Micrococcus luteus DNA (72% G+C); chicken DNA (43% G+C), Clostridium perfringens DNA (29% G+C); or poly(A-dT.poly(dA-dT) have been examined by circular dichroism, thermaldetenaturation, electron microscopy, and DNAse I digestion. Circular dichroism spectra of all particles show a typically suppressed ellipticity at 260--280 nm and a prominent alpha-helix signal at 222 nm. All particles show biphasic melting except nu 1 (dA-dT), which show three prominent melting transitions at ionic strength less than or equal to 1 mM. DNAse I digestion of nu 1 (dA-dT) produces a ladder of DNA fragments fiffering in lengthy by one base residue. nu 1 (dA-dT) contain 146 base pairs of DNA and exhibit an average DNA helix pitch of 10.4-10.5 bases per turn. There appear to be two regions of different DNA pitch wihtin nu 1 (dA-dT). It is suggested that the two regions of DNA pitch might correspond to the two regions of the melting profiles.