A possible structure for calf satellite DNA I.

Calf satellite DNA I (p = 1.715) has been hydrolysed by a number or restriction endonucleases. It consists of a repeating unit of 1460 nucleotide pairs within which the sites of Eco R II Mbo I, Sac I, Alu I, Ava II and Hha I were localised in comparison with those of Eco R I and Hind II. The distribution of the Hpa II, Sac I, Hha I, Hinf I and Mbo II sites within calf satellite DNA I, as well as that of several restriction endonuclease sites within calf satellite DNA III (p = 1.705) allowed me to define subsatellite fractions. Furthermore, some of the sites of the CpG containing restriction enzymes Hpa II and Hha I are lacking. The possible implications of these results are discussed.     

Structure and stability of Z* DNA

The structure and stability of the left handed Z* DNA aggregate was examined by spectroscopic methods and by electron microscopy. Poly(dGdC), upon heating in the presence of Mn++, forms a large aggregate which may be sedimented at 12,000 X g, with a circular dichroism spectrum characteristic of left handed DNA. Aggregation gives rise to turbidity changes at visible wavelengths, providing a convenient means of monitoring the transition in solution. The wavelength dependence of turbidity is consistent with the scattering behavior of a long thin rod. Electron microscopy shows that Z* DNA is a large fibrous structure of indeterminant length, with a uniform diameter of approximately 20 nm. The results obtained in solution and under the requisite conditions for electron microscopy are mutually consistent. Poly(dGdC) preparations with average lengths of 60, 240, 500, and 2000 base pairs all form Z* DNA. Poly(dGm5dC) forms Z* DNA in the presence of Mn++ without heating, but poly(dAdC)-poly(dGdT) and calf thymus DNA cannot be induced to the Z* form under any conditions tried. Kinetic studies, monitored by turbidity changes, provide evidence that the formation of Z* DNA proceeds by a nucleated condensation mechanism. Dissolution of the Z* aggregate results from the chelation of Mn++ or by the addition of the intercalator ethidium bromide. The allosteric conversion of Z* DNA to an intercalated, right handed form by ethidium is demonstrated by kinetic studies, equilibrium binding studies and circular dichroism spectroscopy. Electron microscopy provides a striking visualization of the dissolution of the Z* aggregate by ethidium.     

Direct visualization of the sites of DNA methylation in human,and mosquito chromosomes

Human and mosquito fixed chromosomes were digested with restriction endonucleases that are inhibited by the presence of 5-methylcytosine in their restriction sites (Hha I, Hin PI, Hpa II), and with endonucleases for which cleavage is less dependent on the state of methylation (Taq I, Msp I). Methylation-dependent enzymes extracted low DNA amounts from human chromosomes, while methylation-independent enzymes extracted moderate to high amounts of DNA. After DNA demethylation with 5-azacytidine the isoschizomers Hpa II (methylation-dependent) and Msp I (methylation-independent) extracted 12-fold and 1.4-fold amounts of DNA from human chromosomes, respectively. These findings indicate that human DNA has a high concentration of Hpa II and Msp I restriction sites (CCGG), and that the internal C of this sequence is methylated in most cases, while the external cytosine is methylated less often. All the enzymes tested released moderate amounts of DNA from mosquito chromosomes whether or not the DNA was demethylated with 5-azacytidine. Hpa II induced banding in the centromere chromosome regions. After demethylation with 5-azacytidine this banding disappeared. Mosquito DNA has therefore, moderate to high frequencies of nonmethylated CpG duplets. The only exception is the centromeric DNA, in which the high levels of C methylation present produce cleavage by Hpa II and the appearance of banding. Centromere regions of human chromosomes 1 have a moderately low concentration of Hpa II-Msp I restriction sites.     

Inhibition of the B to Z transition in poly(dGdC).poly(dGdC) by covalent attachment of ethidium: kinetic studies

The photoaffinity analogue ethidium monoazide was used to prepare samples of poly(dGdC).poly(dGdC) containing covalently attached ethidium. The effects of both noncovalently and covalently bound ethidium on the kinetics of the NaCl-induced B to Z transition in poly(dGdC).poly(dGdC) was examined using absorbance and fluorescence spectroscopy to monitor the reaction. Covalently and noncovalently attached ethidium were equal in the extent to which they reduce the rate of the B to Z transition. By using fluorescence to selectively monitor the fate of noncovalently bound ethidium over the course of the transition, we found that ethidium completely dissociates as the reaction proceeds, but at a rate that lags behind the conversion of the polymer to the Z form. These experiments provide evidence for the redistribution of noncovalently bound ethidium over the course of the B to Z transition, leading to the development of biphasic reaction kinetics. The observed kinetics suggest that the primary effect of both covalently and noncovalently bound ethidium is on the nucleation step of the B to Z transition. The reduction in the rate of the B to Z transition by noncovalently or covalently bound ethidium may be quantitatively explained as resulting from the reduced probability of finding a drug-free length of helix long enough for nucleation to occur. As necessary ancillary experiments, the defined length deoxyoligonucleotides (dGdC)4, (dGdC)5, and (dGdC)6 were synthesized and used in kinetic experiments designed to determine the nucleation length of the B to Z transition, which was found to be 6 bp. The activation energy of the B to Z transition was demonstrated to be independent of the amount of covalently bound ethidium and was found to be 21.2 +/- 1.1 kcal mol-1. Covalent attachment of ethidium was observed to increase the rate of the reverse Z to B transition, presumably by locking regions of the polymer into a right-handed conformation and thereby providing nucleation sites from which the Z to B conversion may propagate.     

Structure and Stability of Z* DNA

Abstract

The structure and stability of the left handed Z* DNA aggregate was examined by spectroscopic methods and by electron microscopy. Poly(dGdC), upon heating in the presence of Mn⁺⁺, forms a large aggregate which may be sedimented at 12,000 X g, with a circular dichroism spectrum characteristic of left handed DNA Aggregation gives rise to turbidity changes at visible wavelengths, providing a convenient means of monitoring the transition in solution. The wavelength dependence of turbidity is consistent with the scattering behavior of a long thin rod. Electron microscopy shows that Z* DNA is a large fibrous structure of indeterminant length, with a uniform diameter of approximately 20 nm. The results obtained in solution and under the requisite conditions for electron microscopy are mutually consistent Poly(dGdC) preparations with average lengths of 60,240,500, and 2000 base pairs all form Z* DNA Poly(dGm⁵dC) forms Z* DNA in the presence of Mn⁺⁺ without heating, but poly(dAdC)-poly(dGdT) and calf thymus DNA cannot be induced to the Z* form under any conditions tried. Kinetic studies, monitored by turbidity changes, provide evidence that the formation of Z* DNA proceeds by a nucleated condensation mechanism. Dissolution of the Z* aggregate results from the chelation of Mn⁺⁺ or by the addition of the intercalator ethidium bromide. The allosteric conversion of Z* DNA to an intercalated, right handed form by ethidium is demonstrated by kinetic studies, equilibrium binding studies and circular dichroism spectroscopy. Electron microscopy provides a striking visualization of the dissolution of the Z* aggregate by ethidium.     

Inhibition of the B to Z transition in poly(dGdC).poly(dGdC) by covalent attachment of ethidium: equilibrium studies

The effects of covalent modification of poly(dGdC).poly(dGdC) and poly(dGm5dC).poly(dGm5dC) by ethidium monoazide (a photoreactive analogue of ethidium) on the salt-induced B to Z transition are examined. Earlier studies have shown ethidium monoazide to bind DNA (in the absence of light) in a manner identical to that of the parent ethidium bromide. Photolysis of the ethidium monoazide-DNA complex with visible light results in the covalent attachment of the photoreactive analogue to the DNA. This ability to form a covalent adduct was utilized to probe the effects of an intercalating irreversibly bound adduct on the salt-induced B to Z transition of the poly(dGdC).poly(dGdC) and poly(dGm5dC).poly(dGm5dC) polynucleotides. In the absence of drug, the salt-induced transition from the B to Z structure occurs in a highly cooperative manner. In contrast, this cooperativity is diminished as the concentration of covalently attached drug is increased. The degree of inhibition of the B to Z transition is quantitated as a function of the concentration of covalently attached drug. At a concentration of one drug bound per four base pairs for poly(dGdC).poly(dGdC) and seven base pairs for poly(dGm5dC).poly(dGm5dC), total inhibition of this transition is achieved. Lower concentrations of bound drug were effective in the partial inhibition of this transition. The effects of the covalently bound intercalator on the energetics of the B to Z transition were determined and demonstrated that the adduct is effective in locking the alternating copolymer in a right-handed conformation under high salt conditions.     

Identification of Thiobacillus ferrooxidans strains based on restriction fragment length polymorphism analysis of 16S rDNA.

The 16S rDNA sequences from ten strains of Thiobacillus ferrooxidans were amplified by PCR. The products were compared by performing restriction fragment length polymorphism (RFLP) analysis with restriction endonucleases Alu I, Hap II, Hha I, and Hae III. The RFLP patterns revealed that T. ferrooxidans could be distinguished from other iron- or sulphur-oxidizing bacteria such as T. thiooxidans NB1-3, T. caldus GO-1, Leptospirillum ferrooxidans and the marine iron-oxidizing bacterium strain KU2-11. The RFLP patterns obtained with Alu I, Hap II, and Hae III were the same for nine strains of T. ferrooxidans except for strain ATCC 13661. The RFLP patterns for strains NASF-1 and ATCC 13661 with Hha I were distinct from those for other T. ferrooxidans strains. The 16S rDNA sequence of T. ferrooxidans NASF-1 possessed an additional restriction site for Hha I. These results show that iron-oxidizing bacteria isolated from natural environments were rapidly identified as T. ferrooxidans by the method combining RFLP analysis with physiological analysis.     

The heterogeneity of rat-liver mitochondrial DNA

Two types of mitochondrial DNA (mtDNA) can be distinquished in an inbred strain of rats of the Wistar type. The population of DNA molecules of the liver of one single rat is homogeneous. This was shown for a number of 100 animals and confirms the data of other investigators. The two types of mitochondrial DNA, designated A and B, differ in their number of cleavage sites for the restriction endonucleases Eco RI (2sites), Hind II (1 site) and Hha I (1 site). No differences were found for the restriction enzymes Bam HI, Hap II, Hind III and Hpa I. The degree of sequence divergence of the two types of DNA is calculated to be roughly 5% on the basis of these observations. From 20 rats part of the liver was taken and the mtDNA was characterized. Heterologous and homologous crosses between type A and type B rats were made. Analysis of the offspring revealed strictly maternal inheritance of the A and B mtDNA traits. For purposes of base-sequence analysis and RNA.DNA hybridization the strain could easily be "purified" genetically.     

Methylation of somatic vs germ cell DNAs analyzed by restriction endonuclease digestions.

Bacterial restriction endonucleases containing the dinucleotide CpG in their cleavage sequences were used to compare the methylation patterns of primarily repeated DNA sequences in (1) bovine somatic cell native DNAs vs bovine sperm cell native DNA and (2) native vs renatured bovine liver and sperm cell DNAs. The restriction patterns of sperm native DNA differ markedly from those of somatic cell native DNAs when using Hpa II, Hha I, and Ava I but not when using the enzymes Eco RI and Msp I. Digestion patterns of germ cell renatured DNA differed significantly from those of germ cell native DNA when using Hpa II but not when using Msp I or Eco RI. The results may not be due to artifacts of renaturation of the DNAs. The results are consistent with the concept that germ cell DNA may be strand asymmetrically hemimethylated. The data also suggest that methylation of the 5'-cytosine in the sequence CCGG renders this site insensitive to cleavage by Msp I.     

Formation of psi (+) and psi (-) DNA

DNA molecules can be organized into ordered aggregates of opposite handedness by complexation with polylysine and other polypeptides; we have investigated the conditions under which ψ(+) and ψ(?) structures are produced with double-helical synthetic polynucleotides. Both poly(dGdC)·poly(dGdC) and poly(dAdT)·poly(dAdT) readily form ψ(?) structures with polylysine, although the method of preparation can alter the CD spectra. The GC copolymer, which is more susceptible to conversion into A or Z conformers, forms ψ(+) structures with lysine–alanine copolypeptides more readily than the AT copolymer. Nucleotide base modifications that favor the Z structure, such as bromination and methylation, also favor ψ(+) formation, and the Co(NH₃)₆Cl₃ reagent that readily induces the Z structure also leads to ψ(+). Thus, the production of the ψ(+) structure seems to be frequently correlated with susceptibility to A or Z formation, although there are some cases in which the B conformer also leads to ψ(+). Polyethylene glycol generally produces a ψ(?) structure; the differentiation between ψ(+) and ψ(?) structures seems to require electrically charged polymers.     

A family of moderately repetitive sequences in mouse DNA.

When mouse DNA is digested to completion with restriction endonuclease Eco R1, a distinct band of 1.3 kb segments comprising about 0.5-3% of the genome is observed upon agarose gel electrophoresis. This DNA is not tandemly repeated in the genome and is not derived from mouse satellite DNA. Restriction endonuclease analysis suggested that the 1.3 kb segments are heterogeneous. Specific sequences were selected from the 1.3 kb segments and amplified by cloning in plasmid pBR322. Southern transfer experiments indicated that three separately cloned mouse DNA inserts hybridized predominantly to the Eco R1 1.3 kb band and to the conspicuous subsegments generated by secondary restriction endonuclease cleavage of the sucrose gradient purified 1.3 kb segments. Segments were also excised by Hha I (Hha I segments) from the chimeric plasmids containing mouse DNA inserts and subjected to restriction endonuclease and cross-hybridization analysis. It was found that the three Hha I segments were different, although two of them exhibited partial sequence homology. Cot analysis indicated that each of the Hha I segments are repeated about 10(4) times in the mouse genome. These findings indicate that a family of related but non-identical, moderately repetitive DNA sequences, rather than a single homogeneous repeat, is present in the 1.3 kb Eco R1 band.     

Bacillus subtilis bacteriophages SP82, SPO1, and phie: a comparison of DNAs and of peptides synthesized during infection.

The genomes of Bacillus subtilis phages phie, SPO1, and SP82 were compared by DNA-DNA hybridization, analysis of DNA fragments produced by digestion with restriction endonucleases, comparison of the arrays of peptides synthesized during infection, and phage neutralization. DNA-DNA hybridization experiments indicated that about 78% of the SP82 DNA was homologous with SPO1 DNA, whereas 40% of the phie DNA was homologous to either SPO1 or SP82 DNA. Agarose gel electrophoresis was used to compare the molecular weights of DNA fragments produced by cleavage of SP82, SPO1, and phie DNAs with the restriction endonucleases Hae III, Sal I, Hpa II, and Hha I. Digestion of the DNAs with Hae III and Sal I produced only a few fragments, whereas digestion with Hpa II and Hha I yielded 29 to 40 fragments, depending on the DNA and the enzyme. Comparing the Hpa II fragments, 51% of the SP82 fragments had mobilities which matched those of SPO1 fragments, 32% of the SP82 fragments matched the phie fragments, and 34% of the SPO1 fragments matched the phie fragments. Comparing the Hha I digestion products, 62% of the SP82 fragments had mobilities matching the SPO1 fragments, 24% of the SP82 fragments matched the phie fragments, and 22% of the SPO1 fragments matched the phie fragments. Analysis of peptides by electrophoresis on one-dimensional sodium dodecyl sulfate-polyacrylamide slab gels showed that approximately 70 phage-specific peptides were synthesized in the first 24 min of each infection. With mobility and the intervals of synthesis as criteria, 66% of the different SP82 peptides matched the SPO1 peptides, 34% of the SP82 peptides matched the phie peptides, and 37% of the SPO1 peptides matched the phie peptides. Phage neutralization assays using antiserum to SP82 yielded K values of 510 for SP82, 240 for SPO1, and 120 for phie.     

Methylated and unmethylated DNA compartments in the sea urchin genome.

Sea urchin (Echinus esculentus) DNA has been separated into high and low molecular weight fractions by digestion with the mCpG-sensitive restriction endonucleases Hpa II, Hha I and Ava I. The separation was due to differences in methylation at the recognition sequences for these enzymes because an mCpG-insensitive isoschizomer of Hpa II (Msp I) digested Hpa II-resistant DNA to low molecular weight, showing that many Hpa II sites were in fact present in this fraction; and because 3H-methyl methionine administered to embryos was incorporated into the high molecular weight Hpa II-, Hha I- and Ava I-resistant fraction, but not significantly into the low molecular weight fraction. The fraction resistant to Hpa II, Hha I and Ava I amounted to about 40% of the total DNA. It consisted of long sequence tracts between 15 and well over 50 kg in length, in which many sites for each of these enzymes were methylated consecutively. The remaining 60% of the genome, (m-), was not significantly methylated. Methylated and unmethylated fractions were considered to be subfractions of the genome because enriched unique sequences from one fraction cross-reassociated poorly with the other fraction and specific sequences were found in either (m+) or (m-) but not in both (see below). Similar (m+) and (m-) compartments were found in embryos, germ cells and adult somatic tissues. Furthermor, we found no evidence for changes in the sequence composition of (m+) or (m-) between sperm, embryo or intestine DNAs, although low levels of exchange would not have been detected. Using cloned Echinus histone DNA, heterologous 5S DNA and ribosomal DNA probes, we have found that each of these gene families belongs to the unmethylated DNA compartment in all the tissues examined. In particular, there was no detectable methylation of histone DNA either in early embryos, which are thought to be actively transcribing the bulk of histone genes, or in sperm and gastrulae, in which most histone genes are not being transcribed. In contrast to these gene families, sequences complementary to an internally repetitious Echinus DNA clone were found primarily in the methylated DNA compartment.     

Restriction endonucleases from Bifidobacteria

The sitespecific restriction endonucleases were found in four strains among the twelve strains of anaerobic bacteria of generum Bifidobacterium. Two of the restriction endonucleases studied, BadI from B. adolescentis LVA1 and BbfI from B. bifidum LVA3, are isoshizomers of XhoI and recognize the nucleotide sequence CTCGAG. The restriction endonucleases Bbf7411I from B. bifidum 7411 and Bla7920I from B. lactentis 7920 recognize and hydrolize the nucleotide sequence TCCGGA having the specifity analogous to the one of restriction endonuclease CauB3I. Like CauB3I, these restriction endonucleases are unable to hydrolyize DNA if the adenine residues in the recognition site are methylated.     

Electron microscopy of SV40 DNA cross-linked by anti-Z DNA IgG.

Electron microscopy has revealed the specific binding of bivalent anti-Z DNA immunoglobulin G (IgG) to different sites on supercoiled Form I SV40 DNA. The anti-Z IgG links together left-handed regions located within individual or on multiple SV40 DNA molecules at the superhelix density obtained upon extraction. Velocity sedimentation, electrophoresis, and electron microscopy all show that two or more Z DNA sites in the SV40 genome can be intermolecularly cross-linked with bivalent IgG into high mol. wt. complexes. The formation and stability of the intermolecular antibody-DNA complexes are dependent on DNA superhelix density, as judged by three criteria: (1) relaxed circular (Form II) DNA does not react; (2) release of torsional stress by intercalation of 0.25 microM ethidium bromide removes the antibody; and (3) linearization with specific restriction endonucleases reverses antibody binding and DNA cross-linking. Non-immune IgG does not bind to negatively supercoiled SV40 Form I DNA, nor are complexes observed in the presence of competitive synthetic polynucleotides constitutively in the left-handed Z conformation; B DNA has no effect. Using various restriction endonucleases, three major sites of anti-Z IgG binding have been mapped by electron microscopy to the 300-bp region containing nucleotide sequences controlling SV40 gene expression. A limited number of minor sites may also exist (at the extracted superhelix density).     

Characterization of intermediate conformational states in the B↔Z transitions of poly(dG?dC) · poly(dG?dC)

Time-dependent uv absorption and CD spectrum changes in salt-induced conformational B → Z and Z → B transitions of poly(dG? dC) · poly(dG? dC) were measured. This polynucleotide does not convert directly from a right-handed double-helical B form to a left-handed double-helical Z form, but goes through an intermediate, B* form, with the B → B* transition proceeding nearly instantaneously, and then transforms gradually to the Z form. Uv absorption spectra of these B and B* forms are nearly identical, but their CD spectra are quite different. The CD spectrum of the B* form is identical with that obtained for DNA in high salt solutions and is similar to a spectrum which for some time was thought to be a C form. These B and B* forms have the same number of base pairs per turn [Sprecher, C.A., Baase, W.A. & Johnson, Jr., W.C. (1979) Biopolymers 18 , 1009–1019]. Kinetic measurements showed that uv absorption and CD intensities at fixed wavelengths do not change in a simple exponential curve. However, both the uv absorption spectrum change in the B → Z transition and the CD spectrum change in the B* → Z transition, respectively, have isosbestic points. In the B → Z transition, no hyperchromicity can be observed. These results suggest that this B* form unfolding or premelting process is a rate-determining step in the B* → Z transition and makes it easy for the unfolded or premelted polynucleotide to almost immediately fold into the Z form. The double helix does not dissociate into single strands and transforms from the B* form to the Z form point-by-point along the chain in a soliton-like manner of with a small amount of open states in which the bases are unpaired. Also, in the Z → B transition, the polynucleotide does not convert directly from the Z to the B form, but goes through a B*-like form. In this transition, the uv-absorption spectra also have an isosbestic point. The reaction velocity in the Z → B transition is much faster than that in the B → Z transition. Possibly, the positive CD band between 265 and 310 nm in the B form comes from a n-π* transition due to an interaction of the bases with sugarphosphate groups.     

Type I restriction endonucleases are true catalytic enzymes

Type I restriction endonucleases are intriguing, multifunctional complexes that restrict DNA randomly, at sites distant from the target sequence. Restriction at distant sites is facilitated by ATP hydrolysis-dependent, translocation of double-stranded DNA towards the stationary enzyme bound at the recognition sequence. Following restriction, the enzymes are thought to remain associated with the DNA at the target site, hydrolyzing copious amounts of ATP. As a result, for the past 35 years type I restriction endonucleases could only be loosely classified as enzymes since they functioned stoichiometrically relative to DNA. To further understand enzyme mechanism, a detailed analysis of DNA cleavage by the EcoR124I holoenzyme was done. We demonstrate for the first time that type I restriction endonucleases are not stoichiometric but are instead catalytic with respect to DNA. Further, the mechanism involves formation of a dimer of holoenzymes, with each monomer bound to a target sequence and, following cleavage, each dissociates in an intact form to bind and restrict subsequent DNA molecules. Therefore, type I restriction endonucleases, like their type II counterparts, are true enzymes. The conclusion that type I restriction enzymes are catalytic relative to DNA has important implications for the in vivo function of these previously enigmatic enzymes.