An Iterative Approach to Placing Counterions Around DNA

Abstract

An iterative approach, in which the effect of placing counter ions around DNA influences the electrostatic potential that the other subsequently approaching ions feel, has been used to place sodium ions around polynucleotides. The main focus of this report is to study the sequence and structure dependence on the distribution of ions around DNA, particularly that of tightly bound ions. The interesting results of the calculations are that there is significant sequence dependence on the electrostatic potentials in the B form of DNA, whereas relatively less difference in A form. In the case of Z form, the cations bridge the inter-stand phosphates along the minor groove.     

Dequalinium vesicles form stable complexes with plasmid DNA which are protected from DNase attack.

Upon sonication, the antimicrobial and antineoplastic compound dequalinium forms vesicles (DQAsomes, Weissig et al., 1998). Dequalinium (1,1'-(1,10-decamethylene-bis-[aminoquinaldinium])-chloride) was shown to be a fluorophore with an emission maximum at 366 nm. Addition of DNA results in a characteristic quenching of its intrinsic fluorescence. After density gradient centrifugation a band of dequalinium (DQA) tightly associated with DNA is located between the DNA and DQA bands. DQA/DNA-complexes containing plasmid DNA at a molar ratio of DQA/DNA 6:1 are completely protected against DNase activity. Addition of negatively-charged lipids release intact DNA in the same manner as from cationic lipid/DNA complexes. As regards biological effects, DQAsomes show a differential cytotoxicity for normal and sarcoma cell lines. In vitro incubation with fluorescein-labeled oligodeoxynucleotides (5'-fluorescein-[GATC]5) showed an increased uptake of the tagged oligodeoxynucleotide if complexed with dequalinium. We hypothesize that the DQA/DNA complexes are well-suited for 'DQAsomal gene transfer' in vitro and in vivo. Noteworthy, they display an intrinsic antitumor activity manifested by differential cytotoxicity for normal and sarcoma cells.     

A.T and C.C+ base pairs can form simultaneously in a novel multistranded DNA complex

Previous experiments have established that in certain synthetic oligomeric DNA sequences, including mixtures of d(AACC)5 with d(CCTT)5, adenine-thymine (A.T) base pairs form to the exclusion of neighboring protonated cytosine-cytosine (C.C+) base pairs [Edwards, E., Ratliff, R., & Gray, D. (1988) Biochemistry 27, 5166-5174]. In the present work, circular dichroism and other measurements were used to study DNA oligomers that represented two additional classes with respect to the formation of A.T and/or C.C+ base pairs. (1) One class included two sets of repeating pentameric DNA sequences, d(CCAAT)3-6 and d(AATCC)4,5. For both of these sets of oligomers, an increase in the magnitude of the long-wavelength positive CD band centered at about 280 nm occurred as the pH was lowered from 7 to 5 at 0.1 and 0.5 M Na+, indicating that C.C+ base pairs formed. Even though it may have been possible for these oligomers to form duplexes with two antiparallel A.T base pairs per pentamer, no A.T base pairing was detected by monitoring the CD changes at 250 nm. Thus, spectral data showed that as few as 40% C.C+ base pairs were stable in two sets of oligomers in which A.T base pairs did not form adjacent to, or in place of, C.C+ base pairs. (2) Another class of oligomer was represented by d(C4A4T4C4), which was studied by CD, HPLC, and centrifugation experiments. We confirmed previous work that this sequence was able to form both types of base pairs as the pH and temperature were lowered [Gray, D., Cui, T., & Ratliff, R. (1984) Nucleic Acids Res. 12, 7565-7580].(ABSTRACT TRUNCATED AT 250 WORDS)     

BII nucleotides in the B and C forms of natural-sequence polymeric DNA: A new model for the C form of DNA

A combination of solid-state (31)P and (13)C NMR, X-ray diffraction, and model building is used to show that the B and C forms of fibrous macromolecular DNA consist of two distinct nucleotide conformations, which correspond closely to the BI and BII nucleotide conformations known from oligonucleotide crystals. The proportion of the BII conformation is higher in the C form than in the B form. We show structural models for a 10(1) double helix involving BI nucleotides and a 9(1) double helix involving BII nucleotides. The 10(1) BI model is similar to a previous model of B-form DNA, while the 9(1) BII model is novel. The BII model has a very deep and narrow minor groove, a shallow and wide major groove, and highly inclined bases. This work shows that the B to C transition in fibers corresponds to BI to BII conformational changes of the individual nucleotides.     

Apoptotic cell nuclei favour aggregation and fluorescence quenching of DNA dyes

 Apoptotic cell nuclei are known to stain hyperchromatically with absorption dyes and dimly with many DNA fluorochromes. We hypothesised that both optical phenomena have the same cause - the ability of apoptotic chromatin to aggregate cationic dyes. This hypothesis was tested using prednisolone-primed rat thymus, which is known to contain apoptotic cells. The apoptotic cells were classified as early and late, based on their morphology, in thin and semithin sections and in thymus imprints on slides. Direct reaction for DNA strand breaks (TUNEL) indicated the presence of breaks in both categories of cells, with more intense labelling in late apoptosis. The chromatin ultrastructure of early apoptotic cells initially retained the supranucleosomal order of packaging which characterises control cells, whereas the dense chromatin of late apoptotic cells possessed the degraded structure. Absorption spectra of the toluidine blue-stained early apoptotic cell chromatin revealed a metachromatic shift, indicating a change of DNA conformation and polymerisation of the dye. When the staining was performed by acridine orange (preceded by a short acid treatment), a paradoxical several-fold increase of fluorescence intensity at a several-fold dilution of the dye was found. The simultaneous reduction of the ratio of red to green components of fluorescence confirmed that the concentration-dependent fluorescence quenching was due to aggregation of the dye. The results suggest that the enhanced affinity of the chromatin of early apoptotic cells for cationic dyes is associated with conformational relaxation rather than degradation of DNA. In late apoptotic cells, the very dense packaging of degraded DNA promotes further aggregation of dyes. The results suggest alternative methods for detection and discrimination of early and late apoptotic cells. Accepted: 12 February 1997     

Rat liver DNA ligases. Catalytic properties of a novel form of DNA ligase.

A novel form of rat liver DNA ligase (molecular mass 100 kDa) can be differentiated from DNA ligase I by several biochemical parameters. It is a more heat-labile enzyme and unable to join blunt-ended DNA, even in the presence of poly(ethylene glycol) concentrations which stimulate such joining by DNA ligase I and T4 DNA ligase. It also lacks the AMP-dependent nicking/closing reaction, which is a property of all other DNA ligases tested so far, including DNA ligase I from rat liver. Both rat liver DNA ligases were inhibited by deoxyadenosinetriphosphate, however this inhibition was competitive with respect to ATP, for DNA ligase I (Ki 22 microM) and non-competitive for the 100-kDa DNA ligase (Ki 170 microM). These results support the idea that, when compared with other DNA ligases, the novel form of DNA ligase has a unique AMP-binding site, may have an absolute requirement for single-strand breaks and, furthermore, may have an altered reaction mechanism to that which is conserved from bacteriophage to mammalian DNA ligase I.     

Enantioselective binding of ofloxacin to B form DNA.

The binding affinity and binding mode of S- and R-ofloxacin, one of the quinolone antibiotics, to B form calf thymus DNA were studied in this work. The binding affinity of S-ofloxacin measured by both Stern-Volmer and Benesi-Hilderbrand methods was greater by a factor of 5 compared to R-enantiomer and the CD spectrum of the former is largely altered while that of the latter remained the same in the presence of DNA, indicating the enantiospecific binding of this drug to DNA. The binding geometry of both S- and R-ofloxacin calculated from the reduced linear dichroism was similar to norfloxacin, which is partially intercalated from the minor groove.     

Antibiotics which can alter the rotational orientation of nucleosome core DNA.

Four well-characterised DNA-binding ligands have been tested for effects on reconstituted nucleosome core particles containing the 160 bp tyrT DNA fragment. Two, netropsin and berenil, were found to change the rotational orientation of the DNA on the surface of the protein as judged by marked alterations in the pattern of fragments produced by exposure to DNAase I. Qualitatively their effects were very similar to those previously reported for the related antibiotic distamycin, suggesting that the phenomenon of induced rotation may be a characteristic property of ligands which bind in the narrow groove of the DNA helix. Two intercalators did not produce the effect but, at high concentrations, caused gross disruption of the nucleoprotein structure with apparent release of DNA from the histone octamer. At moderate concentrations little or no effect was detectable with nogalamycin, suggestive of failure to bind as a result of constraints on local opening of the DNA helix. With moderate concentrations of actinomycin, protection of GpC sequences was clearly visible together with some evidence of increase in helix pitch, but no sign of altered phasing of DNA within the nucleosome core particles.     

Analysis of the ATPase subassembly which initiates processive DNA synthesis by DNA polymerase III holoenzyme.

The gamma complex (gamma delta delta' chi psi) subassembly of DNA polymerase III holoenzyme transfers the beta subunit onto primed DNA in a reaction which requires ATP hydrolysis. Once on DNA, beta is a "sliding clamp" which tethers the polymerase to DNA for highly processive synthesis. We have examined beta and the gamma complex to identify which subunit(s) hydrolyzes ATP. We find the gamma complex is a DNA dependent ATPase. The beta subunit, which lacks ATPase activity, enhances the gamma complex ATPase when primed DNA is used as an effector. Hence, the gamma complex recognizes DNA and couples ATP hydrolysis to clamp beta onto primed DNA. Study of gamma complex subunits showed no single subunit contained significant ATPase activity. However, the heterodimers, gamma delta and gamma delta', were both DNA-dependent ATPases. Only the gamma delta ATPase was stimulated by beta and was functional in transferring the beta from solution to primed DNA. Similarity in ATPase activity of DNA polymerase III holoenzyme accessory proteins to accessory proteins of phage T4 DNA polymerase and mammalian DNA polymerase delta suggests the basic strategy of chromosome duplication has been conserved throughout evolution.     

Polypurine DNAs and RNAs form secondary structures which may be tetra-stranded.

Polypurine DNAs and RNAs containing at least 33% guanine form a stable secondary structure at neutral pH and moderate ionic strengths. The tm's of the polymers increase with increasing guanine content. To eliminate possible structures three novel polymers, d(Gn2A)n, d(Gm6A)n and d(IA)n, as well as the random copolymer rr(G,A)n were were studied. Both d(Gn2A)n and d(IA)n can form a secondary structure whereas d(Gm6A)n and r(G,A)n cannot. Model building suggested two possible structures, one a duplex and the other a tetra-stranded polymer. The latter is considered to be the more likely, since previous X-ray diffraction studies have shown that rGn and rIn are tetra-stranded. Circular dichroism spectra are also consistent with such an interpretation.     

DNA sequence of the 5'' terminus containing the replication origin of parvovirus replicative form DNA.

The nucleotide sequence of the 5' terminus of the parvovirus H-1 was determined. There are two orientations of the 242-base-pair terminal palindrome in native replicative form DNA, one inverted with respect to the other. Adjacent to the terminal palindrome is an AT-rich region that is noncoding and contains a 55-base-pair tandem repeat. The addition mutant of H-1, DI-1, was also sequenced in this region and shown to have three copies of the tandem repeat sequence. Similarly, the related parvovirus H-3 contains only one copy of this repeat sequence. This region contains the replication origin for parvovirus replicative form DNA replication. Some of the implications of these results are discussed.     

The circular intracellular form of Epstein-Barr virus DNA is amplified by the virus-associated DNA polymerase.

Selective DNA extraction and hybridization procedures were used to estimate the relative number of covalently closed circular viral genomes in cultures of Epstein-Barr virus (EBV)-transformed cells. In virus-producing P3HR-1 cultures that were exposed for 11 days to phosphonoacetic acid or to acyclovir, the content of covalently closed circular EBV DNA was reduced ca. 70% relative to a control culture without drug. The EBV plasmid content of Raji, a virus nonproducer cell line, was not reduced by exposure to these compounds. When P3HR-1 cultures were exposed to 12-O-tetradecanoylphorbol-13-acetate, the number of circular genomes per cell increased. These findings indicate that two enzyme activities synthesize circular EBV DNA and that the virus-associated DNA polymerase synthesizes most of the circular EBV DNA in a virus producer culture. It is suggested that the circular genomes synthesized by the viral enzyme are intermediates in the syntheses of linear virus DNA.     

Site-specific mutagenesis method which completely excludes wild-type DNA from the transformants.

A highly efficient site-specific mutagenesis method has been devised to exclude wild-type DNA from incorporation into the transformed cells. Two complementary oligonucleotides, corresponding to a target sequence of a DNA molecule and containing an insertion mutation which created an endonuclease restriction site, were synthesized. By using the wild-type DNA molecule flanked by two restriction sites on each side of the target region as a template, the two oligonucleotide primers were extended, enriched, and isolated. The extended products, in turn, were used as templates in a polymerase chain reaction to obtain a mutagenized double-stranded DNA fragment which was conveniently cloned into plasmids by using the flanking restriction sites. Escherichia coli cells transformed by these plasmids were subject to large-scale analysis. One hundred percent of the transformants examined by colony hybridization, restriction enzyme analysis, and DNA sequencing were found to contain the mutant DNA sequence.     

Site-specific mutagenesis method which completely excludes wild-type DNA from the transformants.

A highly efficient site-specific mutagenesis method has been devised to exclude wild-type DNA from incorporation into the transformed cells. Two complementary oligonucleotides, corresponding to a target sequence of a DNA molecule and containing an insertion mutation which created an endonuclease restriction site, were synthesized. By using the wild-type DNA molecule flanked by two restriction sites on each side of the target region as a template, the two oligonucleotide primers were extended, enriched, and isolated. The extended products, in turn, were used as templates in a polymerase chain reaction to obtain a mutagenized double-stranded DNA fragment which was conveniently cloned into plasmids by using the flanking restriction sites. Escherichia coli cells transformed by these plasmids were subject to large-scale analysis. One hundred percent of the transformants examined by colony hybridization, restriction enzyme analysis, and DNA sequencing were found to contain the mutant DNA sequence.