Signatures of Protein-DNA Recognition in Free DNA Binding Sites

One obstacle to achieving complete understanding of the principles underlying sequence-dependent recognition of DNA is the paucity of structural data for DNA recognition sequences in their free (unbound) state. Here, we carried out crystallization screening of 50 DNA duplexes containing cognate protein binding sites and obtained new crystal structures of free DNA binding sites for three distinct modes of DNA recognition: anti-parallel β strands (MetR), helix-turn-helix motif + hinge helices (PurR), and zinc fingers (Zif268). Structural changes between free and protein-bound DNA are manifested differently in each case. The new DNA structures reveal that distinctive sequence-dependent DNA geometry dominates recognition by MetR, protein-induced bending of DNA dictates recognition by PurR, and deformability of DNA along the A-B continuum is important in recognition by Zif268. Together, our findings show that crystal structures of free DNA binding sites provide new information about the nature of protein-DNA interactions and thus lend insights towards a structural code for DNA recognition.     

Affinity purification of plasmid DNA by temperature-triggered precipitation

This report describes a new plasmid DNA purification method, which takes advantage of the DNA-binding affinity and specificity of the bacterial metalloregulatory protein MerR, and of the temperature responsiveness of elastin-like proteins (ELPs). Upon increasing the temperature, ELP undergoes a reversible phase transition from water-soluble forms into aggregates, and this property was exploited for the precipitation of plasmid DNA containing the MerR recognition sequence by a simple temperature trigger. In one purification step, plasmid DNA was purified from E. coli cell lysates to a better purity than that prepared by a standard alkaline purification method, with no contaminating chromosomal DNA and cellular proteins. This protein-based approach, in combination with the reversible phase transition feature of ELP, makes the outlined method a promising candidate for large-scale purification of plasmid DNA for sensitive applications such as nonviral gene therapy or DNA vaccines.     

Affinity purification of plasmid DNA by temperature-triggered precipitation

This protocol presents a new method to purify plasmid DNA using temperature-triggered precipitation. The principle is based on the specific DNA-binding affinity of a bacterial metalloregulatory (MerR) protein to its cognate DNA sequence and the temperature responsiveness of elastin-like protein (ELP). A bifunctional ELP-MerR fusion protein is created to enable the precipitation of plasmid DNA, designed to contain the MerR recognition sequence, by a simple temperature trigger. The protocol covers all stages of the process from the design of ELP-MerR fusion proteins and MerR-binding plasmids, to the isolation of plasmid DNA from Escherichia coli cultures after boiling lysis, the subsequent temperature-triggered precipitation of plasmid DNA-fusion protein complexes and final elution of plasmid DNA by mild heating. This protocol is well suited to laboratory research-scale applications, producing plasmid DNA of better purity and similar yield as one of the most commonly used laboratory methods, standard alkaline lysis (known as the midiprep procedure). The protocol takes approximately 30 min to obtain pure plasmid DNA from cell cultures using the temperature-triggered precipitation method.     

Entropic Profiles of DNA Sequences Through Chaos-game-derived Images

A new method to determine entropic profiles in DNA sequences is presented. It is based on the chaos-game representation (CGR) of gene structure, a technique which produces a fractal-like picture of DNA sequences. First, the CGR image was divided into squares 4^-m in size (m being the desired resolution), and the point density counted. Second, appropriate intervals were adjusted, and then a histogram of densities was prepared. Third, Shannon's formula was applied to the probability-distribution histogram, thus obtaining a new entropic estimate for DNA sequences, the histogram entropy , a measurement that goes with the level of constraints on the DNA sequence. Lastly, the entropic profile for the sequence was drawn, by considering the entropies at each resolution level, thus providing a way to summarize the complexity of large genomic regions or even entire genomes at different resolution levels. The application of the method to DNA sequences reveals that entropic profiles obtained in this way, as opposed to previously published ones, clearly discriminate between random and natural DNA sequences. Entropic profiles also show a different degree of variability within and between genomes. The results of these analyses are discussed in relation both to the genome compartmentalization in vertebrates and to the differential action of compositional and/or functional constraints on DNA sequences.     

Protein-DNA interactions determine the shapes of DNA toroids condensed in virus capsids

DNA toroids that form inside the bacteriophage capsid present different shapes according to whether they are formed by the addition of spermine or polyethylene glycol to the bathing solution. Spermine-DNA toroids present a convex, faceted section with no or minor distortions of the DNA interstrand spacing with respect to those observed in the bulk, whereas polyethylene glycol-induced toroids are flattened to the capsid inner surface and show a crescent-like, nonconvex shape. By modeling the energetics of the DNA toroid using a free-energy functional composed of energy contributions related to the elasticity of the wound DNA, exposed surface DNA energy, and adhesion between the DNA and the capsid, we established that the crescent shape of the toroidal DNA section comes from attractive interactions between DNA and the capsid. Such attractive interactions seem to be specific to the PEG condensation process and are not observed in the case of spermine-induced DNA condensation.     

Protein-DNA interaction within one cloned chloroplast DNA replication origin of Chlamydomonas

Summary A partially purified algal protein mixture which supports in vitro DNA replication consists of soluble proteins and proteins extracted from thylakoid membrane. The membrane extract is essential for the specific initiation of replication at a displacement loop (D-loop) site previously mapped by electron microscopy. D-loop site and its flanking sequences have been cloned and sequenced. In this study, fragment-retention assays using various subclones of the sequenced region indicate that some proteins in the membrane extract bind strongly and specifically with a 494 bp restriction fragment which partially overlaps the D-loop site. Protein gel analyses of the protein-DNA complex identify three DNA-binding polypeptides with apparent molecular weights of 18, 24 and 26 kDa, respectively. Treatment with chloramphenicol, an inhibitor of chloroplast protein synthesis, for 1 h has no obvious effect on the contents of the 24 or 26 kDa polypeptides but significantly reduces the content of the 18 kDa polypeptide in the membrane extract.     

Affinity adsorption of plasmid DNA

The development of a protein-mediated dual functional affinity adsorption of plasmid DNA is described in this work. The affinity ligand for the plasmid DNA comprises a fusion protein with glutathione-S-transferase (GST) as the fusion partner with a zinc finger protein. The protein ligand is first bound to the adsorbent by affinity interaction between the GST moeity and gluthathione that is covalently immobilized to the base matrix. The plasmid binding is then enabled via the zinc finger protein and a specific nucleotide sequence inserted into the DNA. At lower loadings, the binding of the DNA onto the Fractogel, Sepharose, and Streamline matrices was 0.0078 +/- 0.0013, 0.0095 +/- 0.0016, and 0.0080 +/- 0.0006 mg, respectively, to 50 microL of adsorbent. At a higher DNA challenge, the corresponding amounts were 0.0179 +/- 0.0043, 0.0219 +/- 0.0035, and 0.0190 +/- 0.0041 mg, respectively. The relatively constant amounts bound to the three adsorbents indicated that the large DNA molecule was unable to utilize the available zinc finger sites that were located in the internal pores and binding was largely a surface adsorption phenomenon. Utilization of the zinc finger binding sites was shown to be highest for the Fractogel adsorbent. The adsorbed material was eluted with reduced glutathione, and the eluted efficiency for the DNA was between 23% and 27%. The protein elution profile appeared to match the adsorption profiles with significantly higher recoveries of bound GST-zinc finger protein.     

Enhancement of DNA vaccine potency by electroporation in vivo

The potential of electric current-mediated delivery technology to enhance DNA delivery and DNA vaccine potency was evaluated. Higher levels of reporter gene expression were observed in muscle cells of mice inoculated with luciferase or beta-galactosidase DNA followed by the application of electrical current, compared with DNA injected with no current. Similarly, substantially higher levels of immune responses (up to 20-fold) were demonstrated in mice vaccinated with HIV gag DNA and electric current. These enhanced responses were observed after one or two inoculations, and were maintained for at least 12 weeks. Therefore, the present studies demonstrate the utility of electroporation for enhancement of DNA vaccine potency in animals.     

Protein-DNA interactions in vitro with 5'-flanking DNA fragments from the chicken vitellogenin gene

The expression of the vitellogenin gene in the liver of oviparous animals is under strict control of estrogen. We have studied the interaction of proteins extracted from nuclei of different estrogen responsive tissues with two fragments (-728 to -470 and -625 to -470) of the upstream region of the chicken vitellogenin gene, using the gel-retardation technique. We found a complex pattern of retarded bands using nuclear extracts from laying hen liver, rooster liver and MCF-7 cells. The patterns observed display differences in the position and intensities of some of the bands, depending on the source of the extract used. The possible significance of these findings will be discussed.     

Enhancement of the Affinity of Nucleoli and Chromosomes for Zinc by Treatment with Chromic Acid

Cultured mammalian cells and wet touch preparations from human organs were fixed for 10 min in 5:85:10 acetic-alcohol-formalin; placed in 5% aqueous CrO₃ for 30 min at 22-25 C; washed in running water 1 min; placed in 2 mM zinc acetate in 0.14 M veronal-acetate buffer, pH 6.5, at 37 C, 30 mm; rinsed 5 sec in 50% acetone; and stained 10 min in a solution dithizone. This results in selective staining of the nucleoli of interphase cells, and of the chromosomes of mitotic cells.     

Synthesis of new bivalent peptides for applications in the Affinity Enhancement System

The feasibility of two-step radioimmunotherapy (RIT) of cancer by the Affinity Enhancement System (AES) has been demonstrated in experimental and clinical studies. This technique, associating a bispecific antibody and a bivalent peptide radiolabeled with iodine-131, has been developed to reduce toxicity and to improve therapeutic efficacy compared to one-step targeting methods. The use of AES with different beta-emitters such as rhenium-188, samarium-153, or lutetium-177 or alpha-emitters such as actinium-225 or bismuth-213 is now considered. Thus three new peptides, designed to allow for the coupling of a variety of bifunctional chelating agents BCA, were synthesized by associating two glycyl-succinyl-histamine (GSH) arms, which are recognized by the 679 monoclonal antibody (mAb-679), with different binding agents, such as p-nitrophenylalanine or N,N-bis(carboxymethyl)-4-N'-(9-fluorenylmethyloxycarbonyl)aminobenzylamine. Immunoreactivity and serum stability evaluation were performed for each synthesized peptide. One of the three peptides (LM218) proved to be more stable than the others, and three different BCAs were coupled to LM218 (CITC-DTPA, CITC-TTHA, and CITC-CHXA'DTPA). One of these products, LM218-BzTTHA was radiolabeled with indium-111 without loss of immunoreactivity toward the mAb-679. These new peptides will allow pretargeted RIT with a large variety of radionuclides, to adapt the choice of the radionuclide (LET, half-life, penetrating emission) to the nature and size of targeted tumors.     

Enhancement of Thermostability of Firefly Luciferase from Luciola lateralis by a Single Amino Acid Substitution

We constructed firefly luciferase mutants from Luciola lateralis in which Ala at position 217 was replaced by each of three hydrophobic amino acid residues (lie, Leu, and Val). These mutants were superior to the wild-type in thermostability. Especially, the purified Ala217Leu mutant still maintained over 70% of the initial activity after 60 min at 50°C. This mutant is the most thermostable firefly luciferase obtained.     

Enhancement of bleomycin-mediated DNA damage by epidermal microsomal enzymes

The role of epidermal microsomal enzymes in catalyzing bleomycin-mediated chain breakage in calf-thymus DNA and in DNA isolated from neonatal rat epidermis was studied. Aerobic incubation of bleomycin with epidermal microsomes, epidermal or calf-thymus DNA and NADPH caused substantial chain breakage of the DNA which was dependent upon concentrations of drug, microsomal protein and NADPH. The reactive oxygen scavenger superoxide dismutase, the metal chelator EDTA, and cytochrome c each inhibited the enzyme-mediated chain breakage reaction. Scavengers of hydrogen peroxide and hydroxyl radicals, including catalase and benzoate and inhibitors of microsomal cytochrome P-450-dependent monooxygenases such as 1-benzylimidazole, metyrapone and alpha-naphthoflavone, had no inhibitory effects on bleomycin-mediated DNA chain breakage. In contrast, ascorbic acid significantly enhanced DNA damage by bleomycin. These studies indicate that mammalian epidermis possesses membrane-bound enzyme activity capable of enhancing bleomycin-mediated chain breakage of DNA and that oxidation/reduction of adventitious iron and generation of reactive oxygen participate in the reaction. These responses in the epidermis could directly relate to the mechanism of action of intralesional injections of bleomycin which are used quite effectively in the management of recalcitrant human warts. Either epidermal or wart virus DNA or both could be targets for this pharmacologic effect of the drug which is augmented by epidermal microsomal enzymes.     

Purification of Avian Myeloblastosis Virus DNA Polymerase by Affinity Chromatography on Polycytidylate-Agarose

Polycytidylic acid [poly(rC)] covalently linked to cyanogen bromide-activated agarose is an effective affinity matrix for the RNA-dependent DNA polymerase from avian myeloblastosis virus. Poly(rC)-agarose is capable of binding large quantities of avian myeloblastosis DNA polymerase, which is then eluted by using a linear KCl gradient of increasing concentration. The DNA polymerase isolated from crude, detergent-disrupted virions by a single pass through columns of poly(rC)-agarose appears nearly homogeneous (approximately 90% pure) as determined by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. Complete recovery of input enzymatic activity was obtained. Results suggest that polyribonucleotide columns may provide a high-yield, rapid method for the purification of oncornaviral DNA polymerase.     

Enhancement and Cultural Characteristics of Leucomycin Production by Streptomyces kitasatoensis in the Presence of Magnesium Phosphate

Stimulation of leucomycin production with Streptomyces kitasatoensis by magnesium phosphate (MgP), and a concomitant decrease of ammonium ion concentration in the culture supernatant have been reported from this laboratory. Cultural characteristics of the MgP-supplemented culture were further examined in relation to the stimulation of leucomycin production. When the leucomycin titer increased 4- to 5-fold in the presence of MgP, mycelial growth level and assimilation rate of a major carbon source (glycerol) increased about 2-fold. MgP-grown mycelia were not appreciably different in shape or in carbon and nitrogen contents from the control mycelia. Magnesium and phosphate ions were released from MgP into the medium, while the addition of magnesium or phosphate salts to the control medium was rather inhibitory for leucomycin production. The pH values of MgP-supplemented cultures were lower than those of the control culture. When modifications of the basal media and culture conditions were done so as to permit the pH-change almost identical with that of the control media, the leucomycin titer was still higher in the presence of MgP than in its absence. When uric acid, an insoluble compound, was used as the sole nitrogen source, the leucomycin production increased to a high level even in the absence of MgP, which was comparable to that seen when MgP was added to the ammonium lactate-containing medium. The ammonium ion concentration in the culture supernatant of the uric acid-containing medium was maintained at a considerably low level. While in the ammonium lactate-containing medium, leucomycin production was low and ammonium ion concentration remained at a high level. Thus, it was demonstrated that the stimulation of leucomycin production by MgP is related to the lowering of ammonium ion level, but not to the other factors examined such as growth level, carbon or phosphate regulation and pH-effect.