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T D Tullius

Publications and source records attributed to T D Tullius.

48 records · Page 3Linked to original sources
PubMed1988

Structural details of an adenine tract that does not cause DNA to bend.

Runs of adenines (adenine tracts) have been implicated as the main determinant of sequence-directed DNA bending. The most widely used experimental test for bending relies on the observation that bent DNA migrates more slowly than straight DNA on a polyacrylamide electrophoresis gel. It was shown recently that the polymer (GTTTTAAAAC)n runs with normal mobility on a gel, whereas (GAAAATTTTC)n runs more slowly and thus appears to be strongly bent. The observation that these similar sequences, which differ only in the order of the adenine and thymine tracts, adopt such different shapes offers a stringent test of theories to explain DNA bending. Although the wedge model for DNA bending has recently been elaborated to explain the gel mobilities of these molecules, we wished to determine experimentally the structural basis for the difference in bending. We report here measurements of the frequency of cleavage by the hydroxyl radical at each nucleotide of cloned versions of the two polymers (see Fig. 1). We show that the TTTTAAAA sequence does not display the cleavage pattern that is associated with bent DNA, whereas the AAAATTTT sequence does. The observed sequence dependence of the cleavage pattern of an adenine tract is at odds with current models for DNA bending, which assume that adenine tracts always adopt the same conformation.

Adenine↗
PubMed1988

A Holliday recombination intermediate is twofold symmetric.

Four-arm Holliday structures are ephemeral intermediates in genetic recombination. We have used an oligodeoxynucleotide system to form immobile DNA junctions, which are stable analogs of Holliday structures. We have probed the equilibrium structure of a junction by means of hydroxyl radicals generated by the reaction of iron(II)EDTA with hydrogen peroxide. The hydroxyl radical cleavage pattern shows twofold symmetry throughout the molecule. Strong protection from hydroxyl radical attack is evident on two strands near the branch site, and weaker protection may be seen four or five residues 3' to the branch site on the other two strands. No other position appears significantly distinct from double-helical DNA controls. From these data, we conclude that the Holliday junction is a twofold symmetric complex whose four arms form two stacking domains.

Free Radicals↗
PubMed1988

Mapping functional regions of transcription factor TFIIIA.

Functional deletion mutants of the trans-acting factor TFIIIA, truncated at both ends of the molecule, have been expressed by in vitro transcription of a cDNA clone and subsequent cell-free translation of the synthetic mRNAs. A region of TFIIIA 19 amino acids or less, near the carboxyl terminus, is critical for maximal transcription and lies outside the DNA-binding domain. The elongated protein can be aligned over the internal control region (ICR) of the Xenopus 5S RNA gene with its carboxyl terminus oriented toward the 5' end of the gene and its amino terminus oriented toward the 3' end of the gene. The nine "zinc fingers" and the linkers that separate them comprise 80% of the protein mass and correspond to the DNA-binding domain of TFIIIA. The zinc fingers near the amino terminus of the protein contribute more to the overall binding energy of the protein to the ICR than do the zinc fingers near the carboxyl end. The most striking feature of TFIIIA is its modular structure. This is demonstrated by the fact that each zinc finger binds to just one of three short nucleotide sequences within the ICR.

Animals↗
PubMed1987

The unusual conformation adopted by the adenine tracts in kinetoplast DNA.

To determine the structural features responsible for the curvature of kinetoplast DNA, we studied 13 adenine tracts in Crithidia fasciculata kinetoplast DNA. The structures of the A tracts were analyzed by cutting the DNA with hydroxyl radical. Reactivity of hydroxyl radical toward the DNA backbone progressively decreased in the 5'----3' direction of each A tract. The cutting pattern of the T-rich strand was offset by 1 or 2 bp from the pattern on the A-rich strand. An A tract in a restriction fragment from plasmid pBR322 had the same cutting pattern as the kinetoplast A tracts. We interpret these experiments to show that in A tracts the width of the minor groove decreases smoothly from the 5'----3' end of the A tract.

Adenine↗
PubMed1986

Hydroxyl radical "footprinting": high-resolution information about DNA-protein contacts and application to lambda repressor and Cro protein.

A method has been developed for making "footprints" of proteins bound to DNA. The hydroxyl radical, generated by reduction of hydrogen peroxide by iron(II), is the reagent used to cut the DNA. Hydroxyl radical breaks the backbone of DNA with almost no sequence dependence, so all backbone positions may be monitored for contact with protein. In addition to defining the DNA sequence in contact with the protein, hydroxyl radical footprints embody structural information about the DNA-protein complex. For example, hydroxyl radical footprints of the bacteriophage lambda repressor and Cro protein show directly that these proteins are bound to only one side of the DNA helix. Additional contacts of lambda repressor and Cro protein with DNA, not observed by other chemical footprinting methods, are revealed by hydroxyl radical footprinting.

Binding Sites↗
PubMed1985

Iron(II) EDTA used to measure the helical twist along any DNA molecule.

A new method has been devised to measure the number of base pairs per helical turn along any DNA molecule in solution. A DNA restriction fragment is adsorbed onto crystalline calcium phosphate, fragmented by reaction with iron(II) EDTA, and subjected to electrophoresis on a denaturing polyacrylamide gel. A modulated cutting pattern results, which gives directly the helical periodicity of the DNA molecule. A 150-base pair sequence directly upstream of the thymidine kinase gene of the type 1 herpes simplex virus was found to have an overall helical twist of 10.5 base pairs per turn, which is characteristic of the B conformation of DNA. In addition, purines 3' to pyrimidines showed lower than expected reactivity toward the iron cutting reagent, which is evidence for sequence-dependent variability in DNA conformation.

Base Sequence↗
PubMed1985

A selenomethionine-containing azurin from an auxotroph of Pseudomonas aeruginosa.

The production and spectroscopic properties of an L-selenomethionine-containing homolog of Pseudomonas aeruginosa azurin are described. The amino acid substitution was carried out by developing an L-methionine-dependent bacterial strain from a fully functional ATCC culture. Uptake studies monitored using L-[75Se]methionine indicated that L-selenomethionine was incorporated into the protein synthetic pathway of Pseudomonas bacteria in a manner analogous to L-methionine. Several batches of bacteria were grown, and one sample of isolated and purified selenoazurin (azurin in which methionine was substituted by selenomethionine) was found (by neutron activation analysis) to contain 5.2 +/- 0.8 seleniums/copper. Correspondingly, a residual 0.35 methionines, relative to 6.0 in the native protein, were found by amino acid analysis in this azurin sample. The redox potential and extinction coefficient of this selenoazurin were found to be 333 +/- 1 mV (pH 7.0, I = 0.22) and 5855 +/- 160 M-1 cm-1 at 626 +/- 1 nm, respectively. Visible electronic, CD, and EPR spectra are reported and Gaussian curve fitting to the former spectrum allowed assignment of the selenomethionine Se----Cu(II) transition to a band found at 18034 cm-1, based upon an observed 450 cm-1 shift to the red from the analogous band position in the native protein. The data are consistent with a relatively more covalent copper site stabilizing the reduced, Cu(I), form in the selenoprotein. A role for the methionine as a modulator of the blue copper site redox potential by metal----ligand back bonding from Cu(I) is discussed in terms of a ligand sphere which limits the valence change at copper to much less than 1 during a redox cycle.

Azurin↗
PubMed1982

Ethidium bromide changes the nuclease-sensitive DNA binding sites of the antitumor drug cis-diamminedichloroplatinum(II).

Exonuclease III has been shown previously to reveal the binding sites of the antitumor drug cis-diamminedichloroplatinum(II) on DNA. Pretreatment of the same DNA with the intercalator ethidium bromide causes new platinum binding sites to be detected by the exonuclease III method. In particular, a 5'd(G6-D-G2)3' sequence in a 165-base-pair restriction fragment of plasmid pBR322 becomes a preferred site for exonuclease III-detectable cis-diamminedichloroplatinum(II)binding. This switching of nuclease-sensitive platinum binding to new sites by the influence of another drug, ethidium bromide, offers an explantation at the molecular level for the phenomenon of synergism in combination chemotherapy.

Base Sequence↗
PubMed1978

Characterization of the blue copper site in oxidized azurin by extended x-ray absorption fine structure: Determination of a short Cu-S distance.

The primary coordination environment of the "blue" copper ion in oxidized azurin has been elucidated by x-ray absorption spectroscopy. The most striking feature is the unambiguous presence of a very short copper-sulfur distance at 2.10 +/- 0.02 A. Nitrogen ligands, presumed to be from imidazoles, are found at 1.97 A. There is some evidence that the copper coordination sphere may be completed by a second sulfur, the distance of which is determined with much less certainty.

Journal Article↗
PubMedDate not supplied

Use of the hydroxyl radical and gel electrophoresis to study DNA structure.

The hydroxyl radical has been used as a chemical probe to study in solution the structure of DNA and DNA-protein complexes. The hydroxyl radical abstracts a deoxyribose hydrogen atom, cleaving one strand of the DNA. The cutting pattern, visualized by separating the cleavage products using gel electrophoresis, shows the reactivity of each backbone position toward the radical. This method has been applied to studies of DNA bending and helical twist. Phased runs of adenines (adenine tracts) cause sequence-directed DNA bending. The hydroxyl radical cleavage of a bent DNA fragment containing short adenine tracts phased with the helix screw gives rise to an unusual cutting pattern. The hydroxyl radical cleavage rate decreases in the 5' to 3' direction along each adenine tract, with a minimum at the 3' end of each adenine tract. The cleavage of the matching thymine tract is similar, but the minimum in the pattern is offset in the 3' direction. This pattern on the autoradiograph of the gel is interpreted to indicate that bending is accompanied by a narrow minor groove in the DNA molecule. Furthermore, hydroxyl radical cleavage results in different cutting patterns for two similar sequences, (CGA4T4)5 and (CGT4A4)5, which have been shown to be bent and relatively straight, respectively. The hydroxyl radical method has also been used to determine the helical repeat of the metallothionein IIA gene to be about 10.5 base pairs per turn. Methods of optimizing the hydroxyl radical reaction for DNA-protein footprinting are discussed. Because individual gel bands give information about cutting frequency at particular positions in the backbone, gel resolution and clear autoradiographs are important to this work.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoradiography↗