Albert M. Bobst

Effect of the methylation of the 2′-hydroxyl groups in polyadenylic acid on its structure in weakly acidic and neutral solutions and on its capability to form ordered complexes with polyuridylic acid☆

Abstract The influence of the methylation of the 2′-hydroxyl groups in poly A on its structure in weakly acidic and neutral solutions and on its capability to form ordered complexes with poly U has been investigated by ultraviolet and circular dichroism spectroscopy. A major effect of the methylation of the backbone is to increase the thermal stability of the double-stranded, acid structure of poly A, while no significant effect on the single-stranded conformation was observed. The dissymmetric structures, as reflected in the circular dichroic spectra, were virtually identical for poly A and poly Am § for the tightly packed, double-stranded, acid structures (pH 4.6, 0.15 m -KCl) on one hand and for the single-stranded conformations (pH 7, 0.15 m -KCl, 25 °C) on the other. Circular dichroic spectra with characteristics intermediate between those of the tightly packed, double-stranded and the single-stranded structures of poly A and poly Am are obtained in the pH range from 5 to 7 at moderate to high ionic strength (for poly Am e.g. at pH 5.7, 0.15 m -NaCl, 4 to 44 °C). From the circular dichroic spectra and absorbance melting profiles it is tentatively concluded that poly A and poly Am form less tightly packed, less ordered double-stranded structures under these conditions. Methylation of the 2′-hydroxyl groups in poly A has virtually no effect on the thermal stability of its double-stranded complex with poly U, but considerably weakens the attachment of the third strand of poly U in poly(Am + 2U). It is concluded that the presence of an unsubstituted 2′-hydroxyl group in poly A is not essential for the stabilization of the double-stranded conformations of poly A in acidic solutions, for the partially-ordered single-stranded conformation of poly A at neutrality or for the double-stranded complex of poly A with poly U.

Dipsticking the major groove of DNA with enzymatically incorporated spin-labeled deoxyuridines by electron spin resonance spectroscopy

Site-specifically spin-labeled deoxyuridine triphosphates with tethers of different lengths were synthesized and then enzymatically incorporated with terminal transferase to form a spin-labeled poly(dT) copolymer. The spin-labeled copolymers were annealed with poly(dA) to form a duplex, which was analyzed by electron spin resonance spectroscopy in a solution of low ionic strength. The spin labels are attached in position 5 of the deoxyuridine and protrude into the major groove. Based on the correlation between tether length of the spin label and the electron spin resonance lineshape, we show that the depth of the major groove of a DNA in its B-form is about 8 A in solution, which is in good agreement with X-ray fiber studies. We also conclude, based on electron spin resonance lineshape simulation data, that the correlation time of the bases in a DNA duplex is of the order of nanoseconds.

Polarography of polynucleotides: II. Conformations of poly(adenylic acid) at acidic pH

Abstract Evidence is presented that poly(riboadenylic acid) (poly(A)) can exist in three different forms at acidic pH values. The “intermediate” form was detected as the only form in a narrow pH range immediately below the pH induced transition of neutral poly(A); at lower pH values, depending how the pH was reached, the “intermediate” form was accompanied by “tightly packed” and/or “frozen” forms; at still lower pH values only the “frozen” and “tightly packed” forms were found. The “tightly packed” form is characterized by total inaccessibility of formaldehyde-reactive and polarographically-reducible sites. In terms of accessibility of these sites and circular dichroic spectra, the “intermediate” form is truly intermediate between the “tightly packed” and neutral forms of poly(A). Virtually all these sites are accessible in the “frozen” form, which was found to be formed only from the neutral poly(A) upon fast acidification to a sufficiently low pH. A structure consisting of mismatched strands and randomly distributed short double stranded regions is suggested for the “frozen” form which can be converted into the “tightly packed” form, e.g. by thermal annealing. Upon acidification of the “intermediate” form only the “tightly packed” form was observed. All three forms are distinguishable by circular dichroic spectra. The “intermediate” and “tightly packed” forms show identical ultraviolet spectra, while the “frozen” form is slightly hyperchromic to the “tightly packed” form.

Fluorescent in situ detection of Encephalitozoon hellem spores with a 6-carboxyfluorescein-labeled ribosomal RNA-targeted oligonucleotide probe.

Abstract A fluorescent in situ hybridization assay has been developed for the detection of the human-pathogenic microsporidian, Encephalitozoon hellem in water samples using epifluorescence microscopy. The assay employs a 19-nucleotide species-specific 6-carboxyfluorescein-labeled oligonucleotide probe, HEL878F, designed to be complementary to the nucleic acid sequence 878–896, a highly variable segment of the 16S ribosomal RNA of E. hellem spores. The specificity of this probe for its ribosomal RNA target site was confirmed using RNA degradation, ribosomal RNA target site competition, and nucleotide base mismatch control probe assays. Furthermore, the specificity of the HEL878F oligonucleotide probe for E. hellem spores was established when it was evaluated on spores from all three species of the genus Encephalitozoon that had been seeded in reagent water and environmental water concentrates. The specificity of the HEL878F oligonucleotide probe was further corroborated when tested on algae, bacteria, and protozoa commonly found in environmental water. The study demonstrates the applicability of a fluorescent in situ hybridization assay using a species-specific fluorescent-labeled oligonucleotide probe for the detection of E. hellem spores in water samples.

Overall and internal dynamics of DNA as monitored by five-atom-tethered spin labels

Electron paramagnetic resonance (EPR) spectra of the two-atom-tethered six-membered ring thymidylate spin label (DUMTA) incorporated into duplexes of different sizes were found to display a helix length dependence and a local-order parameter S = 0.32 +/- 0.01 for B-DNA based on the dynamic cylinder model (Keyes, R. S., and A. M. Bobst. 1995. Detection of internal and overall dynamics of a two-atom-tethered spin-labeled DNA. Biochemistry. 34:9265-9276). This sensitivity to size, which reflects global tumbling, is now reported for the more flexible five-atom-tethered five-membered ring thymidylate spin label (DUAP) that can be readily incorporated enzymatically and sequence specifically into nucleic acids of different sizes. The DUAPs containing B-DNA systems were simulated with the same dynamic cylinder model, giving S = 0.20 +/- 0.01 for the more flexibly tethered spin label. This shows that S is dependent on tether length but not on global motion. An analysis with the same motional model of the B-Z transition in a (dG-dC)n polymer containing the five-atom-tethered six-membered ring cytidylate spin label (DCAT) (Strobel, O. K., R. S. Keyes, and A. M. Bobst. 1990b. Base dynamics of local Z-DNA conformations as detected by electron paramagnetic resonance with spin-labeled deoxycytidine analogues. Biochemistry. 29:8522-8528) revealed an increase in S from 0.15 +/- 0.01 to 0.26 +/- 0.01 in response to the B- to Z-DNA transition. This indicates that S is not only sensitive to tether length, but also to conformational changes in DNA. Both the DUAP- and the DCAT-labeled systems were also simulated with a base disk model. From the DUAP spectral series, the perpendicular component of the correlation time tau perpendicular describing the spin-labeled base diffusion was found to be sensitive to global tumbling, confirming earlier results obtained with DUMTA. The DCAT polymer results demonstrated that tau perpendicular monitors a conformational change from B- to Z-DNA, indicating that tau perpendicular is also sensitive to local base dynamics. These results confirm that the dynamics of five-atom-tethered nitroxides are coupled to the nucleic acid dynamics and, as with two-atom-tethered spin labels, can be characterized by S and tau perpendicular. The analyses of both spin-labeled systems provide good evidence for spin-labeled base motions within double-stranded DNA occurring on the nanosecond time scale, and establish that both labels can be used to monitor changes in global tumbling and local order parameter due to variations in DNA conformation and protein-DNA interactions.

Enzymatic sequence-specific spin labeling of a DNA fragment containing the recognition sequence of EcoRI endonuclease

Deoxyuridine analogs spin labeled in position 5 have been enzymatically incorporated sequence specifically into an oligodeoxyribonucleotide to form a spin‐labeled 26‐mer. The 26‐mer contains the EcoRI‐binding site and two labels which are located symmetrically close to the binding site. The labels are separated from one another far beyond the Heisenberg spin‐exchange distance. The local base motion as determined by ESR spectroscopy is of the order of 4 ns in the oligonucleotide duplex. This is the same value as reported earlier for local T motions in polynucleotide duplexes, thereby providing direct experimental evidence that the ESR line shape of spin levels covalently attached to nucleic acids depends primarily on the local dynamics of the nucleic acid building blocks.

Menadione-Induced Oxidative Stress in Bovine Heart Microvascular Endothelial Cellsa

Objective: Oxidative stress from increased production of reactive oxygen species or decreased efficiency of inhibitory and scavenger systems may contribute to vascular injury. In this study, we developed an in vitro model of vascular injury by menadione‐induced oxidative stress in bovine heart microvascular endothelial cells.

Variability in the nucleic acid binding site size and the amount of single-stranded DNA-binding protein in Escherichia coli

The Escherichia coli single‐stranded DNA binding protein (SSB), essential for DNA replication, recombination and repair, can undergo a thermally induced irreversible conformational change which does not eliminate its biological activity, but changes the number of nucleotides it covers (binding site size) when binding to a single‐stranded nucleic acid lattice. The binding site size of native and conformationally changed SSB was also found to be a function of the molecular mass of the polynucleotide, an observation which is unusual for single‐stranded DNA binding proteins and will greatly affect the affinity relationship of this protein for nucleic acids. A radioimmunoassay used to quantitate in SSB level in cells revealed the number of SSB tetramers to be larger than initial estimates by a factor of as much as six. All these data suggest that the biological role of SSB and its mechanism of action is by far more complex than originally assumed.

INCORPORATION OF THE SPIN TRAP DMPO INTO CULTURED FETAL MOUSE LIVER CELLS

Abstract— The incorporation of four different spin traps into cultured fetal mouse liver cells was investigated using electron spin resonance spectroscopy. The cells were incubated in saline solutions of the traps, washed, and then irradiated in a saline solution containing hydrogen peroxide. The presence of a reproducible ESR signal was taken as evidence of spin trap incorporation. The spin trap DMPO was found to be incorporated, while PBN and 4‐POBN were not. MNP was toxic to the cells.

A spin probe approach for measuring the nucleic acid affinity of gene 32 protein

Abstract A novel and fast procedure for determining by electron spin resonance the affinity of proteins for nucleic acids is described. The assay makes use of nitroxide radicals which are covalently bound to various polynuleotides to the extent of one probe per 75 to 100 nucleotides. As a test example gene 32 protein was used, a protein known to interact with single stranded nucleic acids. Competition experiments with unlabeled nucleic acids made it feasible to directly monitor the gene 32 protein affinity for different nucleic acids. It was observed that DNA single strands are not necessarily favored by this protein for preferential binding. The experimental data also suggest that the difference in the binding constants for most of the complexes is remarkably large; for instance, (dT)n binds at least 3 to 4 orders of magnitude better to gene 32 protein than (dA)n.