High-resolution NMR and UV-melting experiments have been used to study the hairpin formation of partly self-complementary DNA fragments in an attempt to derive rules that describe the folding in these molecules. Earlier experiments on the hexadecanucleotide d(ATCCTA-TTTT-TAGGAT) had indicated that within the loop of four thymidines a wobble T-T pair is formed (Blommers et al., 1987). In the present paper it is shown that if the first and the last thymines of the intervening sequence are replaced by complementary bases, sometimes base pairs can be formed. Thus for the intervening sequences -CTTG- and -TTTA- with the pyrimidine in the 5'-position and the purine in the 3'-position, a base pair is formed leading to a loop consisting of two residues. For the intervening sequences -GTTC- and -ATTT- with the purine in the 5'-position and the pyrimidine in the 3'-position, this turns out not to be the case. It was found that it made no difference when the four-membered sequence was closed by a G-C base pair or an A-T base pair. Replacement of the two central thymidine residues by the more bulky adenine residues limits the hairpin to a four-membered loop scheme. Very surprisingly, it was found from 2D NOE experiments that the T-A base pair, formed in the loop consisting of the -TTTA- sequence, is a Hoogsteen pair. It is argued that the pairing of the bases in this scheme may facilitate the formation of a loop of two residues, since the distance of the C1' atoms in this base pair is 8.6 A instead of 10.4 A found in the canonical Watson-Crick base pair. Combination of the data obtained for the series of DNA fragments studied shows that the results can be explained by a simple, earlier proposed, loop folding principle which assumes that the folding of the four-membered loop is dictated by the stacking of the double-helical stem of the hairpin.

In an effort to identify events initiating up-regulation of epidermal growth factor receptor after single and repeated radiation exposures, we investigated the role of epidermal growth factor receptor, a receptor protein tyrosine kinase, in radiation-induced signal transduction. Human malignant mammary, MCF-7, and squamous, A431, cells showed low baseline phospho-tyrosine levels of epidermal growth factor receptor, permitting reproducible dose-dependent stimulation of epidermal growth factor receptor autophosphorylation after exposure to epidermal growth factor. MCF-7 cells exhibited a mean 2.3-fold increase (95% confidence interval: 1.91, 2.65; P < 0.0001) in levels of epidermal growth factor phosphorylation in response to exposures of 2 Gy, which was substantially less than the epidermal growth factor receptor Y phosphorylation induced by epidermal growth factor. A quantitatively similar radiation response was seen in A431 cells. In the dose range of 1 to 4 Gy, no clear dose response was seen. There was a rapid induction of radiation-induced epidermal growth factor receptor Y phosphorylation, starting within 2 min, with maximum values between 0.5 and 5 min after radiation exposure followed by a slower decline to baseline levels after 20 min. The data presented identify the epidermal growth factor receptor protein tyrosine kinase associated with the plasma membrane as one target for ionizing radiation in the dose range used in radiotherapy.

The 3'-5' cyclic dinucleotide d(pApA) was studied by means of 1H and 13C NMR experiments, UV-melting experiments, and molecular mechanics calculations. The 1H and 13C NMR spectra were analyzed by means of 2-dimensional NMR experiments. J-Coupling analysis of the 1D and 2D 1H and 13C spectra was used to determine the conformation of the ring systems in the molecule. It appeared that at low temperature (283 K) the deoxyribose sugars adopt a N-type conformation. The geometry is best described by an intermediate between the 3(2)T and 3E forms. In addition, we were able to derive all other torsion angles in the phosphate backbone ring system, i.e., alpha +, beta t, gamma +, delta (= 89 degrees), epsilon t, and zeta +. When the molecule is subjected to an energy minimization procedure (using the program AMBER), the sugar ring system retains, practically speaking, the torsion angles found from the NMR experiments, while the torsion angles around the glycosidic bond adopt a value of 175 degrees in the minimum energy conformation. UV-melting experiments indicate that two molecules can form a dimer in which the adenine bases are intercalated. The feasibility of this structure is indicated by molecular mechanics calculations. At higher temperatures the dimer is converted into separate monomers. In the monomer form the sugars exhibit S-pucker 20% of the time. Concomitantly with the conversion of the N- to the S-conformation, the torsion angles alpha and gamma change.

Recently, the imino proton spectrum of yeast tRNAPhe has been assigned by means of the application of the nuclear Overhauser effect (NOE). In the present paper it will be shown that even for tRNA (MW 28000) connectivities between the imino proton spins can be observed using two-dimensional NOE spectroscopy. In this way the imino proton resonances of the D-stem region are assigned. The results are discussed in relation to those obtained by the classical one-dimensional nuclear Overhauser effect. It turns out that in 2D-NOE experiments connectivities from overlapping resonances can be observed which cannot be determined by one-dimensional Overhauser experiments. Moreover, the total assignment of the imino proton spectrum of yeast tRNAPhe is used to relate the three-dimensional crystal structure of the tRNA to its solution structure. It is shown that the principle elements of the X-ray structure, i.e. the hydrogen bonding network and the stacking of the stems upon one another, are also found in solution. This is true for the presence as well as for the absence of magnesium ions. However, in absence of magnesium ions the tRNA structure appears to differ in details from that in the presence of magnesium ions. Finally, the influence of the elongation factor Tu from B.stearothermophilus on the tRNA structure is discussed.

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