No abstract

Circular dichroism spectroscopy was used to study mutants of phage fd gene 5 protein (Y26F, Y34F, and Y41F) in which three of the five tyrosines, Tyr-26, Tyr-34, and Tyr-41, were individually substituted with phenylalanine. The tyrosine 229 nm CD bands of the wild type, Y26F, and Y41F gene 5 proteins decreased in magnitude during complex formation with either fd ssDNA or poly[d(A)]. However, the Y34F gene 5 protein showed no decrease in the 229 nm band during titrations of these nucleic acids. This suggested that Tyr-34 of the wild type gene 5 protein dominated the 229 nm CD changes upon binding to single-stranded DNA. Titrations of poly[d(A)] or fd ssDNA with wild type, Y26F, Y34F, or Y41F gene 5 proteins resulted in perturbations of the nucleic acid near-UV CD bands, specific for the particular nucleic acid, but similar for all four proteins (in 2 mM sodium phosphate buffer, pH 7.0). For both nucleic acids, the addition of protein beyond a certain [protein monomer]/[nucleotide] ratio (0.25 for poly[d(A)] or 0.33 for fd ssDNA) resulted in a partial reversal of the CD change of the nucleic acid. These data are interpreted to mean that, in addition to the two well-known n = 4 and n = 3 stoichiometric modes of binding, there is a third mode of binding in which the nucleic acid is in limited contact with the protein. As shown by salt dissociation studies of complexes with poly[d(A)], the binding affinities, K omega, of the proteins were in the order: wild type > Y26F >> Y34F > or = Y41F (for the n = 4 binding mode in 0.1-0.2 M NaCl). Our data indicate that Tyr-34 plays a more important role in forming a complex with ssDNA than is apparent in current models of the g5p.ssDNA complex. We suggest that the hydroxyl moieties of Tyr-34 and Tyr-41 are both somehow involved in stabilizing the interface of bound protein dimers.

No abstract

Knowledge of the relative stabilities of S-DNA.RNA hybrids of different sequences is important for choosing RNA targets for hybridization with antisense phosphorothioate oligodeoxyribonucleotides (S-DNAs). It is also important to know how hybrid secondary structure varies with sequence, since different structures could influence thermal stability and the activity of RNase H. Our approach has been to study relatively simple sequences consisting of repeating di-, tri-, and tetranucleotides, which allow the maximum resolution of nearest-neighbor effects. Circular dichroism (CD) spectra and melting temperatures were acquired for 16 hybrid sequences that could be formed by mixing S-DNA and RNA oligomers of 24 nucleotides in length. CD spectra of S-DNA.RNA hybrids were sequence-dependent and were similar to those of analogous unmodified hybrids. From singular value decomposition, the major CD spectral component was like that of the A-conformation. Three nearest-neighbor relationships among the hybrid CD spectra were in as good agreement as are such relationships among spectra of duplex RNAs. Tm values ranged from 44.1 degrees C for S-d(ACT)8. r(AGU)8 to 66.6 degrees C for S-d(CCT)8.r(AGG)8 (in 0.15 M K+, phosphate buffer, pH 7). The S-DNA.RNA hybrids had a sequence-dependence of melting temperatures that was approximately the same as that calculated using published data for normal DNA.RNA hybrids [Sugimoto, N., Nakano, S., Katoh, M., Matsumura, A., Nakamuta, H., Ohmichi, T.,Yoneyama, M., & Sasaki, M. (1995) Biochemistry 34, 11211-11216]. In general, sequence-dependent CD spectra and Tm values of S-DNA.RNA hybrids appear to reflect the unique nearest-neighbor interactions of adjacent base pairs, where the S-DNA and RNA strands are in different, but relatively uniform, conformations.

We have found that the CD spectrum of poly [d(C-T)] undergoes a marked change upon protonation, taking on the unmistakable features of the spectrum of poly[d(C) x d(C+)]. The acid poly[d9C-T)] structure appears to be one in which each of the T residues is individually looped out of a helix formed by stacked C x C+ base pairs. We also present data for poly[d(C-A)] and review data for the other four possible repeating DNA dinucleotide sequences. These data show that self-complexes in which up to 50% of the residues are extrahelical may be unexceptional.