Uranyl photoprobing of a four-way DNA junction: evidence for specific metal ion binding.
Metal ions are very important in mediating the folding of nucleic acids, as exemplified by the folding of the four-way DNA junction into the stacked X-conformation. Uranyl ion-mediated photocleavage provides a method for the localization of high-affinity ion binding sites in nucleic acids, and we have applied this to the four-way DNA junction. We have made the following observations. (i) Uranyl ions (UO2(2+)) suppressed the reactivity of junction thymine bases against attack by osmium tetroxide, indicating that the uranyl ion induces folding of the junction into a stacked conformation. (ii) DNA located immediately at the point of strand exchange on the two exchanging strands was hypersensitive to uranyl photocleavage. The relative hypersensitivity was considerably accentuated when the photocleavage was carried out in the presence of citrate ions. This suggests the presence of a tight binding site for the uranyl ion in the junction. (iii) The same positions were significantly protected from uranyl cleavage by the presence of hexamminecobalt (III) or spermidine. These ions are known to induce the folded conformation of the four-way junction with high efficiency, suggesting a direct competition between the ions. By contrast, magnesium ions failed to generate a similar protection against photocleavage. These results suggest that the uranyl, hexamminecobalt (III) and spermidine ions compete for the same high affinity binding site on the junction. This site is located at the centre of the junction, at the point where the exchanging strands pass between the stacked helices. We believe that we have observed the first known example of a metal ion 'footprint' on a folded nucleic acid structure.
Møllegaard
N E
NE
Department of Biochemistry, University of Copenhagen, Denmark.
Murchie
A I
AI
Lilley
D M
DM
Nielsen
P E
PE
D001483:Base Sequence; D002412:Cations / Q000494:pharmacology; D003035:Cobalt / Q000494:pharmacology; D004247:DNA / Q000737:chemistry* / Q000187:drug effects / Q000528:radiation effects; D008027:Light; D008958:Models, Molecular; D015335:Molecular Probes; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry* / Q000528:radiation effects; D009993:Osmium Tetroxide / Q000494:pharmacology; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology; D014502:Uranyl Nitrate / Q000494:pharmacology*
D002412:Cations; D015335:Molecular Probes; D009838:Oligodeoxyribonucleotides; D011838:Radiation-Sensitizing Agents; D014502:Uranyl Nitrate; C032300:hexaamminecobalt(II); D003035:Cobalt; D004247:DNA; D009993:Osmium Tetroxide