DNA is probably the most discussed and least observed of all biological macromolecules. Although its role in biology is a central one, with many examples such as operators and restriction sites where specific base sequences have control functions or interact with specific enzymes, the structures that DNA can adopt have been based until now only on sequence-averaged fibre diffraction patterns. Recent improvements in triester synthesis methods have made possible the preparation of sufficient homogeneous DNA of predetermined sequence for crystallization and X-ray structure analysis. We report here the first single-crystal structure analysis of more than a complete turn of right-handed B-DNA, with the self-complementary dodecamer sequence d(CpGpCpGPApApTpTpCpGpCpG) or CGCGAATTCGCG.
The Watson-Crick imino protons, the backbone phosphodiester resonances, and the antibiotic exchangeable protons have been used as markers to monitor the separate and simultaneous binding of actinomycin and netropsin to the d(C-G-C-G-A-A-T-T-C-G-C-G) self-complementary duplex in aqueous solution. We demonstrate that intercalation ofactinomycin at dG(3'-5')dC sites at either end of the duplex results in a conformational perturbation at the dA-dT tetranucleotide core of the dodecanucleotide duplex. Parallel studies ofthe groove binding of netropsin at dAdT sites in the interior ofthe duplex reveal a conformational perturbation which extends to adjacent dG-dC base pairs in the dodecanucleotide duplex. The NMR markers demonstrate that the d
(C-G-C-G-A-A-T-T-C-G-C-G) duplex can accommodate ac-tinomycin and netropsin simultaneously at adjacent dG-dC and dAdT tetranucleotide blocks along its length with some mutual interaction between neighboring antibiotic binding sites.There have been extensive NMR studies of the binding of antibiotics to DNA duplexes at the oligonucleotide and polynucleotide level in solution. The majority of these efforts have focussed on the binding ofa single antibiotic to the DNA and have provided insights into conformational features of nucleic acid complexes with intercalating, groove-binding, and kinking agents (1). It was ofinterest to extend these NMR investigations to the binding of the two different antibiotics at adjacent sites on the DNA duplex and to investigate their mutual interactions along the helix.
The d(C-G-C-G-A-A-T-T-C-G-C-G) dodecanucleotide du-plex (scheme I) contains more than one turn of helix so that it is sufficiently long to bind more than one antibiotic along its length. Furthermore, it contains a dA-dT tetranucleotide block flanked by dG-dC tetranucleotide blocks in either direction, providing adjacent sites for dAdT-specific and dG-dC-specific antibiotics.The antibiotic actinomycin D [scheme II, R = cyclo(ThrDVal-Pro-Sar-MeVal)] is an intercalative agent which exhibits a sequence specificity for dG(3'-5')dC sites on double helical DNA. Intercalation of actinomycin D results in unwinding of the duplex with the pentapeptide lactone rings extending between 2 and 3 base pairs on either side of the intercalation site (2, 3). Scheme II Netropsin, a peptide antibiotic, exhibits a specificity for dA-dT-rich duplex regions and spans between 2 and 3 base pairs of the DNA duplex. Netropsin binds in the minor groove ofthe DNA, and the complex is stabilized predominantly by hydrogen bonding through the antibiotic peptide protons and electrostatic interactions through the charged ends (4, 5).
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