Mutual interaction between adjacent dG . dC actinomycin binding sites and dA . dT netropsin binding sites on the self-complementary d(C-G-C-G-A-A-T-T-C-G-C-G) duplex in solution.

Abstract

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).

Sequence specificity of actinomycin D and Netropsin binding to pBR322 DNA analyzed by protection from DNase I.

“…Our interpretation of this enhancement is that it is due to a conformational change in the DNA molecule induced by actinomycin D binding. This view is consistent with, data generated from NMR investiga- (20,21) and block copolymer studies (24), which support the argument that the binding of actinomycin D can affect DNA structure away from the primary binding sequence. Netropsin Binding.…”

“…All three sites are rich in G+C and contain the dimer 5'-G-C-3'. This observation is in accord with earlier studies (18)(19)(20)(21)(22)(23). It is interesting to note that other G-C sites are present between the observed binding sites but they are not occupied by actinomycin D. The significance of the 32-bp binding-site periodicity observed is unclear and requires further investigation.…”

Kinetics for exchange of the imino protons of the d(C-G-C-G-A-A-T-T-C-G-C-G) double helix in complexes with the antibiotics netropsin and/or actinomycin

“…Netropain is known to bind with a specificity for A•T regions in the minor groove of DNA duplexes (Zlmmer, 1975;Warteil et al, 1974). It has been shown that in the complex only one netropsin is bound per 12-mer duplex (Patel et al, 1981). Netropsin, which lacks a center of symmetry, removes the 2-fold symmetry of the 12-iner as revealed by up to a 0.02 ppm chemical shift difference for base pairs 3-6 on different ends of the helix (Patel et al, 1981).…”

“…It has been shown that in the complex only one netropsin is bound per 12-mer duplex (Patel et al, 1981). Netropsin, which lacks a center of symmetry, removes the 2-fold symmetry of the 12-iner as revealed by up to a 0.02 ppm chemical shift difference for base pairs 3-6 on different ends of the helix (Patel et al, 1981). However, no measurable difference in the exchange lifetimes for individual base pairs on different ends of the 12-mer helix in the netropsin complex was observed.…”

“…The 12'mer has been shown to form a complex having two actinomycins and one netropsin simultaneously bound at adjacent G'C and AT blocks (Patel et al, 1981). The lifetime in the 12-iner-netropsin-actinomycin complex at 55°C This result could be explained by a cooperative mechanism where all the inilno protons exchange together.…”