Discrimination between G/C Binding Sites by Olivomycin A Is Determined by Kinetics of the Drug-DNA Interaction

Abstract

Olivomycin A (OA) exerts its cytotoxic potency due to binding to the minor groove of the G/C-rich DNA and interfering with replication and transcription. Screening of the complete set of tetranucleotide G/C sites by electrophoretic mobility gel shift assay (EMSA) revealed that the sites containing central GC or GG dinucleotides were able to bind OA, whereas the sites with the central CG dinucleotide were not. However, studies of equilibrium OA binding in solution by fluorescence, circular dichroism and isothermal titration calorimetry failed to confirm the sequence preference of OA, indicating instead a similar type of complex and comparable affinity of OA to all G/C binding sites. This discrepancy was resolved by kinetics analysis of the drug–DNA interaction: the dissociation rate significantly differed between SGCS, SGGS and SCGS sites (S stands for G or C), thereby explaining the disintegration of the complexes during EMSA. The functional relevance of the revealed differential kinetics of OA–DNA interaction was demonstrated in an in vitro transcription assay. These findings emphasize the crucial role of kinetics in the mechanism of OA action and provide an important approach to the screening of new drug candidates.

Differential Impact of Random GC Tetrad Binding and Chromatin Events on Transcriptional Inhibition by Olivomycin A

“…GC tetrads with specific positions of G/C nucleotides have been shown to be critical for OA–DNA complex formation in vitro [ 17 ]. These short DNA repeats in gene promoters are responsible for binding of cognate transcription factors such as Sp1 and others [ 31 , 32 ].…”

“…In the latter construct, each of tetrads 1–5 was mutagenized by replacing G for A ( Figure 1 A). Substitution of the guanine residue in central GC base pairs is expected to completely abrogate the binding of the antibiotic since the amino group of guanine is absolutely required for interaction with the aglycon moieties in the OA dimer [ 17 ]. The restriction site MluI at the 5′ end of the construct remained intact (yellow square in Figure 1 A).…”

“…According to previous results [ 17 ], we mapped GC tetrads as tentative OA binding sites in the region −2500 to +2500 bp around TSS. No dependence between the presence or distribution of GC tetrads and the sensitivity to OA was revealed ( Figure 6 , the top panel SGSS in each gene block).…”

“…Mechanistic studies proved that AA-derived antibiotics demonstrate a strong preference for GC-rich DNA sequences [ 13 , 14 ]. These compounds bind as dimers to the minor groove of the DNA double helix and form stable complexes with three (mithramycin [ 15 ]) or four (chromomycin A3 [ 16 ] and olivomycin A [ 17 ]) consecutive G/C base pairs. Conformational changes upon drug–duplex complex formation are thought to be responsible for alterations of the transcriptional machinery.…”

“…Recent in vitro experiments have demonstrated that OA has similar binding affinity to different G/C tetranucleotide stretches although the rate of dissociation of OA–DNA complexes differs depending on specific G/C sequences. In the cell-free system, the GC context influenced the effects of OA on the traverse of T7 RNA polymerase along the DNA template [ 17 ]. In cell-based studies the AA family of antibiotics was investigated as DNA ligands capable of modulating the function of GC-preferred transcription factors.…”