Site-specific binding constants for actinomycin D on DNA determined from footprinting studies

Jerry Goodisman,Robert Rehfuss,James C Dabrowiak,Brian Ward

doi:10.1021/bi00119a013

Abstract

We report site-specific binding constants for the intercalating anticancer drug actinomycin D (Act-D), binding to a 139-base-pair restriction fragment from pBR 322 DNA. The binding constants are derived from analysis of footprinting experiments, in which the radiolabeled 139-mer is cleaved using DNase I, the cleavage products undergo gel electrophoresis, and, from the gel autoradiogram, spot intensities, proportional to amounts of cleaved fragments, are measured. A bound drug prevents DNase I from cleaving at approximately 7 bonds, leading to decreased amounts of corresponding fragments. With the radiolabel on the 3' end of the noncoding strand (A-label), we measured relative amounts of 54 cleavage products at 25 Act-D concentrations. For cleavage of the 139-mer with the label on the 3' end of the coding strand (G-label), relative amounts of 43 cleavage products at 11 Act-D concentrations were measured. These measurements give information about approximately 120 base pairs of the restriction fragment (approximately 12 turns of the DNA helix); in this region, 14 strong and weak Act-D binding sites were identified. The model used to interpret the footprinting plots is derived in detail. Binding constants for 14 sites on the fragment are obtained simultaneously. It is important to take into account the effect of drug binding at its various sites on the local concentration of probe elsewhere. It is also necessary to include in the model weak as well as strong Act-D sites on the carrier DNA which is present, since the carrier DNA controls the free-drug concentration. As expected, the strongest sites are those with the sequence (all sequences are 5'----3') GC, with TGCT having the highest binding constant, 6.4 x 10(6) M-1. Sites having the sequence GC preceded by G are weak binding sites, having binding constants approximately 1 order of magnitude lower than those of the strong sites. Also, the non-GC-containing sequences CCG and CCC bind Act-D with a binding constant comparable to those of the weak GGC sites. The analysis may reveal drug-induced structural changes on the DNA, which are discussed in terms of the mechanism of Act-D binding.

Highlights

Actinomycin D, actinomycin D (Act-D) (Figure I), is one of the most intensely studied anticancer drugs (Gale et al, 1981)
We report site-specific binding constants for the intercalating anticancer drug actinomycin D (Act-D), binding to a 139-base-pair restriction fragment from pBR 322 DNA
We present measured footprinting plots for actinomycin D binding to DNA, as well as the model and analysis which allow us to derive binding constants for strong and weak sites, as well as other parameters

Summary

Introduction

Actinomycin D, Act-D (Figure I), is one of the most intensely studied anticancer drugs (Gale et al, 1981). The binding mechanism involves intercalation of the phenoxazone ring system of the drug via the minor groove of DNA at GC-rich sites (Gale et al, 1981). Model building and a single-crystal X-ray structural analysis (Sobell 1973) indicated that the specificity of Act-D for this sequence is due to hydrogen bonding between the 2-amino group of guanine of DNA and the threonine moiety located in the cyclic peptide of the drug. Striction fragments and found that GC doublets were strongly preferred only if the 5’-flanking base was a pyrimidine and the 3‘-flanking base was not cytosine. The central GG doublet in the sequence TGGG is a strong Act-D site

Methods

Findings

Discussion

Conclusion