A 3·(ET743)-DNA Complex That Both Resembles an RNA-DNA Hybrid and Mimicks Zinc Finger-Induced DNA Structural Distortions

Abstract

The antitumor ecteinascidin ET743 has been shown to inhibit the transcriptional activation of a number of genes at nanomolar concentrations. Cell sensitivity to subnanomolar concentrations of the drug has also been shown to specifically depend on the transcription-coupled nucleotide excision repair system. ET743 is known to bind covalently to the minor groove of a DNA double helix in regions comprising selected sets of three consecutive base pairs. Following alkylation of a central guanine, the minor groove is widened and the DNA is bent toward the major groove. We have previously shown that in the resulting adduct the DNA triplet containing the covalently modified guanine bears a strong resemblance to a DNA triplet recognized by a C(2)H(2) zinc finger. We now expand this earlier finding and use simulation methods to show that head-to-tail binding of three ET743 molecules to three adjacent optimal binding sites stabilizes a DNA structure whose conformation is intermediate between A- and B-form DNA. Furthermore, despite the increase in roll at the sites of covalent attachment, no net curvature is apparent in this complex due to cancellation of the localized bends over virtually one turn of the helix. Both observations are in good analogy to findings in zinc finger-DNA complexes. Triplets are virtually superimposable both directly and upon shifting the register one base pair. In this latter case, the central guanine in a triplet alkylated by ET743 corresponds to the third nucleic base in the triplet recognized by a zinc finger of transcription factors such as EGR1 or Sp-1. The DNA conformation found in the ET743-DNA complex is also strongly reminiscent of an RNA-DNA hybrid, as found in the RNA polymerase II elongation complex. The possible biological implications of these findings in relation to the antitumor action of ET743 are discussed.

Assessment by Molecular Dynamics Simulations of the Structural Determinants of DNA-binding Specificity for Transcription Factor Sp1

“…The local helical axes of the base-pairs are writhed around an unbent global axis, resembling A-DNA, as discussed. 38 Since our oligonucleotides (14-mers) are much shorter than those used in the experiments, we cannot presently assess the effects of Sp1 binding on the sequences flanking the "GC box" but our results strongly suggest that Sp1-induced bending is more likely to take place at the junctions between GC boxes and intervening sequences than at the GC boxes themselves.…”

“…To illustrate this point, in the refined averaged structures of our modeled complexes the mean values are 7.4(^0.9) Å (mdr1) and 6.5(^0.7) Å (wt1), which indeed represent a substantial increase relative to the same distance measured in standard B-DNA (5.9 Å ). 38 …”

“…37 This similarity was recently proposed to extend beyond a single ET743-binding site when the feasibility of head-to-tail tandem binding of three ET743 molecules to three adjacent optimal-binding sites (TGG CGG CGG) was demonstrated using unrestrained molecular dynamics simulations. 38 This method makes use of recent advances in force-field parameters, simulation algorithms, and computer power to derive reliable trajectories in atomic detail for macromolecular systems, including protein -DNA complexes, 39 exclusively in terms of intermolecular forces and motions. Analyses of these trajectories can provide independent accounts of experimentally observed behavior, including DNA bending 36 and other sequence-dependent structural effects in DNA duplexes, even when starting from incorrectly modeled structures.…”

“…narrower groove width in A/T regions, intrinsic tendency of CpG and TpG steps to bend into the major groove, 36,38 twist/roll preferences 38 and greater rigidity of ApG and GpA steps compared to CpG and GpC steps 70 ). In fact, it is now accepted that the best residues for DNA recognition can depend on the position of a finger in the protein and/or the effect of neighboring fingers so that each zinc finger is not completely independent.…”

Relevance of the Fanconi anemia pathway in the response of human cells to trabectedin

“…Because proteins encoded by these genes are components of the homologous recombination (HR) machinery, which is required for the repair of double-strand breaks (DSBs) associated with replication forks (16), all these data indicate that HR participates in the processing of the DNA lesions induced by trabectedin, including the generation and repair of DSBs (15,17,18). Given the unique mode of binding of trabectedin to DNA and the feasibility of having two or more trabectedin molecules simultaneously targeting adjacent or closely spaced sites on opposing strands (19,20), we reasoned that this type of covalent adduct might functionally mimic an interstrand cross-link (ICL) such as that produced by the potent antitumor and bifunctional cross-linking agent mitomycin C (MMC; Supplementary Fig. S1; 6 ref.…”