Determination of the structural features of (+)-CC-1065 that are responsible for bending and winding of DNA

Abstract

Analysis of the anomalous migration in electrophoretic mobilities of (+)-CC-1065-modified oligomers following ligation reveals that (+)-CC-1065 induces DNA bending and winding of the helix. (+)-CC-1065 is a potent antitumor antibiotic produced by Streptomyces zelensis. This drug selectively bonds covalently to N3 of adenine and lies in the minor groove of DNA, reacting in a highly sequence-selective manner. Structurally, (+)-CC-1065 consists of three subunits: two identical pyrroloindole units (subunits B and C) and a third subunit containing the DNA-reactive cyclopropane ring (subunit A). While the bonding reaction is the main determinant of DNA sequence selectivity of (+)-CC-1065, binding interactions between the inside edge substituents of the B and C subunits and the floor of the minor groove of DNA can modulate or fine tune this sequence selectivity, [Hurley, L. H., Lee, C.-S., McGovern, J. P., Mitchell, M. A., Warpehoski, M. A., Kelly, R. C., & Aristoff, P. A. (1988) Biochemistry 27, 3886-3892]. The A subunit of (+)-CC-1065 is responsible for the bending of DNA, and close van der Waals contacts between the inside edge of (+)-CC-1065 and the floor of the minor groove of DNA cause winding equivalent to about 1 base pair per alkylation site and stiffening of DNA. The magnitude of DNA bending induced by (+)-CC-1065 and related compounds is about 14-19 degrees, which is equivalent to that produced by an adenine-thymine tract of about 5-6 base pairs in length. Experiments using oligomers containing both an adenine tract and a unique (+)-CC-1065 bonding site approximately one helix turn apart demonstrate that the directionality of drug-induced bending is in toward the minor groove and the locus of bending is about 2-3 base pairs to the 5'-side of the covalently modified adenine. A circularization efficiency assay shows that the optimum size of circles produced by (+)-CC-1065 and related drugs is between 168 and 180 base pairs. These results are discussed in relation to the molecular basis of the DNA sequence selectivity of (+)-CC-1065, and the (+)-CC-1065-induced DNA bending is compared with the intrinsic bending associated with adenine tracts. Since (+)-CC-1065 induces effects on local DNA structure that appear similar to those produced naturally by adenine tracts and certain DNA binding proteins, the relevance of this phenomenon to biological effects of (+)-CC-1065 and related drugs is considered.

Devising a Structural Basis for the Potent Cytotoxic Effects of Ecteinascidin 743

“…Since the anomalous mobility was at a maximum when the Et 743 alkylation site was positioned five base pairs (one-half helical turn) away from the center of the A-tract and at a minimum when the separation was 11 base pairs (one complete helical turn), these observations were taken as an indication that the bend caused by Et 743 was in a direction opposite to that of the A-tract, that is, toward the major groove, again in good agreement with the findings from the unrestrained MD simulations. This feature was novel among minor groove DNA monoalkylating agents, since covalent modification of N3 of adenine in AT-rich regions by (þ)-CC-1065 and related compounds is accompanied by bending of the DNA into the minor groove [29]. …”

Recognition of DNA Adducts by Human Nucleotide Excision Repair

“…Anthramycin binds selectively to N 2 of guanine through aminal bonds and forms covalent adducts with essentially no distortion of the DNA helix (21,29). CC-1065 displays a cyclopropyl ring that alkylates DNA at position N 3 of adenine, generating covalent adducts that cause bending and winding of the double helix (22,30). Both anthramycin and CC-1065 adducts enhance duplex stability through noncovalent interactions derived from hydrogen bonds (anthramycin) or van der Waals and hydrophobic forces (CC-1065) within the minor groove of DNA (21,22,30).…”

Mutation spectra induced by adozelesin in the supF gene of human XP-A fibroblasts

“…The drug molecule lies within the minor groove and covers four bases on the 5' side and overlapping one base on the 3' side of a covalently modified adenine (Hurley et al, 1984;Scahill et al, 1990). This covalent DNA modification stabilizes the helix as it bends into the minor groove (Swenson et al, 1982;Reynolds et al, 1985;Lee et al, 1991). The bulky adducts produced by CC-1065 and adozelesin are largely refractory to recognition by nucleotide and base excision repair enzymes (Selby and Sancar, 1988;Tang et al, 1988;Jin et al, 2001).…”