Solution Structure of the Monoalkylated Mitomycin C–DNA Complex

Abstract

Mitomycin C (MC) is a potent antitumor antibiotic which alkylates DNA through covalent linkage of its C-1″ position with the exocyclic N2amino group of guanine to yield the [MC]dG adduct at the duplex level. We report on the solution structure of the monoalkylated MC-DNA 9-mer complex where the [MC]dG5 adduct is positioned opposite dLC14 in the d(A3-C4-[MC]G5-T6).d(A13-C14-G15-T16) sequence context. The solution structure was solved based on a combined NMR-molecular dynamics study including NOE intensity based refinement. The formation of the [MC]dG adduct occurs with retention of the Watson-Crick alignment at the [MC]dG5.dC14 base-pair and flanking pairs in the complex. The MC ring is positioned in the minor groove with its indoloquinone aromatic ring system at a ∼45° angle relative to the helix axis and directed towards the 3′-direction on the unmodified strand. The MC indoloquinone chromphore is asymmetrically positioned in a slightly widened minor groove so that its plane is parallel to and stacked over the d(C14-G15-T16) segment on the unmodified strand with its other face exposed to solvent. The MC five-membered ring adopts an envelope pucker with its C-2″ atom displaced from the main plane and directed away from the unmodified strand. We observe conformational perturbations in the DNA 9-mer duplex on formation of the monoalkylated MC complex. Specifically, the base-pairs are displaced by ∼−3.0 Å towards the mjor groove on positioning the MC in the minor groove. This perturbation is accompanied by base stacking patterns similar to those observed inA-DNA while the majority of the sugars adopt puckers characteristic ofB-DNA. Conformational perturbations as monitored by helix twist, sugar pucker pseudorotation and glycosidic torsion angles are also observed for th 6(T6-C7-I8).d(C11-G12-A13) segment that is adjacent to but does not overlap the MC binding on the 9-mer duplex. We note that the O-10″ atom on the carbamate side-chain of MC forms an intermolecular hydrogen bond with the exocyclic amino group of dG15 in two of the three refined structures of the complex. The solution structure of the complex containing this intramolecular hydrogen bond readily explains both the previously observed d(C-G).d(C-G) sequence requirement for cross-linking and the observed, somewhat less stringent, requirement of the same sequence for the initial monoalkylation step. The structure indicates that the former is determined by the specific alignment of the MC monoadduct in the minor groove, while the specificity of the monoalkylation step likely reflects a specific hydrogen-bond between MC and DNA at the d(C-G).d(C-G) step in the precovalent complex.