SOLUTION STRUCTURE OF THE MONOALKYLATED MITOMYCIN C-DNA COMPLEX

Mitomycin C (MC) is a potent antitumor antibiotic which alkylates DNA through covalent linkage of its C-1 '' position with the exocyclic N-2 amino 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-me r complex where the [MC]dG5 adduct is positioned opposite dC14 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 s ystem at a similar to 45 degrees angle relative to the helix axis and directed towards the 3'-direction on the unmodified strand. The MC ind oloquinone chromphore is asymmetrically positioned in a slightly widen ed 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 ex posed to solvent. The MC five-membered ring adopts an envelope pucker with its C-2 '' atom displaced from the mean plane and directed away f rom the unmodified strand. We observe conformational perturbations in the DNA 9-mer duplex on formation of the monoalkylated MC complex. Spe cifically, the base-pairs are displaced by similar to -3.0 Angstrom to wards the major groove on positioning the MC in the minor groove. This perturbation is accompanied by base stacking patterns similar to thos e observed in A-DNA while the majority of the sugars adopt puckers cha racteristic of B-DNA. Conformational perturbations as monitored by hel ix twist, sugar pucker pseudorotation and glycosidic torsion angles ar e also observed for the d(T6-C7-I8). d(C11-G12-A13) segment that is ad jacent 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 stru cture of the complex containing this intramolecular hydrogen bond read ily explains both the previously observed d(C-G). d(C-G) sequence requ irement for cross-linking and the observed, somewhat less stringent, r equirement of the same sequence for the initial monoalkylation step. T he structure indicates that the former is determined by the specific a lignment of the MC monoadduct in the minor groove, while the specifici ty of the monoalklyation step likely reflects a specific hydrogen-bond between MC and DNA at the d(C-G). d(C-G) step in the precovalent comp lex.