SOLUTION STRUCTURE OF THE COVALENT DUOCARMYCIN A-DNA DUPLEX COMPLEX

Duocarmycin A is an antitumour antibiotic that binds covalently to the minor groove N-3 position of adenine with sequence specificity for th e S'-adenine in a d(A-A-A-A) tract in duplex DNA. The adenine ring bec omes protonated on duocarmycin adduct formation resulting in charge de localization over the purine ring system. We report on the solution st ructure of duocarmycin A bound site specifically to A12 (designated A 12(+)) in the sequence context d(T3-T4-T5-T6). d(A9-A10-A11-A12(+)) w ithin a hairpin duplex. The solution structure was solved based on a c ombined NMR-molecular dynamics study including NOE based intensity ref inement. The A and B-rings of duocarmycin are positioned deep within t he walls of the minor groove with the B-ring (which is furthest from t he covalent linkage site) directed towards the 5'-end of the modified strand. Duocarmycin adopts an extended conformation and is aligned at similar to 45 degrees to the helix axis with its non-polar concave edg es interacting with the floor of the minor groove while its polar edge s are sandwiched within the walls of the minor groove. The T3 .A12(+) modification site pair forms a weak central Watson-Crick hydrogen bon d in contrast to all A . T and G . C pairs, which align through standa rd Watson-Crick pairing in the complex. The helical parameters are con sistent with a minimally perturbed right-handed duplex in the complex with minor groove width and x-displacement parameters indicative of a B-form helix. A striking feature of the complex is the positioning of duocarmycin A within the walls of the minor groove resulting in upfiel d shifts of the minor groove sugar protons, as well as backbone proton and phosphorus resonances in the DNA segment spanning the binding sit e.