STRUCTURE OF A COVALENT DNA MINOR-GROOVE ADDUCT WITH A PYRROLOBENZODIAZEPINE DIMER - EVIDENCE FOR SEQUENCE-SPECIFIC INTERSTRAND CROSS-LINKING

The structure of the interstrand cross-linked adduct formed between a C8-C8'-linked pyrrolobenzodiazepine (PBD) dimer(DSB-120; ahydro-5H-pyr rolo[2,1-c][1,4]benzodiazepin-5-one]) and a self-complementary d(CICGA TCICG)(2) duplex has been determined from high-field 1D- and 2D-NMR da ta using a simulated annealing procedure. The refined structure suppor ts earlier observations from solution NMR experiments and indicates th at the covalently bound molecule spans six DNA base pairs in the minor groove, forming a symmetric cross-link between the spatially separate d internal guanines and with active recognition of an embedded 5'-GATC bonding site. This result confirms that template-directed approaches are useful for the design of Linked. DNA-interactive PBD dimers with v iable DNA cross-linking potential, Further, head-to-head connection of the PBD moieties results in an overall retention of 5'-GA bonding sit e preference for each alkylating PBD subunit. Structural analysis indi cates that cross-link formation results in a localized perturbation of the DNA duplex, attributable in part to a mutual reduction in dynamic mobility or ''covalent clamping'' within the Gua4-Cyt7 base tract. Ho wever, ligand-induced distortion is confined to the Cyt7 and Ino8 resi dues on each strand. The Gua(N2) Gua(N2) cross-link is stabilized by t wo directed H-bonds from the formed aminal residues to N3 acceptor ato ms of adenine bases on the 3'-side of each covalently modified guanine . Evidence for sequence-specific cross-linking with DSB-120 is provide d by extended modeling studies which suggest that recognition of the f avored d(.GATC.) motif is dominated by van der Waals steric factors, a lthough electrostatic and H-bonded interaction terms also play a key r ole. This conclusion supports recent covalent footprinting studies rev ealing that this PBD dimer shows a selectivity for embedded base seque nces of the type 5'-(pu/py)GATC(py/pu).