COMPARATIVE STRUCTURAL-ANALYSIS BY [H-1,P-31]-NMR AND RESTRAINED MOLECULAR-DYNAMICS OF 2 DNA HAIRPINS FROM A STRONG DNA TOPOISOMERASE-II CLEAVAGE SITE

The structural analysis of two single-stranded DNAs d(AGCTTATCATCGATAA GCT) (ATC-19) and d(AGCTTATCGATGATAAGCT) (GAT-19) was performed by NMR and restrained molecular dynamics. These oligonucleotides reproduce t he 15-33 segment of phage pBR322 DNA, which contains a strong cleavage site for topoisomerase II coupled to the antitumor drugs VP-16 and el lipticine. Because of their partial palindromic nature, the two oligon ucleotides ATC-19 and GAT-19 may fold back into stable hairpin structu res, consisting of a stem of eight base-pairs and a loop of three resi dues. NMR assignments and conformational parameters were determined fr om combined 2D NOESY, COSY and H-1-P-31 spectra. Conformations of ATC- 19 and GAT-19 hairpins were calculated using the X-PLOR 3.1 program. S tructures were generated through simulated annealing procedures starti ng from 50 structures with randomized torsion angles. A good convergen ce was observed for ATC-19 molecules, while no consensus was found for GAT-19. Within the GAT-19 loop, the base stacking was poor and no hyd rogen bond could be detected. In contrast, ATC-19 displayed a well-def ined three residue loop stabilized by both extensive base stackings an d hydrogen bonding between the N3 atom of the adenine ring and the ami no group of the cytosine ring. The results confirm our earlier ATC-19 structure obtained by a completely different calculation procedure (JU MNA) and the higher thermal stability of ATC-19 compared to GAT-19. Mo reover, due to its mismatched basepair, the ATC-19 loop may be better described as a single residue loop rather than a three residue loop. C omparison of this loop to those containing sheared purine purine base- pairs revealed striking resemblances, particularly on the backbone ang le combination. Finally, the differences observed between the ATC-19 a nd GAT-19 structures could help toward understanding the sequential cl eavage of DNA strands by topoisomerase II. (C) 1998 Academic Press.