SOLUTION STRUCTURES OF PSORALEN MONOADDUCTED AND CROSS-LINKED DNA OLIGOMERS BY NMR-SPECTROSCOPY AND RESTRAINED MOLECULAR-DYNAMICS

We have used two-dimensional H-1 NMR spectroscopy to determine the sol ution structures of the 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (H MT) furanside mono adducted (MAf) and the photoisomeric HMT interstran d cross-linked (XL) DNA oligonucleotide d(5'-GCGTACGC-3')(2). The dete rmination of the structure was based on total relaxation matrix analys is of the NOESY cross-peak intensities using the program MARDIGRAS. Im proved procedures to consider the experimental ''noise'' in NOESY spec tra during these calculations have been employed. The NOE-derived dist ance restraints were applied in restrained molecular dynamics calculat ions. Twenty final structures each were generated for both the MAf and XL from both A-form and B-form dsDNA starting structures. The root-me an-square (rms) deviations of the coordinates for the 40 structures fo r the MAf and XL were 1.12 and 1.10 Angstrom, respectively. The rmsd o f the MAf with respect to the XL is 2.20 Angstrom. The local DNA struc ture is distorted in both adducts, with the helix unwound by 34 degree s and 25 degrees for the MAf and XL, respectively, and an overall heli cal repeat of 11 base pairs, caused by intercalation of the HMT. The M Af is a photochemical intermediate on the path to interstrand XL. Cons iderable local structural distortion is induced by both adducts, but t he DNA returns to B-form structure within three base pairs ofthe damag e site. There is no significant bend in the helix axis of either the M Af or the XL. We have evaluated the accuracy of the two major methods of converting NOESY data into interproton distances, the isolated spin -pair approximation (ISPA) and the complete relaxation rate matrix ana lysis (RMA). Both methods were evaluated by comparing the resulting ca lculated interproton distances generated to known covalently fixed dis tances in the HMT. The overall structures were evaluated by checking t heir agreement with biophysical evidence from non-NMR techniques. Only the modified RMA method gave correct interproton distances.