Abstract: Six nitroxide spin-labeled psoralen derivative have been synthesized and evaluated as probes for structural and dynamic studies. Sequence specific photoaddition of these derivatives to DNA oligonucleotides resulted in site-specifically cross-linked and spin-labeled oligomers. Comparison of the general line shape features of the observed electron paramagnetic resonance (EPR) spectra of several duplexes ranging in size from 8 to 46 base pairs with simulated EPR spectra indicate that the nitroxide spin-label probe reports the global tumbling motion of the oligomers. While there is no apparent large amplitude motion of the psoralen other than the overall tumbling of the DNA on the time scales investigated, there are some indications of bending and other residual motions. The (A)BC excinuclease DNA repair system detects structural or dynamic features of the DNA that distinguish between damaged and undamaged DNA and are independent of the intrinsic structure of the lesion. NMR studies have shown that psoralen-cross-linked DNA has altered backbone dynamics and conformational populations in the immediate vicinity of the adduct [Emsley et al. (1993) J. Am. Chem. Soc. 115, 7765-7771; Spielmann et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 2345-2349]. We suggested that the signal for recognition of a lesion to be repaired is in the sugar--phosphate backbone and not in the damaged base(s).