Biochemistry zyxwvut 1995, 34, zyxwvu 14801-14814 14801 Spin-Labeled Psoralen Probes for the Study of DNA Dynamics? H. Peter Spielmann,* Dae-Yoon Chi,$ Nathan G. Hunt,” Melvin P. Klein, and John E. Hearst* Structural Biology Division, Lawrence Berkeley Laboratory, and Department of Chemistry, University zyx of California, Berkeley, California 94720 Received January 31, 1995; Revised Manuscript Received July 5, 199P ABSTRACT: Six nitroxide spin-labeled psoralen derivatives 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 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) zyxwvutsr J. Am. Chem. SOC. 115, 7765-7771; Spielmann et al. (1995) Proc. Nutl. Acad. Sci. U.S.A. 92, 2345-23491, 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). Psoralen-damaged DNA serves as an excellent substrate for the study of structural and dynamic motifs that DNA repair enzyme systems may recognize. Psoralens are pho- toreagents which form a well-characterized set of covalent nucleic acid adducts through photochemical addition (Figure 1) (Straub et al., 1981; Kanne et al., 1982a,b). The primary reaction is cyclobutane ring formation between the 5,6 double bond of thymidine in DNA and either the 4’,5’ or 3,4 double bonds of the psoralen. Reaction at the 4’3’ double bond creates a furanside monoadduct (MAf),’ which can react further at a site with opposed and adjacent pyrimidines to create an interstrand cross-link (XL). The XL is a rigid unit formed by the psoralen and the opposite and adjacent pyrimidine bases, linking the two strands together. Reaction at the 3,4 double bond of the psoralen first creates a pyroneside monoadduct (MAP), which cannot form an interstrand cross-link. Psoralen-DNA monoadducts and cross-links are recog- nized and removed by the excision repair system in both zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA ’ This work was supported in part by the National Institutes of Health Grant GM-41911 (J.E.H. and M.P.K.), by postdoctoral fellowship GM- 14966 (H.P.S.), and by the Director, Office of Energy Research, Office of Basic Energy Sciences, Materials Sciences Division of the U.S. Department of Energy, under Contract No. DE-AC03-76SF00098 (J.E.H. and M.P.K.). * Author to whom correspondence should be addressed. Present address: Department of Biochemistry, University of Present address: Division of Nuclear Medicine, Samsung Hospital, ‘I Present address: Department of Pharmaceutical Chemistry, Uni- @ Abstract published in Advance ACS Abstracts, November zyxwvuts 1, 1995. ’ Abbreviations: EPR, electron paramagnetic resonance; MAf, psoralen-DNA furanside monoadduct; XL, psoralen-DNA interstrand cross-link; MAP, psoralen-DNA pyroneside monoadduct; NMR, nuclear magnetic resonance; 2-D NMR, two-dimensional NMR; AMT, zyxwvut 4’-(aminomethyl)-4,5’,8-trimethylpsoralen; HMT, 4’-(hydroxymethy1)- 4,5’,8-trimethylpsoralen; %MOP, 8-methoxypsoralen; bp, base pair. Kentucky, 800 Rose Street, Lexington, KY 40536-0084. 50 Ilwondong Kangnamgu, Seoul, Korea 135-230. versity of California, San Francisco, CA 94143. 0 cn, FIGURE 1: Structures and numbering schemes of HMT (lb) and the most common HMT-nucleoside adducts. bacteria and higher organisms (Sancar & Sancar, 1988; Reardon et al., 1991). There are a large number of structurally unrelated DNA lesion-forming agents that are efficiently removed by the excision repair system (Sancar & Sancar, 1988; Smith, 1988; Van Houten et al., 1987, 1988; Sladek et al., 1989; Smith et al., 1989; Cheng et al., 1991; Reardon et al., 1991; Huang et al., 1994). The sequence dependent microstructures in normal DNA are not recognized as damaged by these systems. It has been proposed that the recognition of lesions in DNA takes place by the recognition of either (or both) structural distortions or dynamic changes in the polymer (Sancar & Sancar, 1988; Lin & Sancar, 1989; Pu et al., 1989). It has also been proposed that DNA damage causes local instability in the helix and that the (A)BC excision repair system recognizes an excessive or unusual deformability (Williams & Gao, 1992). 0006-2960/95/0434- 14801$09.00/0 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 0 1995 American Chemical Society