242

PROBINGDNA STRUCTUREin Vitro

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tercalate. This is seen in the case of tRNA in which Ru(phen)3 z+ preferentially cleaves in the D-T loop region. Studies on larger RNA helices need to be conducted. The transition metal complexes certainly provide new, specific, and unique probes to examine RNA structures. Acknowledgments We are grateful to the National Institute of General Medical Science (GM33309 and GM08346) and to the Ralph M. Parsons Foundation for financial support of research.

[13] P r o b i n g D N A S t r u c t u r e w i t h P s o r a l e n in Vitro By

DAVID W. USSERY, ROBERT W. HOEPFNER,

and

RICHARD R. SINDEN

Introduction: Psoralen and Its Photochemistry Psoralens have been widely used as probes of DNA structure, RNA structure, and DNA-protein interaction. Bacterial and eukaryotic cells are permeable to psoralens which can photobind to DNA inside cells. Psoralen binds preferentially to DNA in cells. The binding of psoralens to DNA is sensitive to superhelical density, alternate DNA conformations, and protein association. Psoralens initially bind noncovalently to DNA by interacalation into the DNA double helix. The intercalative binding is sufficiently weak that relatively few psoralens will bind DNA, resulting in little perturbation of the topology and structure of DNA. On irradiation with near-UV light (320-400 nm), an intercalated psoralen can be photobound to an adjacent pyrimidine base, forming a monoadduct. When the adjacent base in the opposite strand is also a pyrimidine, interstrand crosslinks can form in a second photochemical reaction (Fig. l). 1 Cross-links covalently bind the two strands of the double helix together. The number of psoralens photobound to DNA can be carefully controlled by the incident exposure to UV light. Psoralens bind tightly to proteins and membranes but the binding is not dependent on irradiation with 360 nm light. 2'3 Psoralen monoadducts and cross-links can be easily assayed by a number of methods, which will be described or referenced in this chapter. I R. S. Cole, Biochim. Biophys. Acta 217, 30 (1970). 2 S. Frederiksen and J. E. Hearst, Biochim. Biophys. Acta 563, 343 (1979). 3 j. D. Laskin, E. Lee, E. J. Yurkow, D. L. Laskin, and M. A. Gallo, Proc. Natl. Acad. Sci. U.S.A. 82, 6158 (1985).

METHODS IN ENZYMOLOGY.VOL. 212

Copyright © 1992by AcademicPress, Inc. AIJ rights of reproduction in any form reserved.

PSORALEN PHOTOB[NDING D N A in Vitro

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243

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