Sequence Determinants of the Intrinsic Bend in the Cyclic AMP Response Element † Leslie S. Sloan and Alanna Schepartz* Department of Chemistry, Yale UniVersity, New HaVen, Connecticut 06520-8107 ReceiVed August 13, 1997; ReVised Manuscript ReceiVed January 6, 1998 ABSTRACT: The cyclic AMP response element (CRE site, ATGACGTCAT) is the DNA target for transcription factors whose activities are regulated by cyclic AMP (1). Recently, we discovered that the CRE site is bent by 10-13° toward the major groove (2). Little or no bend is detected in the related AP-1 site (ATGACTCAT), which differs from the CRE site by loss of a single, central, C‚G base pair (2, 3). Here we describe experiments designed to identify which base pairs within the CRE site induce the bent structure in an attempt to understand the origins of the dramatically different conformations of the CRE and AP-1 sites. Our data indicate that the intrinsic CRE bend results from distortion within the TGA sequence found in each CRE half site (ATGAC). These two TGA sequences are located in phase with one another in the CRE sequence but are not (completely) in phase in the AP-1 sequence. This difference in phasing leads to the overall difference in bend as detected by gel (2) and cyclization methods (S. C. Hockings, J. D. Kahn, and D. M. Crothers, unpublished results; M. A. Fabian and A. Schepartz, unpublished results). Our results confirm earlier predictions of altered structure within TG steps, provide insight into the structural reorganizations induced in DNA by bZIP proteins, and lead to a revision of the relationship between the structures of the free and bZIP-bound forms of the CRE and AP-1 sites. The possibility that certain DNA molecules might possess B-form structures that differed from that of the Watson- Crick model (4) was recognized by the discovery of bent DNA in 1982 (5). It was discovered that the kinetoplast minicircles of the Leishmania tarentolae parasite contained tracts of four to five dA‚dT base pairs (called A-tracts) repeated in phase with the DNA helical repeat (6, 7). Fragments of these minicircles displayed anomalously low mobilities in nondenaturing gels when compared with reference DNA sequences of equivalent length. Subsequent experimentation revealed that the anomalous migration of minicircle DNA resulted from a bend of approximately 17° associated with each A-tract (8). When located along the same face of the DNA double helix, the bends produced by each A-tract added constructively to generate a DNA fragment with a significant overall bend. A-tract bending has been studied extensively (for reviews, see refs 9-13) since its discovery because of the significance of DNA structure and structural dynamics for at least three vital biological functions: transcriptional regulation (14), control of replication and recombination (15, 16), and the packaging of DNA into nucleosomes (10, 17, 18). Although a number of intrinsically bent DNA sequences unrelated to A-tracts have been identified (14, 19-26), the magnitudes of the bends in these sequences are, with two exceptions (24, 26), small relative to the bend produced by a single A-tract (∼17°) (8). Recently, we discovered that the cyclic AMP response element (CRE site, ATGACGTCAT), the DNA target for transcription factors whose activities are regulated by cyclic AMP (1), is bent significantly in solution (2). The CRE site consists of two ATGAC half-sites arranged in an inverted repeat and is unrelated to A-tract DNA. The magnitude of the intrinsic CRE bend was estimated at between 10° and 13° toward the major groove (2). Thus the CRE bend is similar in magnitude to that of a single A-tract, but the bend is in the opposite direction (8). Although the bend in the CRE site was detected initially by use of gel electrophoretic methods (27), its existence has been confirmed subsequently by analyzing the rates of minicircle ligation reactions (28, 51) (M. A. Fabian and A. Schepartz, unpublished results) as well as by X-ray crystallography [see reference in Paolella et al. (2)]. Here we employ a related gel electrophoretic method to analyze the extent of bending in a series of oligonucleotides related to the CRE site. Our data indicate that the intrinsic CRE bend (2) is the additive result of two major groove bends that are each found within the TGA sequence in each CRE half-site (ATGAC). These two TGA sequences are located in phase with one another in the sequence ATGACGTCAT but are not (completely) in phase in the related AP-1 sequence (ATGACTCAT). We propose that this difference in phasing leads to the overall difference in bend as detected by gel-based methods. MATERIALS AND METHODS Materials. Enzymes were purchased from New England Biolabs and used with the buffers supplied. Adenosine 5′- [γ- 32 P]triphosphate was purchased from DuPont. Adenosine 5′-triphosphate monomagnesium salt was purchased from Sigma. Phosphoramidites and other reagents employed in solid-phase oligonucleotide synthesis were purchased from Perceptive Biosystems. † This work was supported by the NIH (Grant GM 52544). L.S.S. was supported by an NSF predoctoral fellowship. * Correspondence should be addressed to this author at alanna.schepartz@yale.edu. 7113 Biochemistry 1998, 37, 7113-7118 S0006-2960(97)02009-6 CCC: $15.00 © 1998 American Chemical Society Published on Web 05/02/1998