Common Physical Properties of DNA Affecting Target Site Selection of Sleeping Beauty and other Tc1/mariner Transposable Elements Thomas J. Vigdal 1 , Christopher D. Kaufman 2 , Zsuzsanna Izsva ´k 2,3 Daniel F. Voytas 1 and Zolta ´n Ivics 2 * 1 Department of Zoology and Genetics, Iowa State University Ames, IA, USA 2 Max Delbru ¨ck Center for Molecular Medicine, Berlin Germany 3 Institute of Biochemistry Biological Research Center of the Hungarian Academy of Sciences, Szeged, Hungary Sleeping Beauty (SB) is the most active Tc1/mariner-type transposable element in vertebrates, and is therefore a valuable vector for transposon mutagenesis in vertebrate models and for human gene therapy. We have analyzed factors affecting target site selection of SB in mammalian cells, by generating transposition events from extrachromosomal plasmids to chromosomes. In contrast to the local hopping observed when transposi- tion is induced from a chromosomal context, mapping of 138 unique SB insertions on human chromosomes showed a fairly random genomic dis- tribution, and a 35% occurrence of transposition into genes. Inspection of the DNA flanking the sites of element integration revealed significant differences from random DNA in both primary sequence and physical properties. The consensus sequence of SB target sites was found to be a palindromic AT-repeat, ATATATAT, in which the central TA is the canoni- cal target site. We found however, that target site selection is determined primarily on the level of DNA structure, and not by specific base-pair interactions. Computational analyses revealed that insertion sites tend to have a bendable structure and a palindromic pattern of potential hydro- gen-bonding sites in the major groove of the DNA. These features appear conserved in the Tc1/mariner family of transposons and in other, distantly related elements that share a common catalytic domain of the transposase, and integrate fairly randomly. No similar target site preference was found for non-randomly integrating elements. Our results suggest common factors influencing target site selection of a wide range of transposable elements. q 2002 Elsevier Science Ltd. All rights reserved Keywords: transposon; target site; transposition; bendability; hydrogen bond *Corresponding author Introduction Most transposable elements do not integrate randomly into target DNA, and display some degree of specificity in target site utilization. 1 There is a wide spectrum of specificity in target site selection, hereby defined as the mechanism by which the specific DNA sequences of target sites are chosen. For example, the bacterial Tn7 element is highly specialized to insert into a single sequence motif in the Escherichia coli genome, 1 whereas several other transposons, such as Tn5, can integrate at several locations even within a single gene. 2 Target selection may depend on primary DNA sequence and chromatin structure, which can influence target site utilization by modulating the accessibility of DNA. For some elements, such as Tn7 and the Ty1, Ty3 and Ty5 retrotransposons in yeast, either element-encoded or host-encoded accessory proteins play a role to locate a potential target area. 3–6 In other systems, including the bacterial transposon Tn10 and the Tc1 and Tc3 transposons in Caenorhabditis elegans, target site selection is determined primarily by the transposase itself. 7,8 Sequences responsible for tar- get site selection of Tn10 and retroviruses have been mapped to the core catalytic domain of the 0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved E-mail address of the corresponding author: zivics@mdc-berlin.de Abbreviations used: SB, Sleeping Beauty; MANOVA, multivariate analysis of variance; BB, bad bender. doi:10.1016/S0022-2836(02)00991-9 available online at http://www.idealibrary.com on B w J. Mol. Biol. (2002) 323, 441–452