Tn 5 y IS 50 target recognition ( Escherichia coli y composite transposons y insertion specificity ) IGOR

This communication reports an analysis of Tn5yIS50 target site selection by using an extensive collection of Tn5 and IS50 insertions in two relatively small regions of DNA (less than 1 kb each). For both regions data were collected resulting from in vitro and in vivo transposition events. Since the data sets are consistent and transposase was the only protein present in vitro, this demonstrates that target selection is a property of only transposase. There appear to be two factors governing target selection. A target consensus sequence, which presumably reflects the target selection of individual pairs of Tn5yIS50 bound transposase protomers, was deduced by analyzing all insertion sites. The consensus Tn5yIS50 target site is A-GNTYWRANC-T. However, we observed that independent insertion sites tend to form groups of closely located insertions (clusters), and insertions very often were spaced in a 5-bp periodic fashion. This suggests that Tn5yIS50 target selection is facilitated by more than two transposase protomers binding to the DNA, and, thus, for a site to be a good target, the overlapping neighboring DNA should be a good target, too. Synthetic target sequences were designed and used to test and confirm this model. Transposition is a multistep DNA rearrangement process in which a transposon DNA sequence, defined by precise end sequences, is inserted into a target sequence on the same or a different DNA molecule. This process is catalyzed by an elementspecific protein called a transposase (Tnp). During integration Tnp that is bound to the ends of the transposon binds to target DNA. Tnp then catalyzes a strand transfer of 39-OH ends of the transposon into opposite strands of the target DNA with a shift varying for different transposons of from 2 to 14 bp (1). This shift defines the length of the short direct repeat (SDR) flanking the transposon after its integration into a target DNA. As a first approximation, the SDR andyor surrounding sequences is thought to represent the target sequence recognized by the transposing complex. For transposon Tn5, whose specificity of integration is analyzed in the present work, the SDR is 9 bp (2). Tnp target DNA selection is important to study not only because it is a critical step in transposition (a genetic event thought to occur in all types of organisms) but also because it is an example of a protein–DNA recognition reaction and may give insights into the mechanisms involved in the formation of multimeric protein– DNA complexes. Target-choice specificity varies for different transposons. Bacteriophage Mu appears capable of inserting within any sequence (3), while transposon Tn7 has only one major target site in the Escherichia coli chromosome (4). For IS4, three structurally similar sites in the E. coli chromosome have been described (5). Homology with Tn3-terminal sequences has been found near the sites of Tn3 insertion (6). Tc1 generates a TA duplication upon integration into the target DNA with the TA target being located within the consensus sequence CAYATATRTG (7). Tc1 insertion sites were found to be the same for both in vivo and in vitro systems (8). For composite transposons such as Tn5, Tn9, and Tn10, there are thousands of possible integration sites in the E. coli chromosome, but some preferable sites also have been described. Different explanations for the existence of ‘‘hot sites’’ have been proposed. For Tn10, the consensus of insertion sites has been reported to be a 9-bp imperfect palindrome (9), although the 6–9 nt flanking this sequence also were demonstrated to have a substantial influence (10). A Tn10 in vivo hot target site recently was found to function in vitro (11). For Tn9 (and its constituent IS1) the regions of preferable integration were determined to be AT-rich sequences (12, 13). Frequent Tn9 insertions were also detected in gene promoter regions as well as near the DNAbinding sites for IHF protein (14). There have been several attempts to find a key for Tn5 target-choice preference. A model was proposed suggesting that partial homology between DNA in the area of the insertion site and Tn5-terminal sequences is important (15, 16). However, incorporation of DNA fragments containing the IS50 outside (OE) or inside (IE) end sequences into pBR322 had no effect on the distribution of Tn5 insertions into pBR322 and promoted no new transposon-integration hot sites (17). Preferred target sites were described for the pBR322 plasmid (18). Of 150 independent Tn5 insertions in the tet gene, 55 were found located at only two sites. One of the characteristics frequently found for Tn5 transposition target sites is the occurrence of GC pairs at each end of the SDR (19). The other proposed characteristic of Tn5 transposition is the preferable integration in actively transcribing or highly super-coiled DNA regions, an observation that is not related to its sequence specificity but rather to the preferred topology of the target (20, 21). In this study we used Tn5(defined by two inverted OE sequences) or IS50 (defined by OE and IE sequences)-like structures to study the specificity of Tn5yIS50 target choice. A series of 138 independent in vivo and 384 in vitro insertions (most of which were in the CmR gene derived from pACYC184 plasmid and KmR gene derived from Tn903) were collected, and the precise insertion sites were determined by DNA sequence analysis. The resulting data led to two interesting proposals that were tested by examining the frequency of Tn5 insertion into specifically designed synthetic target sequences. Tn5yIS50 Tnp does recognize a preferred 9-bp sequence as its target but, surprisingly, sequences resembling this consensus target function optimally when embedded in a cluster of overlapping similar sequences. From this we hypothesize that Tn5 Tnp tends to form small The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1998 by The National Academy of Sciences 0027-8424y98y9510716-6$2.00y0 PNAS is available online at www.pnas.org. This paper was submitted directly (Track II) to the Proceedings office. Abbreviations: SDR, short direct repeat(s); Tnp, transposase; OE or IE, outside or inside end of IS50, respectively; Km, kanamycin; Cm, chloramphenicol; Tet, tetracycline; wt, wild type. ‡To whom reprint requests should be addressed. e-mail: reznikoff@ biochem.wisc.edu.