Defining characteristics of Tn5 Transposase non-specific DNA binding

Published online May 22, 2006 2820–2832 Nucleic Acids Research, 2006, Vol. 34, No. 9 doi:10.1093/nar/gkl179 Defining characteristics of Tn5 Transposase non-specific DNA binding Mindy Steiniger, Christian D. Adams, John F. Marko1 and William S. Reznikoff* Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA and 1 Department of Physics, University of Illinois at Chicago, Chicago, IL 60607, USA Received December 6, 2005; Revised February 14, 2006; Accepted March 21, 2006 ABSTRACT INTRODUCTION Understanding the non-specific DNA binding characteristics of proteins involved in DNA recombination, DNA cleavage and gene regulation is essential for complete comprehension *To whom correspondence should be addressed. Tel: +1 608 262 3608; Fax: +1 608 265 2603; Email:  The Author 2006. Published by Oxford University Press. All rights reserved. The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact While non-specific DNA plays a role in target localization for many recombinases, transcription factors and restriction enzymes, the importance of nonspecific DNA interactions for transposases has not been investigated. Here, we discuss non-specific DNA-Tn5 Transposase (Tnp) interactions and suggest how they stabilize the Tnp and modulate Tnp localization of the 19 bp Tnp recognition end sequences (ESes). DNA protection assays indicate that full-length Tnp interacts efficiently with supercoiled DNA that does not contain ESes. These interactions significantly prolong the lifetime of Tnp, in vitro. The balance between non-specific DNA bound and free Tnp is affected by DNA topology, yet, intermolecular transfer of active Tnp occurs with both supercoiled and linear non-specific DNA. Experiments with substrates of varying lengths show that Tn5 Tnp can utilize non-specific DNA to facilitate localization of an intramolecular ES over distances less than 464 bp. Finally, synaptic complex formation is inhibited in the presence of increasing concentrations of supercoiled and linear pUC19. These experiments strongly suggest that Tn5 Tnp has a robust non-specific DNA binding activity, that non-specific DNA modulates ES sequence localization within the global DNA, most likely through a direct transfer mechanism, and that nonspecific DNA binding may play a role in the cis bias manifested by Tn5 transposition. of their reaction mechanisms. The ability to bind DNA nonspecifically can affect reaction rates, target recognition and the activity of proteins. While the role of non-specific DNA binding has been well documented for the Lac Repressor and some restriction enzymes (1,2), this important phenomenon has been observed, but not analyzed, in transposition systems. A review of current literature reveals evidence of nonspecific DNA binding by transposases (Tnp), site-specific recombinases and retroviral integrases. Two of the three domains of the bacteriophage Mu Tnp (MuA) independently bind DNA non-specifically. Filter binding experiments with proteolytic fragments of MuA reveal that the N-terminal 26 amino acids of domain III binds supercoiled DNA nonspecifically (3) and that domain II can interact with linear non-specific DNA (4). The Tn3 Tnp binds non-specific linear restriction fragments (5,6). Finally, many Tnps, including Tn5 Tnp, can integrate their transposons into random DNA sequences (7–10), a function that depends on non-specific DNA interactions. Remarkably, the importance of these interactions to the stability of Tnp or the transposition mechanism, other than integration, has not been carefully studied. Transposition is the process of moving DNA from one location to another. In its simplest form, this process requires a Tnp, DNA flanked by Tnp recognition end sequences (ESes), target DNA and a divalent metal ion (usually Mg2+) (11). The Tn5 transposon is mobilized using a cut-and-paste mechanism in which Tnp first binds two 19 bp inverted ESes, (12–14) see Figure 1. The two Tnp bound ESes then homodimerize to form a synaptic complex, the nucleoprotein complex required for catalysis (15–17). Following synapsis, a water molecule activated by Mg2+ attacks the phosphodiester backbone of one DNA strand at the junction between the ES and flanking DNA resulting in the generation of a 30 hydroxyl group (first strand nicking). This 30 hydroxyl group then attacks the opposite DNA strand creating a hairpin intermediate and releasing the flanking DNA (18). The hairpin is resolved by nucleophilic attack of a second Mg2+-activated water molecule creating a blunt-ended transposon with free 30 hydroxyl groups (11). Following cleavage, the complex Nucleic Acids Research, 2006, Vol. 34, No. 9 Synapsis 2821 with DNA fragments of different lengths. The reaction rate constants determined on these substrates indicate that DNA covalently linked to the ES can modulate ES localization by Tnp possibly through a direct transfer mechanism. Finally, increasing concentrations of linear or supercoiled pUC19 inhibit synaptic complex formation. These data support a model in which non-specific DNA binding regulates active Tnp concentration in vivo. MATERIALS AND METHODS Cleavage Tn5 Tnps used in this study { { Strand transfer Figure 1. The Tn5 reaction mechanism. Tn5 Tnp first binds to 19 bp inverted repeat recognition ESes, followed by homodimerization of these bound Tnps to form a synaptic complex. The donor backbone (dbb) DNA is then cleaved from the transposon (Tn) at the dbb/Tn junction. Following cleavage, the synaptic complex can capture a non-specific DNA target and integrate the Tn resulting in a 9 bp duplication (represented by ‘{’). Tn5 Tnp is shown as a gray oval and the 19 bp ES is represented by a light gray box. captures a non-specific DNA target and inserts the transposon [for a review see, (19)]. In this manuscript, we investigate the effect of Tn5 Tnp non-specific DNA binding on the initial steps of the transposition mechanism leading to ES localization. A previously described hyperactive variant of Tnp containing E54K, M56A and L372P mutations is used in these studies (11,19–22). In this communication, we define non-specific DNA as any DNA that does not contain the Tn5 Tnp ES. First, we use a restriction enzyme blockage assay (REBA) to show that full-length Tn5 Tnp can interact efficiently with many supercoiled pUC19 sites. These interactions allow Tnp to remain active up to 95 times longer than in the absence of (...truncated)