Published online 16 June 2004
Nucleic Acids Research, 2004, Vol. 32, No. 10 E83
© 2004 Nucleic Acids Research, Vol. 32 No. 10 © Oxford University Press 2004; all rights reserved
A high-throughput assay for Tn5 Tnp-induced DNA cleavage
Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706, USA
* To whom correspondence should be addressed. Tel: +1 608 262 3608; Fax: +1 608 262 3453; Email: reznikoff{at}biochem.wisc.edu
Received March 24, 2004; Revised April 22, 2004; Accepted May 15, 2004
Transposition causes genomic instability by mobilizing DNA elements. This phenomenon is mechanistically related to other DNA rearrangements, such as V(D)J recombination and retroviral DNA integration. A conserved active site architecture within the transposase/integrase superfamily catalyzes these distinct phenomena. The Tn5 transposase (Tnp) falls within this protein class, and many intermediates of the Tn5 transposition reaction have been characterized. Here, we describe a method for the rapid identification of Tn5 Tnp small molecule effectors. This high-throughput screening strategy will aid in the identification of compounds that perturb Tnp-induced DNA cleavage. This method is advantageous, since it identifies effectors that specifically inhibit catalysis without inhibiting Tnp–DNA binding interactions. Effectors identified using this method will serve as a valuable aid both in the isolation and characterization of metal-bound reaction intermediates and in co-crystallization studies involving the effector, Tnp and DNA, to identify the structural basis of the interaction. Furthermore, since Tn5 Tnp shares a similar active site architecture to other transposase/integrase superfamily members, this strategy and any effectors identified using this method will be readily applicable to these other systems.