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Journal of Bacteriology, October 2007, p. 7436-7441, Vol. 189, No. 20
0021-9193/07/$08.00+0     doi:10.1128/JB.00524-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Site-Directed Mutagenesis Studies of Tn5 Transposase Residues Involved in Synaptic Complex Formation{triangledown} ,{dagger}

Soheila Vaezeslami, Rachel Sterling, and William S. Reznikoff*

Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706

Received 5 April 2007/ Accepted 1 August 2007

Transposition (the movement of discrete segments of DNA, resulting in rearrangement of genomic DNA) initiates when transposase forms a dimeric DNA-protein synaptic complex with transposon DNA end sequences. The synaptic complex is a prerequisite for catalytic reactions that occur during the transposition process. The transposase-DNA interactions involved in the synaptic complex have been of great interest. Here we undertook a study to verify the protein-DNA interactions that lead to synapsis in the Tn5 system. Specifically, we studied (i) Arg342, Glu344, and Asn348 and (ii) Ser438, Lys439, and Ser445, which, based on the previously published cocrystal structure of Tn5 transposase bound to a precleaved transposon end sequence, make cis and trans contacts with transposon end sequence DNA, respectively. By using genetic and biochemical assays, we showed that in all cases except one, each of these residues plays an important role in synaptic complex formation, as predicted by the cocrystal structure.

* Corresponding author. Mailing address: Bay Paul Center, Marine Biological Laboratory, 7 MBL St., Woods Hole, MA 02543. Phone: (508) 289-7253. Fax: (508) 457-4727. E-mail: reznikoff{at}biochem.wisc.edu

{triangledown} Published ahead of print on 10 August 2007.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

Journal of Bacteriology, October 2007, p. 7436-7441, Vol. 189, No. 20
0021-9193/07/$08.00+0     doi:10.1128/JB.00524-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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