Nucleic Acids Research Advance Access published online on September 12, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn577
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Molecular Biology |
Phosphate coordination and movement of DNA in the Tn5 synaptic complex: role of the (R)YREK motif
1Department of Biochemistry, University of Wisconsin at Madison, 433 Babcock Drive, Madison, WI 53706, USA and 2Laboratory of Molecular Biology (affiliated with the University of Gdansk), Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Kladki 24, Gdansk 80-822, Poland
To whom correspondence should be addressed. Tel: +1 508 289 7253; Fax: +1 508 457 4727; Email: breznikoff{at}mbl.edu, wsreznik{at}wisc.edu
Received June 11, 2008. Revised August 27, 2008. Accepted August 28, 2008.
Bacterial DNA transposition is an important model system for studying DNA recombination events such as HIV-1 DNA integration and RAG-1-mediated V(D)J recombination. This communication focuses on the role of protein–phosphate contacts in manipulating DNA structure as a requirement for transposition catalysis. In particular, the participation of the nontransferred strand (NTS) 5' phosphate in Tn5 transposition strand transfer is analyzed. The 5' phosphate plays no direct catalytic role, nonetheless its presence stimulates strand transfer 
30-fold. X-ray crystallography indicates that transposase–DNA complexes formed with NTS 5' phosphorylated DNA have two properties that contrast with structures formed with complexes lacking the 5' phosphate or complexes generated from in-crystal hairpin cleavage. Transposase residues R210, Y319 and R322 of the (R)YREK motif coordinate the 5' phosphate rather than the subterminal NTS phosphate, and the 5' NTS end is moved away from the 3' transferred strand end. Mutation R210A impairs the 5' phosphate stimulation. It is posited that DNA phosphate coordination by R210, Y319 and R322 results in movement of the 5' NTS DNA away from the 3'-end thus allowing efficient target DNA binding. It is likely that this role for the newly identified RYR triad is utilized by other transposase-related proteins.