Tn5 transposase active site mutations suggest position of donor backbone DNA in synaptic complex.
Tn5 transposase (Tnp), a 53.3-kDa protein, enables the movement of transposon Tn5 by a conservative mechanism. Within the context of a protein and DNA synaptic complex, a single Tnp molecule catalyzes four sequential DNA breaking and joining reactions at the end of a single transposon. The three amino acids of the DDE motif (Asp-97, Asp-188, and Glu-326), which are conserved among transposases and retroviral integrases, have been shown previously to be absolutely required for all catalytic steps. To probe the effect of active site geometry on the ability to form synaptic complexes and perform catalysis, single mutations at each position of the DDE motif were constructed. The aspartates were changed to glutamates, and the glutamate was changed to an aspartate. These mutants were studied by performing in vitro binding assays using short oligonucleotide substrates simulating the natural substrates for the synaptic complex formation and subsequent transposition steps. The results indicate that the aspartate to glutamate mutations restrict synaptic complex formation with substrates resembling the natural transposon prior to transferred strand nicking. This suggests a structural model in which the donor backbone DNA, prior to nicking, occupies the same space that is invaded by the longer side chains present in the aspartate to glutamate mutants. Additionally, catalytic assays support the previous proposal that the active site coordinates two divalent metal ions.
Peterson
Gregory
G
Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Reznikoff
William
W
D001483:Base Sequence; D017931:DNA Primers; D004269:DNA, Bacterial / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D013379:Substrate Specificity; D013569:Synapses / Q000201:enzymology / Q000378:metabolism*; D019895:Transposases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D004269:DNA, Bacterial; D019895:Transposases