Purification and characterization of TnsC, a Tn7 transposition protein that binds ATP and DNA.
The bacterial transposon Tn7 encodes five transposition genes tnsABCDE. We report a simple and rapid procedure for the purification of TnsC protein. We show that purified TnsC is active in and required for Tn7 transposition in a cell-free recombination system. This finding demonstrates that TnsC participates directly in Tn7 transposition and explains the requirement for tnsC function in Tn7 transposition. We have found that TnsC binds adenine nucleotides and is thus a likely site of action of the essential ATP cofactor in Tn7 transposition. We also report that TnsC binds non-specifically to DNA in the presence of ATP or the generally non-hydrolyzable analogues AMP-PNP and ATP-gamma-S, and that TnsC displays little affinity for DNA in the presence of ADP. We speculate that TnsC plays a central role in the selection of target DNA during Tn7 transposition.
Gamas
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Department of Microbiology and Immunology, University of California, San Francisco 94143.
Craig
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D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D004247:DNA / Q000378:metabolism*; D004251:DNA Transposable Elements / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D010957:Plasmids / Q000235:genetics
D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C074940:TnsC protein, E coli; D000266:Adenylyl Imidodiphosphate; C022571:adenosine 5'-O-(3-thiotriphosphate); D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D004247:DNA