Tn10 transposition and circle formation in vitro.
We describe a cell-free system that promotes Tn10 transposition and transposon circle formation, a related intramolecular event. Tn10 circle formation in vitro has been characterized in detail, and is shown to require a supercoiled substrate and to proceed in the absence of ATP. The reaction requires Tn10 transposase protein, and either of two E. coli proteins, integration host factor (IHF) and HU, which are small DNA binding proteins that change the conformation of DNA. Tn10 is composed of inverted repeats of insertion sequence IS10. Pair-wise combinations of the IS10 "outside" and "inside" ends mediate distinct classes of rearrangements in vivo, and they exhibit different reaction requirements in vitro. In contrast to the Tn10 reaction, which involves two outside ends, circle formation with two inside ends proceeds with a transposase fraction alone, in the absence of added host factors, and is inhibited by methylation of the dam site within each terminus.
Morisato
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Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts 02138.
Kleckner
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N
D000251:Adenosine Triphosphatases / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D005591:Chemical Fractionation; D004265:DNA Helicases; D004251:DNA Transposable Elements; D004270:DNA, Circular / Q000378:metabolism*; D004278:DNA, Superhelical / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D034982:Integration Host Factors; D009713:Nucleotidyltransferases / Q000378:metabolism; D010957:Plasmids; D019895:Transposases
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004270:DNA, Circular; D004278:DNA, Superhelical; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D034982:Integration Host Factors; C040330:histone-like protein HU, bacteria; D009713:Nucleotidyltransferases; D019895:Transposases; D000251:Adenosine Triphosphatases; D004265:DNA Helicases