Identification and characterization of a pre-cleavage synaptic complex that is an early intermediate in Tn10 transposition.
The Tn10 transposition reaction has been reconstituted in vitro on short linear substrate fragments encoding transposon ends. This permits the direct detection of protein-DNA complexes formed during transposition by gel retardation analysis. We demonstrate that a stable synaptic complex containing transposase and a pair of transposon ends forms rapidly and efficiently, prior and prerequisite to the double-strand cleavages involved in transposon excision. These observations extend the general analogies between the Tn10 and Mu transposition reactions, and also reveal significant differences between the two cases. The speed and simplicity of synaptic complex formation in the Tn10/IS10 reaction is suitable for a modular insertion sequence. In contrast, the relative slowness and complexity of this process in the Mu is necessary to permit transposition immunity and control of transposition by Mu repressor protein, two features specifically important for a temperate bacteriophage. Further dissection of the reaction leads to a tentative working model for events preceding the first double-strand cleavage.
Sakai
J
J
Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.
Chalmers
R M
RM
Kleckner
N
N
D010583:Bacteriophage mu / Q000235:genetics* / Q000502:physiology; D002118:Calcium / Q000494:pharmacology; D002413:Cations, Divalent / Q000494:pharmacology; D004251:DNA Transposable Elements; D004926:Escherichia coli; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D016296:Mutagenesis; D009713:Nucleotidyltransferases / Q000378:metabolism*; D011485:Protein Binding; D012097:Repressor Proteins / Q000378:metabolism; D015183:Restriction Mapping; D013753:Tetracycline Resistance / Q000235:genetics*; D019895:Transposases
D002413:Cations, Divalent; D004251:DNA Transposable Elements; D012097:Repressor Proteins; D008345:Manganese; D009713:Nucleotidyltransferases; D019895:Transposases; D008274:Magnesium; D002118:Calcium