Tn 10 transposition in vivo: temporal separation of cleavages at the two transposon ends and roles of terminal basepairs subsequent to interaction of ends.
During Tn10 transposition, the transposon is fully excised from the donor site by double strand cleavages at the two ends of the element prior to integration at a new target site. Results presented here demonstrate that an interaction between the two transposon ends is required for double strand cleavage at either end. Furthermore, despite this essential interaction of ends, subsequent cleavages at the two ends can occur at observably distinct times prior to occurrence of strand transfer at either end. Moreover, the time between cleavages at the two ends is exaggerated by the presence of an appropriate mutation at one end of the element. Biological rationales for this constellation of mechanistic features are suggested. Additional results demonstrate that mutations at the three terminal basepairs of Tn10 confer defects subsequent to interaction of ends, in confirmation of inferences from genetic analysis. More specifically, mutations in bp 1-3 confer strong defects during conversion of the full excision intermediate to a complete strand transfer product; mutations in bp 1 and 2 also confer more subtle defects subsequent to interaction of ends but prior to full excision. Such defects might reflect roles for these basepairs in the chemical steps of transposition per se, the positioning of terminal residues for those chemical steps, and/or the coupling of cleavage(s) to subsequent conformational changes.
Haniford
D
D
Department of Biochemistry, University of Western Ontario, London, Canada.
Kleckner
N
N
D004252:DNA Mutational Analysis; D004251:DNA Transposable Elements / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D015321:Gene Rearrangement / Q000235:genetics*; D010641:Phenotype; D011995:Recombination, Genetic / Q000235:genetics*; D013329:Structure-Activity Relationship; D013489:Suppression, Genetic
D004251:DNA Transposable Elements; D004269:DNA, Bacterial