IS911-mediated transpositional recombination in vitro.
A cell-free system is described that accomplishes an unusual type of transposition/recombination involving the bacterial insertion sequence IS911. Using a plasmid substrate carrying a derivative of IS911, we show that bacterial cell extracts enriched for the IS911 transposase, OrfAB, carry out a single-strand cleavage and transfer reaction. This results in the formation of a figure-eight molecule in which a single strand of the element is circularized, faithfully reproducing an event previously detected in vivo. Moreover, when presented with a figure-eight substrate, OrfAB is capable of "reversing" strand transfer. This activity is equivalent to the "disintegration" reaction carried out by retroviral integrases. We demonstrate that the domain of OrfAB responsible for this catalytic activity is located in the carboxy-terminal region of the protein, since a peptide composed of this region retains disintegration activity. The OrfAB-mediated excision-circularization process previously observed in vivo was proposed to proceed via a figure-eight intermediate by circularization of the second transposon strand. The absence of transposon circles in cell-free reaction suggests either that the figure-eight form is not an intermediate or that additional host factors are required that are eliminated from the cell extract. Two types of model, replicative and non-replicative, are discussed to explain how the figure-eight molecule could be processed into the transposon circle.
Polard
P
P
Laboratoire de Microbiologie et Génétique Moléculaires CNRS UPR9007, Toulouse, France.
Ton-Hoang
B
B
Haren
L
L
Bétermier
M
M
Walczak
R
R
Chandler
M
M
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D004254:DNA Nucleotidyltransferases / Q000378:metabolism; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D019426:Integrases / Q000378:metabolism; D008954:Models, Biological; D009154:Mutation; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D011995:Recombination, Genetic; D012190:Retroviridae / Q000201:enzymology; D012762:Shigella dysenteriae / Q000235:genetics; D019895:Transposases
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C085896:OrfB protein, E coli; D004254:DNA Nucleotidyltransferases; D019426:Integrases; D019895:Transposases