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Mechanism of DNA-mediated transposition

  • Page ID
    370
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    Figure 9.13).
    1. The transposase encoded by a transposable element makes four nicks initially. Two nicks are made at the target site, one in each strand, to generate a staggered break with 5' extensions (3' recessed). The other two nicks flank the transposon; one nick is made in one DNA strand at one end of the transposon, and the other nick is made in the other DNA strand at the other end. Since the transposon has inverted repeats at each end, these two nicks that flank the transposon are cleavages in the same sequence. Thus the transposase has a sequence-specific nicking activity. For instance, the transposase from TnA binds to a sequence of about 25 bp located within the 38 bp of inverted terminal repeat (Figure 9.10). It nicks a single strand at each end of the transposon, as well as the target site (Figure 9.13). Note that although the target and transposon are shown apart in the two-dimensional drawing in Figure 9.13, they are juxtaposed during transposition.
    2. At each end of the transposon, the 3' end of one strand of the transposon is joined to the 5' extension of one strand at the target site. This ligation is also catalyzed by transposase. ATP stimulates the reaction but it can occur in the absence of ATP if the substrate is supercoiled. Ligation of the ends of the transposon to the target site generates a strand-transfer intermediate, in which the donor and recipient replicons are now joined by the transposon.
    Figure 9.14.Three-dimensional structure of the Tn5 transposase in complex with Tn5 transposon DNA. A. The dimer of the Tn5 transposase is shown bound to a fragment of duplex DNA from the end of the transposon. Alpha helices are green cylinders, beta sheets are yellow-brown, flat arrows and protein loops are blue wires. The DNA is a duplex of two red wires, one for each strand. B. The DNA is shown without the protein and with the nucleotides labeled. The end of the DNA at the top of this panel is oriented into the active site in the middle of the protein in panel A. The structure was determined by Davies DR, Goryshin IY, Reznikoff WS, Rayment I. (2000) “Three-dimensional structure of the Tn5 synaptic complex transposition intermediate.” Science 289:77-85. These images was obtained by downloading the atomic coordinates from the Molecular Modeling Database at NCBI, viewing them with CN3D 3.0 and saving static views as screen shots.

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    This page titled Mechanism of DNA-mediated transposition is shared under a not declared license and was authored, remixed, and/or curated by Ross Hardison.

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