Non-replicative Transposition

Transposition of some transposable elements occurs by a non-replicative, "cut-and-paste" mechanism. First, the transposase makes a double-stranded cut in the donor DNA at the ends of the transposon and makes a staggered cut in the recipient DNA. Each end of the donor DNA is then joined to an overhanging end of the recipient DNA. DNA polymerase fills in the short, overhanging sequences, resulting in a short, direct repeat on each side of the transposon insertion in the recipient DNA. A cartoon of this process is shown below.

Why is non-replicative transposition hard to prove experimentally?

This process results in loss of the transposon from the donor DNA and insertion of the transposon into the recipient DNA. Unlike replicative transposon, the transposon is not "duplicated" in the process. However, because transposition often occurs after replication of the donor DNA molecule, it is not possible to determine if a transposon moves by a non-replicative mechanism by simply looking for loss of the transposon at the original site. For example, in the cartoon shown below the transposon moves by non-replicative transposition, but because of DNA repair or recombination a copy of the transposon is still observed in the original site.

Non-replicative transposition followed by either (a) repair or (b) loss of the donor strand.

Evidence that non-replicative transposition occurs in vivo.

(Bender, J., and N. Kleckner. 1986. Cell 45: 801-815)

Because DNA repair and recombination can obscure the simplest feature of non-replicative transposition, solid experimental evidence for this mechanism of transposition was difficult to obtain. The definative experiment proving this mechanism for transposon Tn10 was done by Bender and Kleckner. The experiment involves several steps:

  1. First they constructed two derivatives of Tn10 that carry the lac genes -- one Tn10 with the wild-type lacZ gene and one Tn10 with a mutated lacZ gene. Tn10 encodes tetracycline (Tet) resistance.
  2. Then these Tn10 insertions were separately recombined onto a strain of phage lambda with amber mutations in the N and P genes (which prevent lytic growth in a supo host) and a temperature-sensitive mutation in the cI gene (which prevents lysogeny at 37 C).
  3. DNA was purified from phage with the Tn10(lacZ+) and phage with the Tn10(lacZ-). This DNA was denatured, yielding single-stranded DNA that is homologous except for the mutations in the lacZ gene.
  4. The single-stranded DNA was then annealed. Annealing of the two different phage DNAs should yield approximately 1/4 homoduplex of lac+ phage DNA, 1/4 homoduplex of lac- phage DNA, and 1/2 heteroduplex of lac+/lac- phage DNA. The annealed DNA was then packaged into lambda phage particles.
  5. An E. coli del(lac) supo recipient was infected with the phage particles, and the infected cells were plated on medium with Tet and Xgal. Lac+ cells will form blue colonies and Lac- cells will form white colonies.

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Last modified October 15, 2003