General Information/Influence of transposition mechanisms on genome impact

The way in which strand cleavages and transfers occur during transposition also affects the outcome of the transposition events and therefore impinges on genome structure. For IS with DDE Tpases, Tn3 and IS6 family members generate fusions or cointegrates between the donor and target replicons by a process of replicative transposition, presumably by Target Primed Replicative Transposition (TPRT) (Fig.17.1 and Fig.17.2 top).

However, in the event of intramolecular transposition, this type of mechanism is expected to give rise to inversions with a copy of the IS at each junction or inversions with a single IS copy remaining and a second copy segregating with a circularized deletion (Fig.17.2 botton; see ). Note that similar effects are also known to occur by homologous recombination between two inverted or directly repeated IS copies in a replicon. Other known mechanisms such as cut-and-paste, or copy-and-paste (Donor Primed Replicatice Transposition; DPRT) would not generate this type of genomic rearrangement but could contribute to genomic modifications in other ways such as “nearly precise excision” or by using alternative sequences which resemble their IR.

Fig.17.1. Replicative Transposition (DDE) of TE such as Tn3 and bacteriophage Mu. The figure shows the transposition mechanism of replicative TE that uses a DDE Tpase. The transposon is represented as a yellow line. Flanking sequences in the donor molecule is blue. Flanking sequences in the target molecule are green. Red circles indicate 3′OH moieties generated by Tpase-catalyzed hydrolysis at the transposon end(s). Red boxes indicate target DNA flanks that are duplicated on insertion. Top to bottom: Tpase catalyzed cleavage at the 3′ transposon ends using H2O as the nucleophile. Liberated 3′OH attack the target in a staggered manner to create a branched molecule in which the transposon bridges both donor and target molecules. Replication proceeds, probably using the 3′OH liberated in the flanking target DNA, to generate a second transposon copy (newly replicated DNA is shown as a dotted orange line). If the donor and target DNA are circular molecules, this fuses the two, resulting in a cointegrate where the donor and target DNA are joined at each junction by a TE copy. [Not shown Recombination between the two directly repeated TE ‘resolves’ the cointegrate into the original donor molecule and a target that now contains a copy of the TE. Recombination may use the host homologous recombination system but in the case of the Tn3 family, TnpR, one of several alternative site-specific recombinases, promotes recombination at a specific DNA sequence, the res site.]

Fig.17.2. Inter- and Intra-molecular replicative transposition.Top. Inter-molecular transposition. Cleavage at both TIRs of the IS results in nicks on both strands, generating 3-OH groups that attack the target site, leading to the formation of a Shapiro intermediate. DNA replication generates the cointegrate containing a duplication of the IS and the target site. The cointegrate can be subsequently resolved into a plasmid identical to the original donor plasmid and a modified target plasmid carrying a copy of the IS flanked by TSDs arranged as direct repeats. Bottom. Intra-molecular transposition. When an IS targets a target site in the same replicon, cleavages at both TIRs generate 3=-OH groups that can either attack the target site on the same strand (cis) or the opposite strand (trans). In the cis pathway, DNA between the IS and target site (dashed lines) becomes circularized and contains one IS copy and target site. In the trans pathway, DNA between IS and the target site is instead inverted (“a b” becomes “b a”), bracketed by the original IS, and a new copy in an inverted orientation. The target site is also duplicated but in an inverted orientation, and each TSD is associated with one IS copy.

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