Although replicative and conservative transpositions seem quite different, the actual mechanisms of the transposase steps are closely related. In both cases the target sequence is opened by a staggered cut (Fig. 15.09). In both cases, the transposase cuts at the junction between the ends of the transposon and the host DNA. However, in conservative transposition, both strands are cut whereas in replicative transposition only one strand is cut. In either case, the free 3'-ends of the transposon are joined to the 5'-ends of the opened target sequence. This sequence of events moves a conservative transposon to its new position while it creates a cointegrate in the case of the complex transposon.
The next step is again very similar. The host cell enzymes fill in the single-stranded regions using the free 3'-ends of the opened target sequence as primers. In conservative transposition the new DNA is merely a handful of nucleotides and this step just duplicates the target sequence. In the case of replicative transposition, the single-stranded regions are longer and this step duplicates the transposon itself.
This similarity is illustrated by the transposon Tn7, which normally operates by the conservative mechanism. Tn7 is unusual in having a transposase consisting of two proteins. TnsA makes single-stranded nicks at the 5'-ends of Tn7, and TnsB carries out the nicking and joining at the 3'-ends of Tn7, therefore, TnsA and TnsB create a double-stranded cut when both are expressed. Mutants of Tn7 exist that have a defective TnsA protein and no longer cut the 5'-strand. However, TnsB continues to cut and rejoin the 3'-strand, forming cointegrates as in replicative transposition. Therefore TnsB resembles the transposase of complex transposons. TnsA protein, which cuts Tn7 free of its original site, has a structure similar to a type II restriction endonuclease (see Ch. 22).
Was this article helpful?