Dealing with the second strand. The color code is as follows: transposon DNA (green); flanking donor DNA (blue); target phosphates destined to be removed from the final liberated transposon (filled blue circles with a white “P”); phosphates destined to remain as 5′ transposon ends (open blue circles); the preferred stereoisomer, Sp or Rp, where known, is indicated within the circles; liberated 3′OH groups involved in strand joining reactions (open red circles); 3′OH destined to be removed from the liberated transposon (filled red circles); H2O is the attacking nucleophile in the hydrolysis reactions. (a) The Mu and Tn3 cleavage reactions. Note that the preferred stereoisomer has been demonstrated only for Mu and not for Tn3. (b) Tn7 cleavage reactions. Cleavage of the transferred strand (top of panel) is shown occurring prior to cleavage of the non-transferred strand (middle) leading to liberation of the transposon from flanking donor DNA (bottom of panel), although this order of cleavage reactions has not been demonstrated experimentally. The two types of cleavage are catalyzed by different enzymes. (c) Retroviral “processing” reaction, equivalent to cleavage of the transferred strand. An initial transcription step from the integrated provirus is indicated. The RNA genome is then encapsidated with a second copy and undergoes reverse transcription following infection to generate the double strand DNA integration intermediate. The intermediate is flanked by only short fragments of donor material and does not require second strand processing for insertion. (d) Transposition by the members of the IS630 family and the Tc1/Mariner superfamily is initiated by cleavage of the non-transferred strand (top of panel) at several bases within the transposon end (middle) leaving these bases attached to the liberated flanks following cleavage of the transferred strand (bottom). (e) For IS911, IS2, IS3 and other members of the IS3 family, single-end hydrolysis occurs (top). The liberated 3′OH then directs a strand transfer reaction to the same strand several bases 5′ to the other end of the element. This results in the formation of a single-strand circle which is then resolved into a transposon circle by replication from the free 3′OH (filled red circle). Single-strand hydrolysis at each 3′ end within the circle generates a linear transposon which can then undergo integration. (f ) The IS4 family and piggyBac have similar mechanisms. Following initial nucleophilic attack on the Rp target phosphate, the liberated 3′OH attacks an Sp phosphate in a trans-strand transfer reaction to generate a hairpin intermediate, liberating the transposon from its flanking donor DNA and inverting the target phosphate to its Rp configuration. These then become the substrates for a second hydrolysis. Note that the stereochemistry has been analyzed only in the case of Tn10. (g) Hermes and V(D)J transposition occur by initial cleavage of the non-transferred strand (top). The liberated 3′OH on the donor flank then attacks the opposite strand (middle) to generate hairpin structure on the donor flank (bottom). The stereochemistry has been analyzed for V(D)J only. Modified and reprinted from Turlan and Chandler (2000), with permission from Elsevier.