Protein-DNA interactions define the mechanistic aspects of circle formation and insertion reactions in IS2 transposition

In IS2, Step I reactions are physically and chemically asymmetric; the left imperfect, inverted repeat (IRL), the exclusive recipient end, lacks donor function. In SC I, different protection patterns of the cleavage domains (CDs) of the right imperfect inverted repeat (IRR; extensive in cis) and IRL (selective in trans) at the single active cognate IRR catalytic center (CC) are related to their donor and recipient functions. In SC II, extensive binding of the IRL CD in trans and of the abutted IRR CD in cis at this CC represents the first phase of the complex. An MCJ substrate precleaved at the 3' end of IRR revealed a temporary transition state with the IRL CD disengaged from the protein. We propose that in SC II, sequential 3' cleavages at the bound abutted CDs trigger a conformational change, allowing the IRL CD to complex to its cognate CC, producing the second phase. Corroborating data from enhanced residues and curvature propensity plots suggest that CD to CD interactions in SC I and SC II require IRL to assume a bent structure, to facilitate binding in trans.Different transpososomes are assembled in each step of the IS2 transposition pathway. Recipient versus donor end functions of the IRL CD in SC I and SC II and the conformational change in SC II that produces the phase needed for symmetrical IRL and IRR donor attacks on target DNA highlight the differences.IS2, a 1.3 kb transposable element, is a member of the large and widespread IS3 family of insertion sequences (IS) ([1,2] see also ISfinder: http://www-is.biotoul.fr/is.html webcite). Transposition mechanisms in the IS3 family can be described as a two-step copy-and-paste process [3], in contrast to both classical cut-and-paste and replicative paradigms [4-6]. Although transposases of two IS3 family members, IS911 [7-9] and IS2 [10,11] were originally shown to facilitate transposition by catalyzing the two distinct reactions whose steps are shown in Figure 1A, there is strong evidence for the existence o