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

Abstract

BackgroundTransposition in IS3, IS30, IS21 and IS256 insertion sequence (IS) families utilizes an unconventional two-step pathway. A figure-of-eight intermediate in Step I, from asymmetric single-strand cleavage and joining reactions, is converted into a double-stranded minicircle whose junction (the abutted left and right ends) is the substrate for symmetrical transesterification attacks on target DNA in Step II, suggesting intrinsically different synaptic complexes (SC) for each step. Transposases of these ISs bind poorly to cognate DNA and comparative biophysical analyses of SC I and SC II have proven elusive. We have prepared a native, soluble, active, GFP-tagged fusion derivative of the IS2 transposase that creates fully formed complexes with single-end and minicircle junction (MCJ) substrates and used these successfully in hydroxyl radical footprinting experiments.ResultsIn 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.ConclusionsDifferent 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.

Evidence that the insertion events of IS2 transposition are biased towards abrupt compositional shifts in target DNA and modulated by a diverse set of culture parameters

“…This region in the gfp gene is also shown to adopt an intrinsically bent conformation (Fig. 3b) in line with previous findings from our group (Lewis et al 2012). The insertion produced a duplication of a 5 bp sequence (CCCCA) and resulted in the formation of a hybrid promoter with an outwardly directed −35 hexamer from IS2 and a resident −10 hexamer from gfp (Fig.…”

“…It was only recently that a GFPtagged approach was successfully used to obtain, under native conditions, preparations of full length, soluble, and active IS2 TPase that was usually insoluble when prepared under native conditions, and refractory to whole structure-function or biophysical studies when solubilized (Lewis et al 2011). This paved the way for generating hydroxyl radical footprinting data, which together with curvature propensity data obtained from several target regions, provided for the first time support for a structure-dependent mechanism for IS2 transposition (Lewis et al 2012). Both minicircle junction and IS2 target sites were shown to adopt similar intrinsically curved structures, supporting the idea that a particular configuration is needed to regulate transposition by promoting the assembly of the transpososome (Lewis et al 2012).…”

“…In this study, we present evidence that composition asymmetry directly affects the bendability and configuration of the target region, which in turn, likely modulates IS2 transposition in a way which we do not yet fully understand. Since both the IS minicircle junction and target sites adopt bent structures that share identical curvature profiles (Lewis et al 2012), it seems plausible that a similar conformation could facilitate the transposition process within the synaptic complex. Bending results in a compaction of the DNA structure that might contribute to an increased level of sequence discrimination and a proper alignment of the target DNA within the catalytic domains of the transpososome.…”

“…This paved the way for generating hydroxyl radical footprinting data, which together with curvature propensity data obtained from several target regions, provided for the first time support for a structure-dependent mechanism for IS2 transposition (Lewis et al 2012). Both minicircle junction and IS2 target sites were shown to adopt similar intrinsically curved structures, supporting the idea that a particular configuration is needed to regulate transposition by promoting the assembly of the transpososome (Lewis et al 2012). In fact, a growing body of evidence has yielded similar observations in other MGEs such as IS10 (Kobori et al 2009), IS231 (Hallet et al 1994), Tn10 (Pribil and Haniford 2003) , bacteriophage Mu, and Tn5 (reviewed in (Nagy and Chandler 2004)).…”

“…We have shown in a recent study that IS2 preferentially transposes into intrinsically curved regions (Lewis et al 2012). Because DNA composition asymmetries have been previously linked with transitions between rigid and flexible regions (Pedersen et al 1998), we sought to understand in more detail if a similar relation could exist and explain the intrinsic curvature observed at IS2 target sites.…”