Research

Protein-DNA Interactions Define the Mechanistic Aspects of Circle formation and Insertion Reactions in IS2 Transposition

Leslie A Lewis, Mekbib Astatke, Peter T Umekubo, Shaheen Alvi, Robert Saby, Jehan Afrose, Pedro H Oliveira, Gabriel A Monteiro and Duarte M. F. Prazeres

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Mobile DNA 2012, 3:1 doi:10.1186/1759-8753-3-1

Published: 26 January 2012

Abstract (provisional)

Background

Transposition in the IS3, IS30, IS21 and IS256 insertion sequence(IS) families utilizes an unconventional 2-step pathway. The Step I figure-of-eight intermediate, produced from asymmetric single strand cleavage and joining circularization reactions, is converted into a double stranded minicircle; its junction, the MCJ,[abutted left(IRL)and right(IRR)ends] is the substrate for symmetrical transesterification attacks on target DNA in Step II. This suggests intrinsically different synaptic complexes (SC) for each step. Because transposases of these ISs bind poorly to cognate DNA, comparative biophysical analyses of SC I and SC II have proven elusive. Here we utilize successfully, a native, soluble, active, GFP-tagged fusion derivative of the IS2 transposase that creates fully formed complexes with single-end and MCJ substrates, in hydroxyl radical footprinting experiments.

Results

IS2 Step I reactions are physically and biochemically asymmetric with IRL the recipient end, lacking donor function. In SC I, different protection patterns of the cleavage domains (CDs) of IRR (extensive in cis) and IRL (selective in trans) at one active catalytic center (the IRR CC), are related to their donor and recipient functions. In SC II, an MCJ substrate showed extensive protection of both CDs, IRL in trans and the abutted IRR CD in cis - 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 of the abutted CDs bound at the same CC, trigger a conformational change, allowing the IRL CD to complex to its cognate CC - 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.

Conclusions

Different transpososomes are assembled in each step of the IS2 transposition pathway. Recipient versus donor functions of the IRL CD in SC I and SC II and the proposed conformational change in SC II that precedes the symmetrical IRL and IRR donor attacks on target DNA, highlight the differences.