Control of IS911 target selection: How OrfA may ensure IS dispersion

Institut Pasteur, Lutetia Parisorum, Île-de-France, France
Molecular Microbiology (Impact Factor: 4.42). 04/2007; 63(6):1701-9. DOI: 10.1111/j.1365-2958.2007.05615.x
Source: PubMed


IS911 transposition involves a closed circular insertion sequence intermediate (IS-circle) and two IS-encoded proteins: the transposase OrfAB and OrfA which regulates IS911 insertion. OrfAB alone promotes insertion preferentially next to DNA sequences resembling IS911 ends while the addition of OrfA strongly stimulates insertion principally into DNA targets devoid of the IS911 end sequences. OrfAB shares its N-terminal region with OrfA. This includes a helix-turn-helix (HTH) motif and the first three of four heptads of a leucine zipper (LZ). OrfAB binds specifically to IS911 ends via its HTH whereas OrfA does not. We show here: that OrfA binds DNA non-specifically and that this requires the HTH; that OrfA LZ is required for its multimerization; and that both motifs are essential for OrfA activity. We propose that these OrfA properties are required to assemble a nucleoprotein complex committed to random IS911 insertion. This control of IS911 insertion activity by OrfA in this way would assure its dispersion.

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Available from: Catherine Guynet, Oct 22, 2014
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    • "Instead, it has been shown to modify the stoichiometry of complexes formed with the 1-149 truncated forms of OrfAB [26]. In addition, in IS911 OrfA is involved with both heteromultimerization with OrfAB [41], as well as with its own homomultimerization and with the ability to stimulate minicircle insertion in vitro into target DNA not associated with the IS911 ends [28]. It is likely that these heteromultimers may also exist in our preparations, which consist of a mixture of OrfA and OrfAB [31]. "
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    ABSTRACT: Transposition 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. 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.
    Full-text · Article · Jan 2012 · Mobile DNA
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    • "OrfA does not bind IS911 ends but can form heteromultimers with OrfAB (Haren et al., 2000). It is not required but stimulates integration of the circular IS911 transposition intermediate (Rousseau et al., 2007; Ton-Hoang et al., 1997). To allow us to focus directly on OrfAB activity alone we abolished OrfA expression by eliminating the requirement for frameshifting in OrfAB expression. "
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    ABSTRACT: Transposable elements are important in genome dynamics and evolution. Bacterial insertion sequences (IS) constitute a major group in number and impact. Understanding their role in shaping genomes requires knowledge of how their transposition activity is regulated and interfaced with the host cell. One IS regulatory phenomenon is a preference of their transposases (Tpases) for action on the element from which they are expressed (cis) rather than on other copies of the same element (trans). Using IS911, we show in vivo that activity in cis was ~200 fold higher than in trans. We also demonstrate that a translational frameshifting pause signal influences cis preference presumably by facilitating sequential folding and cotranslational binding of the Tpase. In vitro, IS911 Tpase bound IS ends during translation but not after complete translation. Cotranslational binding of nascent Tpase permits tight control of IS proliferation providing a mechanistic explanation for cis regulation of transposition involving an unexpected partner, the ribosome.
    Preview · Article · Dec 2011 · Molecular cell
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    • "Arrows represent strand cleavage and transfer reactions that take place within the synaptic complex (SCA) or paired end complex (PEC). IS911 insertion is drawn as a nontargeted pathway [16,31,32]. In the synaptic complex B (SCB), proteins are represented as grey ellipses without implying OrfAB stoichiometry or the presence or absence of OrfA in the complex. "
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