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Chapter 20 : Transposon Tn10

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Abstract:

This chapter summarizes one’s current understanding of the molecular mechanism of Tn/IS transposition and describes how double-strand cleavages are made in other complex systems including V(D)J recombination. Additional information regarding the precise role of integration host factor (IHF) in transpososome assembly has come from experiments conducted with use of the short, linear outside-end fragment assay in which transpososomes are easily detected. A mutational study on a particular insertion hotspot called HisG1 demonstrated that only the first 5 to 6 bp of the flanking sequence influenced target site utilization. Mutants of this class exhibit an altered target specificity (ATS) phenotype. Stereoselectivity was observed for each of the four chemical steps in Tn transposition. First, strand nicking, hairpin formation, hairpin resolution, and target strand transfer exhibited Rp, Sp, Rp, and Rp stereoselectivities, respectively. The stereoselectivity of hairpin resolution (Rp) has only been worked out in the Tn system. Finally, target strand transfer in Mu transposition and HIV integration exhibits Rp-Ps stereoselectivity. From the stereochemical model, predictions for the organization of DNA binding determinants in the active site can be made. In addition, in Tn transposition hairpin resolution must necessarily precede target strand transfer to expose a 3'-OH terminus for joining to the target DNA.

Citation: Haniford D. 2002. Transposon Tn10, p 457-483. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch20

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Figures

Image of Figure 1
Figure 1

Structure of TnThe direction of transcription is indicated by arrows. IS-Left encodes a truncated form of transposase that is nonfunctional. Half-arrows indicate nearly perfect inverted repeat sequences at the outside (O) and inside (I) ends of IS-Right. Binding sites for transposase and IHF are indicated by hatched and open rectangles, respectively. IS-Left and IS-Right flank, genes encoding resistance to tetracycline; jemA, a predicted sodium-dependent glutamate permease; jemB, unknown function. jemC has some homology to a family of bacterial transcriptional regulators that repress the arsenic and mercury resistance operons. This figure was adapted from ( ) with permission from the publisher.

Citation: Haniford D. 2002. Transposon Tn10, p 457-483. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch20
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Image of Figure 2
Figure 2

Mechanisms of DNA transposition. In nonreplicative transposition, as performed by Tn transposon sequences (thick lines) are completely separated from flanking donor sequences (thin lines) at an early stage of the reaction. In contrast, in replicative transposition, as conducted by bacteriophage Mu, the donor DNA remains attached to the transposon subsequent to strand transfer into the target DNA (dashed lines). In retroviral integration cleavage of the viral DNA generated by reverse transcription resembles DNA cleavage in the Mu reaction. Strand transfer in all three systems occurs by the same chemical mechanism.

Citation: Haniford D. 2002. Transposon Tn10, p 457-483. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch20
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Image of Figure 3
Figure 3

Nonreplicative transposition can generate different types of transposition products. In addition to the formation of simple inserts, Tn can generate both deletions and inversions as alternative reaction products; sites of insertion are indicated by a bubble not attached to a box. Inversions of the type shown generate new composite transposons, e.g., Tn-dcba; adapted from reference 74 with permission of the publisher.

Citation: Haniford D. 2002. Transposon Tn10, p 457-483. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch20
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Image of Figure 4
Figure 4

An alternate model for cointegrate and adjacent deletion formation through nonreplicative transposition. Synapsis of transposon ends present on sister chromatids can lead to the formation of products that are characteristic of replicative DNA transposition. If the target for insertion is on a separate DNA molecule from the transposon ends (i.e., intermolecular) a cointegrate can be formed. Alternatively, if the target is on the same molecule, either an adjacent deletion is formed or a duplicative inversion, depending on the particular orientation in which the transposon ends attack the target. Reprinted from ( ) with permission of the publisher.

Citation: Haniford D. 2002. Transposon Tn10, p 457-483. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch20
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Image of Figure 5
Figure 5

Chemical steps in selected double-strand DNA cleavage reactions. Note that first-strand cleavage reactions in Tn/ Tn and Tam1/VDJ recombination are on opposite strands, and this leads to a transposon end hairpin in the former and a donor flank hairpin in the latter. The second chemical step in IS/IS transposition resembles that in the reactions above in that it is a transesterification; however, unlike hairpin formation it is an intrastrand event. Adapted from ( ) with permission of the publisher.

Citation: Haniford D. 2002. Transposon Tn10, p 457-483. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch20
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Image of Figure 6
Figure 6

Transpososomes in Tn transposition. In vitro reactions using short, linear outside end fragments provide a convenient means of generating Tn transpososomes. Target capture of and strand transfer into a short linear target fragment are shown. Transpososome assembly does not require the presence of a divalent metal ion but reaction chemistry does. For simplicity transposase is shown bound to only one site on each transposon end fragment and only the equivalent of the b- PEC is shown. Also, the relative arrangement of the two transposon ends within each of the transpososomes is not known. PEC, paired ends complex; SEBC, single-end break complex; DEBC, double-end break complex; TCC, target capture complex; STC, strand transfer complex. Adapted from ( ) with permission of the publisher.

Citation: Haniford D. 2002. Transposon Tn10, p 457-483. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch20
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Image of Figure 7
Figure 7

Comparison of OP-Cu footprints for HisG1 and LacZ* Tn insertion hotspots. Positions of OP-Cu protections are indicated by black circles, and OP-Cu hypersensitivities are indicated by arrows. Black rectangles show the positions of the target core half-sites. Residues in the 9-bp target core are indicated in bold. The numbering scheme for both bases and phosphates is shown.

Citation: Haniford D. 2002. Transposon Tn10, p 457-483. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch20
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Image of Figure 8
Figure 8

Model for sequence-specific target capture. A transposase dimer is shown binding to the target core of a Tn insertion hotspot (left side). Subsequent to the initial contact with the core, the target DNA is shown to form two kinks, the formation of which allows a second region of transposase to contact flanking DNA (right side). Kink formation may be stabilized by the binding of a divalent metal ion to the DNA at the position of the kink and/or the establishment of the kink may be necessary to set up a high-affinity metal ion binding site. Kink formation may also activate the scissile phosphate for nucleophilic attack. It is also possible that the initial contact with the core alters the conformation of transposase so that it is better able to facilitate kink formation (not shown).

Citation: Haniford D. 2002. Transposon Tn10, p 457-483. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch20
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Image of Figure 9
Figure 9

Linear map of Tn transposase showing the relative positions of the major trypsin cleavage sites, the DDE motif, and positions of various classes of mutations. ATS, altered target site specificity mutations; SEM, suppressor of end mutations; TS, transpososome stability mutations. The YREK sequence is part of a highly conserved motif present in the IS family ( ) and a basic residue is usually situated between the E and K ( ). In Tn transposase this basic residue is R296, which is also the position of a TS mutation. α and β refer to different proteolytic fragments generated by trypsin digestion.

Citation: Haniford D. 2002. Transposon Tn10, p 457-483. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch20
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Image of Figure 10
Figure 10

Possible arrangement of DNA strands in the active site as predicted from the stereochemical model shown in Color Plate 40. See text for details.

Citation: Haniford D. 2002. Transposon Tn10, p 457-483. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch20
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