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Alternative mechanisms for tn5 transposition.

Ahmed A - PLoS Genet. (2009)

Bottom Line: Bacterial transposons are known to move to new genomic sites using either a replicative or a conservative mechanism.The behavior of transposon Tn5 is anomalous.In vitro studies indicate that it uses a conservative mechanism while in vivo results point to a replicative mechanism.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada. asada@ualberta.ca

ABSTRACT
Bacterial transposons are known to move to new genomic sites using either a replicative or a conservative mechanism. The behavior of transposon Tn5 is anomalous. In vitro studies indicate that it uses a conservative mechanism while in vivo results point to a replicative mechanism. To explain this anomaly, a model is presented in which the two mechanisms are not independent--as widely believed--but could represent alternate outcomes of a common transpositional pathway.

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Related in: MedlinePlus

Steps in the formation of Tn5-promoted adjacent deletions.The plasmid p4.1 (A) carrying Tn5 was used for the selection of deletions conferring galactose-resistance (GalR). Using the replicative mechanism, Tn5 would be nicked at its termini to produce 3′ ends that would attack the target DNA sequence and join the 5′ ends from the same strand. This would result in the formation of a Shapiro intermediate containing replication forks at both ends of the transposon (B). After replication is completed, two deletion circles would be formed (C), only one of which would carry the origin of replication (ori) and survive. Thus a series of overlapping deletions starting from a fixed site at the right transposon terminus and extending to various sites in the gal region and beyond can be selected positively as GalR colonies. This has been the basis for the development of vectors for DNA sequencing [23]. The Shapiro intermediate can also be formed at individual IS elements (for instance, IS50L) to produce deletions extending from an inside end of the transposon. However, the majority (95%) of deletions in Tn5 start from the outside end. If Tn5 transposed solely by the conservative mechanism, both outside ends of the transposon would be cleaved by double-strand breaks; so, no viable deletion products would be formed after strand-transfer since the plasmid backbone would have been cut at the other end too. That such deletions are actually recovered in large numbers suggests that Tn5 can also utilize the replicative mechanism for its transposition. The plasmid, p6A.1, which carries Tn10 instead of Tn5, behaves in a different manner. It produced deletions solely from an inside end, and none from the outside end [6]. This behavior is to be expected since Tn10 uses the conservative mechanism, and double-strand cuts made at the outside ends would generate inviable deletion products. On the other hand, double-strand cuts made at the two inside ends of Tn10 would generate viable products. The 3′ ends from the inside ends would attack the target sequence and join 5′ ends from the same strand to produce two deletion circles, only one of which would carry ori and survive. This is actually found to be the case. (If the 3′ ends from the inside ends joined the 5′ ends from the opposite strand, the result would be a deletion-inversion as described in the legend to Figure 2.) Hence, the difference between the formation of transposon-promoted deletions and inversions is very narrow and depends on the topology of strand attacks: same-strand attacks produce two deletion circles; opposite-strand attacks produce an inversion circle [24].
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pgen-1000619-g003: Steps in the formation of Tn5-promoted adjacent deletions.The plasmid p4.1 (A) carrying Tn5 was used for the selection of deletions conferring galactose-resistance (GalR). Using the replicative mechanism, Tn5 would be nicked at its termini to produce 3′ ends that would attack the target DNA sequence and join the 5′ ends from the same strand. This would result in the formation of a Shapiro intermediate containing replication forks at both ends of the transposon (B). After replication is completed, two deletion circles would be formed (C), only one of which would carry the origin of replication (ori) and survive. Thus a series of overlapping deletions starting from a fixed site at the right transposon terminus and extending to various sites in the gal region and beyond can be selected positively as GalR colonies. This has been the basis for the development of vectors for DNA sequencing [23]. The Shapiro intermediate can also be formed at individual IS elements (for instance, IS50L) to produce deletions extending from an inside end of the transposon. However, the majority (95%) of deletions in Tn5 start from the outside end. If Tn5 transposed solely by the conservative mechanism, both outside ends of the transposon would be cleaved by double-strand breaks; so, no viable deletion products would be formed after strand-transfer since the plasmid backbone would have been cut at the other end too. That such deletions are actually recovered in large numbers suggests that Tn5 can also utilize the replicative mechanism for its transposition. The plasmid, p6A.1, which carries Tn10 instead of Tn5, behaves in a different manner. It produced deletions solely from an inside end, and none from the outside end [6]. This behavior is to be expected since Tn10 uses the conservative mechanism, and double-strand cuts made at the outside ends would generate inviable deletion products. On the other hand, double-strand cuts made at the two inside ends of Tn10 would generate viable products. The 3′ ends from the inside ends would attack the target sequence and join 5′ ends from the same strand to produce two deletion circles, only one of which would carry ori and survive. This is actually found to be the case. (If the 3′ ends from the inside ends joined the 5′ ends from the opposite strand, the result would be a deletion-inversion as described in the legend to Figure 2.) Hence, the difference between the formation of transposon-promoted deletions and inversions is very narrow and depends on the topology of strand attacks: same-strand attacks produce two deletion circles; opposite-strand attacks produce an inversion circle [24].

Mentions: Both processes start by nicking (short vertical arrows) of the transposon ends to expose the 3′-OH termini (A). At some point (see below), the target DNA is also cleaved to provide short protruding 5′-PO4 ends. In replicative transposition (left), strand-transfer takes place by joining the 3′ ends to 5′ ends of the target DNA in a concerted cleavage and joining reaction to form the “Shapiro intermediate” (B). As a result of replication of the intermediate, the donor and recipient replicons become fused to form a cointegrate (C) carrying one directly repeated copy of the transposon at each junction. Consequently, the cointegrate is an unstable structure that is resolved by recA-dependent generalized recombination (as in Tn5; A. Ahmed, unpublished results) or tnpR-specified site-specific recombination (as in Tn3 [22]). The donor and recipient replicons are thereby separated, each harboring one copy of the transposon (D). If the target DNA is located within the donor replicon itself (intramolecular transposition), maturation of the Shapiro intermediate produces a replicative inversion (as shown in Figure 2) or an adjacent deletion (Figure 3). This process is highly efficient in transposons like Mu and Tn3 [2],[9]. In conservative transposition (right), the 3′ ends engage in hairpin formation at both ends of the transposon (E) [7]. Following hairpin resolution (F), the free 3′ ends of the excised transposon are joined to 5′ ends from the target DNA (G), and the gaps are filled to complete the insertion process. The fate of the donor DNA containing a large gap (G) is not known: it could be degraded or undergo double-strand gap repair to regenerate the transposon sequence. This process is highly efficient in transposons like Tn10 [4],[8]. In Tn5, hairpin formation is not efficient (i.e., is leaky), so that a small proportion of the initial 3′ nicks remains free to engage in strand-transfer. Hence, the transposon displays properties of both conservative and replicative transposition concomitantly [5],[6]. These reactions are carried out by the respective transposases, which, by oligomerization, bring the end sequences of the transposon together to form a synaptic complex. For clarity, however, the transposon is shown as a straight line. The donor DNA sequence is shown in black, transposon DNA sequence is in red, and the recipient DNA sequence is in green. Replication and gap repair are indicated by dashed lines. The crossover event that resolves the cointegrate (C) is indicated by “x.”


Alternative mechanisms for tn5 transposition.

Ahmed A - PLoS Genet. (2009)

Steps in the formation of Tn5-promoted adjacent deletions.The plasmid p4.1 (A) carrying Tn5 was used for the selection of deletions conferring galactose-resistance (GalR). Using the replicative mechanism, Tn5 would be nicked at its termini to produce 3′ ends that would attack the target DNA sequence and join the 5′ ends from the same strand. This would result in the formation of a Shapiro intermediate containing replication forks at both ends of the transposon (B). After replication is completed, two deletion circles would be formed (C), only one of which would carry the origin of replication (ori) and survive. Thus a series of overlapping deletions starting from a fixed site at the right transposon terminus and extending to various sites in the gal region and beyond can be selected positively as GalR colonies. This has been the basis for the development of vectors for DNA sequencing [23]. The Shapiro intermediate can also be formed at individual IS elements (for instance, IS50L) to produce deletions extending from an inside end of the transposon. However, the majority (95%) of deletions in Tn5 start from the outside end. If Tn5 transposed solely by the conservative mechanism, both outside ends of the transposon would be cleaved by double-strand breaks; so, no viable deletion products would be formed after strand-transfer since the plasmid backbone would have been cut at the other end too. That such deletions are actually recovered in large numbers suggests that Tn5 can also utilize the replicative mechanism for its transposition. The plasmid, p6A.1, which carries Tn10 instead of Tn5, behaves in a different manner. It produced deletions solely from an inside end, and none from the outside end [6]. This behavior is to be expected since Tn10 uses the conservative mechanism, and double-strand cuts made at the outside ends would generate inviable deletion products. On the other hand, double-strand cuts made at the two inside ends of Tn10 would generate viable products. The 3′ ends from the inside ends would attack the target sequence and join 5′ ends from the same strand to produce two deletion circles, only one of which would carry ori and survive. This is actually found to be the case. (If the 3′ ends from the inside ends joined the 5′ ends from the opposite strand, the result would be a deletion-inversion as described in the legend to Figure 2.) Hence, the difference between the formation of transposon-promoted deletions and inversions is very narrow and depends on the topology of strand attacks: same-strand attacks produce two deletion circles; opposite-strand attacks produce an inversion circle [24].
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
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pgen-1000619-g003: Steps in the formation of Tn5-promoted adjacent deletions.The plasmid p4.1 (A) carrying Tn5 was used for the selection of deletions conferring galactose-resistance (GalR). Using the replicative mechanism, Tn5 would be nicked at its termini to produce 3′ ends that would attack the target DNA sequence and join the 5′ ends from the same strand. This would result in the formation of a Shapiro intermediate containing replication forks at both ends of the transposon (B). After replication is completed, two deletion circles would be formed (C), only one of which would carry the origin of replication (ori) and survive. Thus a series of overlapping deletions starting from a fixed site at the right transposon terminus and extending to various sites in the gal region and beyond can be selected positively as GalR colonies. This has been the basis for the development of vectors for DNA sequencing [23]. The Shapiro intermediate can also be formed at individual IS elements (for instance, IS50L) to produce deletions extending from an inside end of the transposon. However, the majority (95%) of deletions in Tn5 start from the outside end. If Tn5 transposed solely by the conservative mechanism, both outside ends of the transposon would be cleaved by double-strand breaks; so, no viable deletion products would be formed after strand-transfer since the plasmid backbone would have been cut at the other end too. That such deletions are actually recovered in large numbers suggests that Tn5 can also utilize the replicative mechanism for its transposition. The plasmid, p6A.1, which carries Tn10 instead of Tn5, behaves in a different manner. It produced deletions solely from an inside end, and none from the outside end [6]. This behavior is to be expected since Tn10 uses the conservative mechanism, and double-strand cuts made at the outside ends would generate inviable deletion products. On the other hand, double-strand cuts made at the two inside ends of Tn10 would generate viable products. The 3′ ends from the inside ends would attack the target sequence and join 5′ ends from the same strand to produce two deletion circles, only one of which would carry ori and survive. This is actually found to be the case. (If the 3′ ends from the inside ends joined the 5′ ends from the opposite strand, the result would be a deletion-inversion as described in the legend to Figure 2.) Hence, the difference between the formation of transposon-promoted deletions and inversions is very narrow and depends on the topology of strand attacks: same-strand attacks produce two deletion circles; opposite-strand attacks produce an inversion circle [24].
Mentions: Both processes start by nicking (short vertical arrows) of the transposon ends to expose the 3′-OH termini (A). At some point (see below), the target DNA is also cleaved to provide short protruding 5′-PO4 ends. In replicative transposition (left), strand-transfer takes place by joining the 3′ ends to 5′ ends of the target DNA in a concerted cleavage and joining reaction to form the “Shapiro intermediate” (B). As a result of replication of the intermediate, the donor and recipient replicons become fused to form a cointegrate (C) carrying one directly repeated copy of the transposon at each junction. Consequently, the cointegrate is an unstable structure that is resolved by recA-dependent generalized recombination (as in Tn5; A. Ahmed, unpublished results) or tnpR-specified site-specific recombination (as in Tn3 [22]). The donor and recipient replicons are thereby separated, each harboring one copy of the transposon (D). If the target DNA is located within the donor replicon itself (intramolecular transposition), maturation of the Shapiro intermediate produces a replicative inversion (as shown in Figure 2) or an adjacent deletion (Figure 3). This process is highly efficient in transposons like Mu and Tn3 [2],[9]. In conservative transposition (right), the 3′ ends engage in hairpin formation at both ends of the transposon (E) [7]. Following hairpin resolution (F), the free 3′ ends of the excised transposon are joined to 5′ ends from the target DNA (G), and the gaps are filled to complete the insertion process. The fate of the donor DNA containing a large gap (G) is not known: it could be degraded or undergo double-strand gap repair to regenerate the transposon sequence. This process is highly efficient in transposons like Tn10 [4],[8]. In Tn5, hairpin formation is not efficient (i.e., is leaky), so that a small proportion of the initial 3′ nicks remains free to engage in strand-transfer. Hence, the transposon displays properties of both conservative and replicative transposition concomitantly [5],[6]. These reactions are carried out by the respective transposases, which, by oligomerization, bring the end sequences of the transposon together to form a synaptic complex. For clarity, however, the transposon is shown as a straight line. The donor DNA sequence is shown in black, transposon DNA sequence is in red, and the recipient DNA sequence is in green. Replication and gap repair are indicated by dashed lines. The crossover event that resolves the cointegrate (C) is indicated by “x.”

Bottom Line: Bacterial transposons are known to move to new genomic sites using either a replicative or a conservative mechanism.The behavior of transposon Tn5 is anomalous.In vitro studies indicate that it uses a conservative mechanism while in vivo results point to a replicative mechanism.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada. asada@ualberta.ca

ABSTRACT
Bacterial transposons are known to move to new genomic sites using either a replicative or a conservative mechanism. The behavior of transposon Tn5 is anomalous. In vitro studies indicate that it uses a conservative mechanism while in vivo results point to a replicative mechanism. To explain this anomaly, a model is presented in which the two mechanisms are not independent--as widely believed--but could represent alternate outcomes of a common transpositional pathway.

Show MeSH
Related in: MedlinePlus