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Chapter 8 : Topological Behavior of Plasmid DNA

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Abstract:

Chromosome topology is a fundamental property relevant to a wide range of biological processes including DNA replication, RNA transcription, genetic recombination, transposition, and DNA repair. One aim of this chapter is to summarize the understanding of plasmid topological behavior, and also point out experimental situations in which plasmid topology can be misinterpreted. In enteric bacteria, four distinct topoisomerases are able to change the linking status of plasmid DNA molecules. The known enzymes that alter linking number include Topo I, DNA gyrase, Topo III, and Topo IV. DNA gyrase and Topo IV are related enzymes that break both strands of DNA simultaneously and are classified as type II enzymes. Tests of all four enzymes indicate that Topo III does not normally contribute to the in vivo topology of plasmid DNA. Topo IV can remove negative supercoils from plasmid DNA in vivo, and topological balance inside living cells involves at least DNA gyrase, Topo I, and Topo IV. Intramolecular triplex DNA (H-DNA) may form at sequences containing long stretches of polypurine-polypyrimidine. In the H-form, half of either the purine- or pyrimidine-rich strand becomes unpaired and its complement becomes triple-stranded by forming Hoogsteen base pairs with purines in the major groove of the Watson-Crick base-paired segment. The ability to distinguish between constrained and unconstrained supercoiling is often necessary to fully explain topological changes that can be measured in plasmid DNA.

Citation: Patrick Higgins N, Vologodskii A. 2004. Topological Behavior of Plasmid DNA, p 193-202. In Funnell B, Phillips G (ed), Plasmid Biology. ASM Press, Washington, DC. doi: 10.1128/9781555817732.ch8

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Image of Figure 1
Figure 1

The simplest knots (a) and catenanes (b). DNA molecules are capable of adopting these and many more complex topological states.

Citation: Patrick Higgins N, Vologodskii A. 2004. Topological Behavior of Plasmid DNA, p 193-202. In Funnell B, Phillips G (ed), Plasmid Biology. ASM Press, Washington, DC. doi: 10.1128/9781555817732.ch8
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Image of Figure 2
Figure 2

Diagram of closed circular DNA. The linking number, , of the complementary strands is 20.

Citation: Patrick Higgins N, Vologodskii A. 2004. Topological Behavior of Plasmid DNA, p 193-202. In Funnell B, Phillips G (ed), Plasmid Biology. ASM Press, Washington, DC. doi: 10.1128/9781555817732.ch8
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Image of Figure 3
Figure 3

Typical simulated conformations of supercoiled DNA 4.4 kb in length. The conformations correspond to DNA superhelix density of −0.03 (a) and −0.06 (b). The simulations were performed for close to physiological ionic conditions.

Citation: Patrick Higgins N, Vologodskii A. 2004. Topological Behavior of Plasmid DNA, p 193-202. In Funnell B, Phillips G (ed), Plasmid Biology. ASM Press, Washington, DC. doi: 10.1128/9781555817732.ch8
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Figure 4

Gel electrophoretic separations of topoisomers of pUC19 DNA. The mixture of topoisomers covering the range of Δfrom 0 to −8 was electrophoresed from a single well in 1% agarose from top to bottom. The topoisomer with Δ= 0 has the lowest mobility; it moves slightly more slowly than the opened (nicked) circular DNA (OC). The value of (−Δ) for each topoisomer is shown.

Citation: Patrick Higgins N, Vologodskii A. 2004. Topological Behavior of Plasmid DNA, p 193-202. In Funnell B, Phillips G (ed), Plasmid Biology. ASM Press, Washington, DC. doi: 10.1128/9781555817732.ch8
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Image of Figure 5
Figure 5

Separation of pUC19 DNA topoisomers by two-dimensional gel electrophoresis. Topoisomers 1 to −4 have positive supercoiling; the rest have negative supercoiling. After electrophoresis was performed in the first direction, from top down, the gel was saturated with ligand intercalating into the double helix. Upon electrophoresis in the second direction, from left to right, the 12th and 13th topoisomers turned out to be relaxed. The spot in the top left corner corresponds to the open circular form (OC); the spot in the middle of the gel corresponds to the linear DNA (L).

Citation: Patrick Higgins N, Vologodskii A. 2004. Topological Behavior of Plasmid DNA, p 193-202. In Funnell B, Phillips G (ed), Plasmid Biology. ASM Press, Washington, DC. doi: 10.1128/9781555817732.ch8
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Figure 6

Resolution of knotted forms of plasmid DNA by high-resolution gel electrophoresis from top (left) to bottom (right). Knot types are described in Fig. 1 . Reprinted from reference , with permission from Elsevier.

Citation: Patrick Higgins N, Vologodskii A. 2004. Topological Behavior of Plasmid DNA, p 193-202. In Funnell B, Phillips G (ed), Plasmid Biology. ASM Press, Washington, DC. doi: 10.1128/9781555817732.ch8
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Image of Figure 7
Figure 7

Figure 7. Alternative DNA structures that are stabilized by negative superhelical energy.

Citation: Patrick Higgins N, Vologodskii A. 2004. Topological Behavior of Plasmid DNA, p 193-202. In Funnell B, Phillips G (ed), Plasmid Biology. ASM Press, Washington, DC. doi: 10.1128/9781555817732.ch8
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Figure 8

Two-dimensional gel showing the transition from B- to Z-DNA in plasmid DNA. Reprinted with permission from D. S. Kang and R. D. Wells ( ).

Citation: Patrick Higgins N, Vologodskii A. 2004. Topological Behavior of Plasmid DNA, p 193-202. In Funnell B, Phillips G (ed), Plasmid Biology. ASM Press, Washington, DC. doi: 10.1128/9781555817732.ch8
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Figure 9

Conversion of interwound negative supercoils into catenane links by site-specific recombination. EM reprinted from reference . © 2001 National Academy of Sciences, U.S.A

Citation: Patrick Higgins N, Vologodskii A. 2004. Topological Behavior of Plasmid DNA, p 193-202. In Funnell B, Phillips G (ed), Plasmid Biology. ASM Press, Washington, DC. doi: 10.1128/9781555817732.ch8
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Figure 10

Alternative RNA-DNA structures that could initiate formation of constrained supercoiling in a plasmid containing a fragment of the chicken IgA switch region during transcription with T-7 RNA polymerase (see reference ).

Citation: Patrick Higgins N, Vologodskii A. 2004. Topological Behavior of Plasmid DNA, p 193-202. In Funnell B, Phillips G (ed), Plasmid Biology. ASM Press, Washington, DC. doi: 10.1128/9781555817732.ch8
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Image of Figure 11
Figure 11

Replication intermediates identified in plasmid replication systems. (A) Replication initiated at a unique position leads to dual forks that move toward a terminus of replication. (B) Introduction of positive supercoiling leads to replication fork reversal and formation of a four-way junction. (C) Negative supercoiling, which can be generated by gyrase ahead of the fork, can be converted first into precatenanes (D), which become catenanes (E) upon completion of DNA synthesis. Topoisomerase activity in the replicated region can lead to knotted structures (F).

Citation: Patrick Higgins N, Vologodskii A. 2004. Topological Behavior of Plasmid DNA, p 193-202. In Funnell B, Phillips G (ed), Plasmid Biology. ASM Press, Washington, DC. doi: 10.1128/9781555817732.ch8
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Image of Figure 12
Figure 12

Replication fork reversal in vivo. Reprinted with permission from reference . Copyright 2003 AAAS.

Citation: Patrick Higgins N, Vologodskii A. 2004. Topological Behavior of Plasmid DNA, p 193-202. In Funnell B, Phillips G (ed), Plasmid Biology. ASM Press, Washington, DC. doi: 10.1128/9781555817732.ch8
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Image of Figure 13
Figure 13

Resolution of catenane (CATS) and precatenane links (RI) in plasmid DNA (see Fig. 5 ). Reprinted from reference with permission from Elsevier.

Citation: Patrick Higgins N, Vologodskii A. 2004. Topological Behavior of Plasmid DNA, p 193-202. In Funnell B, Phillips G (ed), Plasmid Biology. ASM Press, Washington, DC. doi: 10.1128/9781555817732.ch8
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Image of Figure 14
Figure 14

Knotting of replication bubbles in vivo. Reprinted from reference with permission from Blackwell.

Citation: Patrick Higgins N, Vologodskii A. 2004. Topological Behavior of Plasmid DNA, p 193-202. In Funnell B, Phillips G (ed), Plasmid Biology. ASM Press, Washington, DC. doi: 10.1128/9781555817732.ch8
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Tables

Generic image for table
Table 1

Constrained and unconstrained supercoiling in K12-derived plasmids

Citation: Patrick Higgins N, Vologodskii A. 2004. Topological Behavior of Plasmid DNA, p 193-202. In Funnell B, Phillips G (ed), Plasmid Biology. ASM Press, Washington, DC. doi: 10.1128/9781555817732.ch8
Generic image for table
Table 2

Nucleoid structural proteins

Citation: Patrick Higgins N, Vologodskii A. 2004. Topological Behavior of Plasmid DNA, p 193-202. In Funnell B, Phillips G (ed), Plasmid Biology. ASM Press, Washington, DC. doi: 10.1128/9781555817732.ch8

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