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Category: Clinical Microbiology
Topological Behavior of Plasmid DNA, Page 1 of 2
< Previous page | Next page > /docserver/preview/fulltext/10.1128/9781555818982/9781555818975_Chap07-1.gif /docserver/preview/fulltext/10.1128/9781555818982/9781555818975_Chap07-2.gifAbstract:
DNA topology is a critical factor in essentially all in vivo chromosomal processes, including DNA replication, RNA transcription, homologous recombination, site-specific recombination, DNA repair, and integration of the abundant and mechanistically distinct forms of transposable elements. Plasmids can be invaluable tools to define the dynamic mechanisms of proteins that shape DNA, organize chromosome structure, and channel chromosome movement inside living cells. The advantages of plasmids include their ease of isolation and the ability to quantitatively measure DNA knots, DNA catenation, hemi-catenation between two DNA molecules, and positive or negative supercoils in purified DNA populations. Under ideal conditions, in vitro and in vivo results can be compared to define the complex mechanism of enzymes that move along and change DNA chemistry in living cells. Many techniques that can be easily done with plasmids are not feasible for the massive chromosome that carries most of the genetic information in Escherichia coli or Salmonella typhimurium. Whereas a large fraction of contemporary chromosomal “philosophy” is based on extrapolation of results from small plasmids such as pBR322 to the 4.6-Mb bacterial chromosome, the comparison is not always valid. One aim of this article is to explain how results derived from small plasmids can be misleading for understanding and interpreting the DNA structure of the large bacterial chromosome.
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The simplest knots (a) and catenanes (b). DNA molecules are capable of adopting these and many more complex topological states.
Diagram of closed circular DNA. The linking number, Lk, of the complementary strands is 18.
Typical simulated conformations of supercoiled DNA 4.4 kb in length. The conformations correspond to a DNA superhelix density of (a) –0.030 and (b) –0.060.
Electrophoretic separation of topoisomers of pUC19 DNA. The mixture of topoisomers covering the range of ΔLk from 0 to –8 was electrophoresed from a single well in 1% agarose that was run from top to bottom. The topoisomer with ΔLk = 0 has the lowest mobility: it moves slightly slower than the open circular form (OC). The value of (–ΔLk) for each topoisomer is shown.
Separation of pUC19 DNA topoisomers by two-dimensional gel electrophoresis. Topoisomers 1 to 4 are positively supercoiled; the rest have negative supercoiling. After electrophoresis in the first direction, from top to bottom, the gel was saturated with ligand intercalating into the double helix. Upon electrophoresis from left to right in the second direction, the 2nd and 13th topoisomers turned out to migrate near the relaxed position in the second dimension. The spot in the top-left corner corresponds to the open circular form (OC); the spot near the middle of the gel corresponds to linear DNA (L).
High-resolution gel electrophoresis of knotted forms of plasmid DNA that was run from left to right. Knot types are described in Fig. 1 (see reference 21 ). Reproduced from the Journal of Molecular Biology with permission from Elsevier.
Alternative DNA structures that are stabilized by negative supercoiling.
Two-dimensional gel showing the transition from B- to left-handed Z-DNA in plasmid DNA. This research was originally published in Kang DS, Wells RD. 1985. B-Z DNA junctions contain few, if any, nonpaired bases at physiological superhelical densities. J Biol Chem 260:7783–7790. © the American Society for Biochemistry and Molecular Biology.
Conversion of interwound negative supercoils into catenanes linked by site-specific recombination. EM reprinted from Spergler SJ, Stasiak A, Cozzarelli NR. 1985. The stereostructure of knots and catenanes produced by phage lambda integrative recombination: implications for mechanism and DNA structure. Cell 42:325–334 with permission from Elsevier.
Alternative RNA-DNA structures that contribute to constrained supercoiling in a plasmid containing a fragment of the chicken IgA immunoglobulin switch region during transcription. The R-loop structure shown in (A) results in a displaced strand of DNA that constrains a ΔLk of about +1 for every 10 bp of RNA/DNA hybrid. (B) shows the structure of an intermolecular triplex in which Hoogsteen base pairing occurs in the major groove of the DNA strand (see reference 159 ).
Proposed mechanism for stable RNA-DNA hybrids that can stimulate repeat instability. Transcription of DNA regions containing CG-rich trinucleotide repeats (red) favors formation of stable RNA-DNA hybrids. The displaced nontemplate DNA strand can adopt non-B DNA structures, such as CTG or CAG hairpins. The unpaired regions of the nontemplate strand are reactive to bisulfite modification. Reprinted from reference 162 with permission from the National Academy of Sciences.
Replication intermediates identified in plasmid replication systems. (A) Replication initiated at a unique position leads to dual forms that move toward the terminus of replication. (B) Introduction of positive supercoils leads to replication fork reversal and formation of a four-way junction. (C) Negative supercoiling, which is generated by gyrase ahead of the fork, can be converted into precatenanes (D), which become catenanes (E) upon completion of DNA synthesis. (F, G) Topoisomerase activity in the replicated region can lead to complex knots.
Model of replication repair. Strand displacement and branch migration create an alternative replication template allowing replication to bypass a lesion (X). Reproduced from reference 174 with permission from Elsevier.
Replication fork reversal in vivo (see Fig. 12 ). Reprinted from reference 178 with permission from the American Association for the Advancement of Science.
Resolution of catenane (CATS) and precatenane links (RI) in plasmid DNA (see Fig. 12 ). Reprinted from reference 182 with permission from Elsevier.
Schematic models for generating hemicatenanes during DNA replication. Three pathways to yield hemicatenane structures are shown. (Left) Lagging strand synthesis encounters a damage site, and the pairing of the lagging strand with the complementary leading strand can produce a pseudo-double Holliday structure. Dissolution of the pseudo-double Holliday structure leads to hemicatenanes and allows replication to bypass the damage site. (Center) Convergence of two replication forks at the final stage of replication can lead to either a single-strand catenane or hemicatenane conjoining two replicated duplexes. Both single-strand catenanes and hemicatenanes can be resolved by a type IA topoisomerase, allowing the segregation of the daughter chromosomes. (Right). Convergent branch migration of a double Holliday junction can generate a hemicatenane. Reproduced from reference 183 with permission from the National Academy of Sciences.
Knotting of replication bubbles in vivo. Reprinted from reference 184 with permission from Wiley.
Constrained and unconstrained supercoiling in E. coli K12-derived strains a
Nucleoid-associated proteins a