Increased production of a knotted form of plasmid pBR322 DNA in Escherichia coli DNA topoisomerase mutants

Shishido, Kazuo; Komiyama, Naoki; Ikawa, Shiro

doi:10.1016/0022-2836(87)90338-x

Cited by 72 publications

(67 citation statements)

References 10 publications

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“…By contrast, for simple trefoil knots, the curvature must be increased by supercoiling-induced tightening in order to stimulate Topo IV binding to knotted domains. Results obtained using living bacterial cells also support our proposal: Several studies report that inhibition of DNA gyrase, which is needed for DNA supercoiling, results in a greatly increased frequency of DNA knotting (28,29). To conclude, our simulations and experimental supporting evidence indicate strongly that supercoiling tightens DNA knots, which leads to their efficient unknotting by type IIA DNA topoisomerases.…”

Section: Discussionsupporting

confidence: 76%

Tightening of DNA knots by supercoiling facilitates their unknotting by type II DNA topoisomerases

Witz

Dietler

Stasiak

2011

Proc. Natl. Acad. Sci. U.S.A.

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Using numerical simulations, we compare properties of knotted DNA molecules that are either torsionally relaxed or supercoiled. We observe that DNA supercoiling tightens knotted portions of DNA molecules and accentuates the difference in curvature between knotted and unknotted regions. The increased curvature of knotted regions is expected to make them preferential substrates of type IIA topoisomerases because various earlier experiments have concluded that type IIA DNA topoisomerases preferentially interact with highly curved DNA regions. The supercoiling-induced tightening of DNA knots observed here shows that torsional tension in DNA may serve to expose DNA knots to the unknotting action of type IIA topoisomerases, and thus explains how these topoisomerases could maintain a low knotting equilibrium in vivo, even for long DNA molecules.Brownian dynamics | DNA topology | DNA structure | DNA configurations D uring normal cell growth, various DNA transactions are facilitated by topoisomerase-mediated passage of one DNA segment through another. These strand passages can inadvertently lead to the formation of DNA knots (1-3), which are highly deleterious for living cells if not removed efficiently (4, 5). Type IIA DNA topoisomerases can use the free energy of ATP hydrolysis (6) to decrease the knotting level significantly below the equilibrium value for DNA rings subject to random passages between colliding segments (7). This topoisomerase (topo) IIA ability has been often assumed to be a physiologically relevant mechanism ensuring efficient DNA unknotting and decatenation (7-10), but doubts on this relevance have been raised as well (11). Particularly problematic is the fact that the original study establishing that type IIA DNA topoisomerases actively unknot DNA also revealed that this ability decreases sharply with increasing DNA length. Although based on only two plasmid sizes, that study showed the generality of this length dependence for several topoisomerases (7) and, therefore, active unknotting would be doomed to be ineffective for long DNA molecules, where random passages lead to very frequent knotting (12). While trying to understand why earlier experimental studies predicted ineffectiveness of type IIA DNA topoisomerases in unknotting of long DNA molecules, we recognized that these studies were performed using torsionally relaxed DNA (7), whereas naturally occurring DNA is frequently under torsional tension (13), the best-known examples of this state being negatively supercoiled bacterial plasmids. To investigate the potential effects of torsional constraints on the activity of type IIA topoisomerases, we have performed Brownian dynamics simulations to examine how negative supercoiling affects the structure of knotted DNA molecules and whether DNA supercoiling can cause knotted regions to be recognized specifically and then unknotted by topoisomerases. We observed that DNA supercoiling results in the tightening of knots, which leads directly to an increase in curvature of the knotted regions, in turn re...

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Section: Discussionsupporting

confidence: 76%

Tightening of DNA knots by supercoiling facilitates their unknotting by type II DNA topoisomerases

Witz

Dietler

Stasiak

2011

Proc. Natl. Acad. Sci. U.S.A.

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“…Significant numbers of DNA knots are found also in biological systems: in Escherichia coli cells harboring mutations in the GyrB or GyrA genes (9), bacteriophages P2 and P4 (10,11), and cauliflower mosaic viruses (12). However, very little information about these systems has been inferred from the observed knots.…”

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confidence: 96%

Knotting probability of DNA molecules confined in restricted volumes: DNA knotting in phage capsids

Arsuaga

Vázquez

Trigueros

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

245

305

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When linear double-stranded DNA is packed inside bacteriophage capsids, it becomes highly compacted. However, the phage is believed to be fully effective only if the DNA is not entangled. Nevertheless, when DNA is extracted from a tailless mutant of the P4 phage, DNA is found to be cyclic and knotted (probability of 0.95). The knot spectrum is very complex, and most of the knots have a large number of crossings. We quantified the frequency and crossing numbers of these knots and concluded that, for the P4 tailless mutant, at least half the knotted molecules are formed while the DNA is still inside the viral capsid rather than during extraction. To analyze the origin of the knots formed inside the capsid, we compared our experimental results to Monte Carlo simulations of random knotting of equilateral polygons in confined volumes. These simulations showed that confinement of closed chains to tightly restricted volumes results in high knotting probabilities and the formation of knots with large crossing numbers. We conclude that the formation of the knots inside the viral capsid is driven mainly by the effects of confinement.

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“…Using a variety of enzymes, knotted DNA have even been generated in a test tube [18]. They form in vivo in non-replicating cells [19][20][21] and during replication, see e.g. [22,23].…”

Section: Introductionmentioning

confidence: 99%

Sedimentation of knotted polymers

et al. 2013

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We investigate the sedimentation of knotted polymers by means of stochastic rotation dynamics, a molecular dynamics algorithm that takes hydrodynamics fully into account. We show that the sedimentation coefficient s, related to the terminal velocity of the knotted polymers, increases linearly with the average crossing number nc of the corresponding ideal knot. To the best of our knowledge, this provides the first direct computational confirmation of this relation, postulated on the basis of experiments in Ref.[1], for the case of sedimentation. Such a relation was previously shown to hold with simulations for knot electrophoresis. We also show that there is an accurate linear dependence of s on the inverse of the radius of gyration R −1 g , more specifically with the inverse of the Rg component that is perpendicular to the direction along which the polymer sediments. When the polymer sediments in a slab, the walls affect the results appreciably. However, R −1 g remains to a good precision linearly dependent on nc. Therefore, R −1 g is a good measure of a knot's complexity.

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Increased production of a knotted form of plasmid pBR322 DNA in Escherichia coli DNA topoisomerase mutants

Cited by 72 publications

References 10 publications

Tightening of DNA knots by supercoiling facilitates their unknotting by type II DNA topoisomerases

Tightening of DNA knots by supercoiling facilitates their unknotting by type II DNA topoisomerases

Knotting probability of DNA molecules confined in restricted volumes: DNA knotting in phage capsids

Sedimentation of knotted polymers

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