Crucial Role for DNA Supercoiling in Mu Transposition

DNA supercoiling plays an indispensable role in an early step of bacteriophage Mu transposition. This step involves formation of a nucleoprotein complex in which the Mu ends synapse and undergo two concerted single-strand cleavages. We describe a kinetic analysis of the role of supercoiling in the Mu-end synapsis reaction as measured by the cleavage assay. We observe a dependence of the reaction rate on superhelical density as well as on the length of Mu donor plasmid DNA. The reaction has a high activation enthalpy (67 kcal/mol). These results imply that the free energy of supercoiling is used directly to lower the activation barrier of the rate-limiting step of the reaction. Only the free energy of supercoiling associated with DNA outside the Mu ends appears to be utilized, implying that the Mu ends come together before the supercoiling energy is used. Our results suggest an essential function for the bacterial sequences attached to the ends of Mu virion DNA.

http://www.pnas.org/cgi/content/abstract/91/2/699

Supercoiling

What’s knotty about DNA? Under an electron microscope DNA looks like a long thin "knotted" strand that is tightly packed inside the cell nucleus. To visualize this, imagine packing 200 km of fishing line inside a basketball without tangling it! Amazingly, the cells in your body do the equivalent of this by supercoiling the DNA. Supercoiling is a very smart form of compact storage that allows for easy manipulation.

To illustrate supercoiling, take a long elastic band, cut it, hold one end tight and twist the other end as many times as possible (about 100 times!). Now without untwisting the elastic band, bring the ends together. You will end up with a supercoiled band, see diagram. When you bring the two end pieces together the elastic band tries to unwind, by untwisting about the centreline. However, this is not possible because you are still holding the ends, so it compromises by writhing around in space (like a well used phone cord).

The mathematical formula Lk=Tw+Wr can be used to describe this process. Lk, the linking number, represents the number of times one strand winds around the other, Tw is the twist or the amount of rotation about the centre line and Wr, the writhe, describes how hard it is to straighten out the curve. When the curve is straightened out the writhe, Wr, is zero and the twist, Tw, is high. You can feel the elastic band trying to untwist. When the elastic band is relaxed it supercoils. The twist Tw is now very small and the writhe Wr is high.

Supercoiling allows for easy manipulation and so easy access to the information coded in the DNA. When a cell is copying a DNA strand it will uncoil a strand, copy it and then recoil it. In order to obtain a more workable interpretation of the stresses in the DNA, David Stump and Peter Watson in the Mathematics Department of the University of Queensland have obtained mathematical formulas for the Twist and Writhe depending on the length of the strand and the angle beta (see figure). This then gives (through the formula above) the Linking number or the number of times one strand winds around the other.These results can then be used to explain the pictures, taken by an electron microscope, of the tiny strands of DNA coiling and uncoiling

http://www.maths.uq.edu.au/~infinity/Infinity7/supercoiling.html

DNA Supercoiling as a Pattern for Understanding Psycho-social Twistedness

The review here of twistedness in DNA provides a technical basis for the discussion in the main paper (Engaging with Questions of Higher Order: cognitive vigilance required for higher degrees of twistedness, 2004).

The insights in the main paper regarding "twistedness" reflect an intuitive understanding of complexity which calls for deeper insight to understand how twistedness works and why it may be vitally important in some psycho-social processes -- as well as being highly problematic in others. Part of the difficulty in approaching this matter is that "twistedness" is in most cases used unthinkingly as a pejorative term to characterize a pattern which is felt to inhibit right-thinking and clarity. The argument here is that, given its importance at every scale in nature, from the organization of nebula to the organization of the human cell, there is a case for distinguishing various forms of twistedness and understanding their function. This could be especially valuable to reconciling apparently irreconcilable understandings in society.

The merit of focusing on the nature and function of twisting in DNA is that it provides a rich natural template. It offers a sense of the degree of complexity that it may be required to master in order to comprehend how twistedness "works" in practice. It might also be argued that, as a process active in every human body and inherent to human life, humans may well have some kind of profound intuitive understanding of how it works and the "rightness" of such working. Some of the very explicit dynamics of this process may also offer patterns for understanding how the inhibiting effects of "twistedness" may be addressed when they are perceived to be a constraint on human development.

Understanding of how DNA works has been much enriched by concepts from topology -- as a branch of mathematics that deals with structural properties that are unchanged by deformations such as stretching and bending. This use of mathematics is especially important because there is no experimental way to observe the dynamics of enzymatic action directly, notably with respect to knotting and coiling of DNA (see De Witt Sumners. Lifting the Curtain: Using Topology to Probe the Hidden Action of Enzymes, 1995; Xiaoyan R. Bao, et al. Behavior of Complex Knots in Single DNA Molecules, 2003).

http://www.laetusinpraesens.org/docs00s/dnahelix.php are very long and thin. There is over a metre of DNA in every human cell in a space of some 0.0006 centimetres diametre. If DNA were constrained to be linear it would not fit into a cell. It must therefore fold many times to fit within the confines of a cell. The DNA is composed of 10** base pairs. This density of packing results in tangles and knots in the DNA that are essential to enable the cell to divide (involving transcription and replication).

http://www.laetusinpraesens.org/docs00s/dnahelix.php

DNA supercoil

In a "relaxed" double-helical segment of DNA, the two strands twist around the helical axis once every 10.4 base pairs of sequence. Adding or subtracting twists, as some enzymes can do, imposes strain. If a DNA segment under twist strain were to be closed into a circle by joining its two ends and then it is allowed to move freely, the circular DNA would contort into new shape, such as a simple figure-eight. Such a contortion is a supercoil.

The simple figure eight is the simplest supercoil, and is the shape a circular DNA assumes to accommodate one too many or one too few helical twists. The two lobes of the figure eight will appear rotated either clockwise or counterclockwise with respect to one another, depending on whether the helix is over or underwound. For each additional helical twist being accommodated, the lobes will show one more rotation about their axis.

The noun form "supercoil" is rarely used in the context of DNA topology. Instead, global contortions of a circular DNA, such as the rotation of the figure-eight lobes above, are referred to as writhe. The above example illustrates that twist and writhe are interconvertible. "Supercoiling" is an abstract mathematical property, and represents the sum of twist and writhe. The twist is the number of helical turns in the DNA and the writhe is the number of times the double helix crosses over on itself (these are the supercoils). The relationship of twist, writhe and supercoiling is expressed as the equation:

S = T + W.

Extra helical twists are positive and lead to positive supercoiling, while subtractive twisting causes negative supercoiling. Many topoisomerase enzymes sense supercoiling and either generate or dissipate it as they change DNA topology. DNA of most organisms is negatively supercoiled.

In part because chromosomes may be very large, segments in the middle may act as if their ends are anchored. As a result, they may be unable to distribute excess twist to the rest of the chromosome or to absorb twist to recover from underwinding--the segments may become supercoiled, in other words. In response to supercoiling, they will assume an amount of writhe, just as if their ends were joined.

Supercoiled DNA forms two structures; a plectoneme or a toroid, or a combination of both. A negatively supercoiled DNA molecule will produce either a one-start left-handed helix, the toroid, or a two-start right-handed helix with terminal loops, the plectoneme. Plectonemes are typically more common in nature, and this is the shape most bacterial plasmids will take. For larger molecules it is common for hybrid structures to form - a loop on a toroid can extend into a plectoneme. If all the loops on a toroid extend then it becomes a branch point in the plectonemic structure.

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Supercoiled structure of circular DNA molecules with low writhe. Note that the helical nature of the DNA duplex is omitted for clarity.


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Supercoiled structure of linear DNA molecules with constrained ends. Note that the helical nature of the DNA duplex is omitted for clarity


http://en.wikipedia.org/wiki/DNA_supercoil

DNA supercoiling

Varying levels of positive and negative supercoiling differently affect the efficiency with which topoisomerase II catenates and decatenates DNA

Type II DNA topoisomerases catalyze the transport of one DNA double helix through another. Here, by using a non-hydrolyzable analog of ATP, I examined the single-step DNA transport preferences of the yeast type II topoisomerase bound to positively and negatively supercoiled DNA rings. I found that negative supercoiling favors decatenation of DNA rings more than positive supercoiling. Conversely, positive supercoiling favors the catenation and knotting of DNA rings more than negative supercoiling. This vectorial effect of DNA supercoiling handedness supports a model in which type II topoisomerases can recognize three DNA segments, and highlights a novel influence of DNA supercoiling in global DNA topology.

http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WK7-457D7V9-6&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=c0fd136de40dfc9683a0e8e90f21d368