MadSci Network: Molecular Biology

Re: what are the changes occur in the structure of supercoiled plasmid DNA?

Date: Wed Feb 27 15:23:29 2002
Posted By: Chris Larson, Research Scientist
Area of science: Molecular Biology
ID: 1014176024.Mb


Supercoiling of DNA is a very difficult concept to understand and work with beyond simply repeating what you have read somewhere, so I understand your frustration. I have never felt totally comfortable with it myself even after years of school and research experience. I will try to give you a good analogy for what it means. Imagine that you had a rubber band that was wider in one direction than the other (in other words, that looked like a ribbon joined into a circle). If you were to cut that rubber band or ribbon, lay it out flat on your desk, hold one end down, twist the other end (like turning a screwdriver) several times, and then somehow reattach the two ends to each other, you would have a perfect model of a piece of circular DNA (a circle made of a ribbon that used to be straight and flat, and was twisted like a screwdriver several times before the ends were attached to make a circle).

Now, remember that normal DNA is two strands annealed to each other (think of a ladder in which the two sides are the backbone and the base pairs are the steps). Normal DNA exists as a right-handed double helix (in other words, imagine holding one end of the ladder fixed and turning the other end in a clockwise direction, exactly the same way in which you would tighten a screw with a screwdriver). To get a another good picture of a piece of circular DNA, think of a very long ladder in which one end is held fixed, the other end is twisted clockwise like a screwdriver, and then the two ends are somehow attached to each other such that the twisting is maintained.

To get supercoiling, what you would do now is take this circular ribbon or ladder (that was already twisted into a helical structure before the two ends were joined to make a circle) and lengthen it in one direction to make what would look like a very tall and thin zero or letter O. Then you would hold one of the ends of this long, thin zero/O/loop fixed and twist the other end of the loop like a screwdriver (of course you have to hold it in place with your hands, otherwise it will fly apart because it is under tension). If you twisted it in one direction (for now it doesn't matter which) it would be negative supercoiling; if you twisted it in the other direction it would be called positive supercoiling. Now you can see why it is called SUPERcoiling: the ladder or ribbon that was twisted into a helix before the ends were joined to make a circle is now twisted AGAIN once (the first twisting is coiling, and the second twisting is supercoiling).

Now if (in your mind) you switch that rubber band or ribbon or ladder into a DNA helix, you have a piece of supercoiled DNA. What "negative supercoiling" means is that the DNA/ladder circular loop is twisted in the opposite screwdriver direction than were the ends of the linear helix/ladder/ribbon/rubber band before they were joined to make a circle.

You can imagine that, once the circular loop of ribbon/ladder/DNA is twisted for that second time, it is under a lot of twisting strain. One way that DNA relieves this strain is to reduce the degree of rotation/winding for each base pair/step. This easiest way to think about this is the ribbon/ladder analogy. You had a linear ribbon and you twisted one end in one screwdriver direction before joining the ends to make a circle; then you introduce negative supercoiling by flattening the circle into an elongated loop and twisting one of the loop in the OPPOSITE screwdriver direction as you did before you joined the ends of the ribbon. The ribbon is under a lot of torsional strain from the second act of twisting, but since the two acts of twisting were in opposite directions it can reduce the strain from the second by slightly reducing the degree of twist that was introduced by the first (the twistings are going in opposite directions and so can "meet in the middle"). I don't know how else to explain this except to say visualize a ribbon going through these 2 steps and it is usually pretty obvious what I mean.

Introduction of positive supercoils means that something reduced the number of twists that were applied in the second act of twisting (when the ends of the flattened, elongated circle were twisted relative to each other in what is called the negative direction).

Take as a given the ethidium bromide (EtBr) can somehow "introduction of positive supercoils;" how does this work? I can't give you a direct answer in case this is classwork, but I can lead you in the right direction. There are 3 things to think about. First, EtBr binds to DNA by inserting itself between 2 base pairs and partially disrupting their interactions with each other. Second, think back to the original twist, the twisting of the ends of a linear ribbon or ladder before it is joined into a circle. Essentially the number of twists that there are reflects the small amount of rotation of one base pair relative to the next one in the chain, and this small amount of rotation between one base pair and the next is caused by the degree of interaction between these two base pairs (which EtBr partially disrupts when it inserts between them...). And third, the second act of twisting, that of the two ends of the flatten loop of ribbon/ladder/DNA, is held in place partially by the binding of proteins called nucleosomes to the circle, and EtBr reduces this protein binding when it inserts into the DNA.

That should help you understand the structural changes in DNA upon supercoiling. The changes in electrophoretic mobility (how fast it runs in a gel) are basically empirical. Again I canít tell you the answer, but I can tell you that negatively and positive supercoils are pulled through the gel by the electric field at different rates (although I have never heard a good explanation of why one is always faster than the other), and you will have to just look it up as to which is which.

Good luck!


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