It was quickly recognized that a DNA double helix could not be unwound indefinitely, any more than you could pull your braids apart from the middle without inflicting great pain. DNA becomes twisted or supercoiled as a result of unwinding, and the strain induced has to be relieved otherwise replication couldn't continue. This is accomplished by two enzymes called topo-isomerases. The name reflects that these enzymes will change the shape or topology of a DNA molecule, but not its chemical structure. Unwinding one turn of the double helix will introduce one supercoil into the DNA. In a circular chromosome, this has the effect of bending a relaxed circular double helix into a figure eight, as suggested on the left here. Unwinding a second turn of the helix introduces another twist or supercoil as shown on the right. There are traces from Karen's theta images, which we can now correlate with different degrees of supercoiling. Unwinding also creates supercoils in linear molecules, that is, in the chromosome of DNA of eukaryotic cells. And topo-isomerases are perched all over the chromosome of DNA to deal with this tension. Here's how topo-isomerases 1 and 2 act to relieve supercoiling by allowing double helical DNA that is too tightly twisted to untwist. Here, topo-isomerase 1 or topo 1, for short, nicks one of the two strands of DNA and allows a controlled unwinding of the cut strand around the uncut strand. And then, religation of the nick after the supercoil is relaxed. Let's look at topo-isomerase 2. Topo 2 nicks both strands of DNA, passing one length of twisted DNA through the cut as shown here, with the result that the twist or coil is relaxed. Since supercoiled or twisted DNA is more compact than relaxed or open-circle DNA in the case of an ecoli chromosome, DNA that is more supercoiled behaves on electrophoretic gels as if it were smaller than the more relaxed topo-isomerase. This allows us to see the effects of topo-isomerases on supercoiled DNA as shown in this slide. Lane 1 shows maximally supercoiled DNA having migrated pretty far down the gel as if it were comprised of small DNA molecules. DNA in the middle and the right lanes has been treated with topo-isomerase 1 for 3 minutes or 30 minutes respectively. As the enzyme caused the DNA to become progressively more relaxed, the DNA behaves as if it were larger and larger. Each band that you see is a next level up or down in the number of supercoils.