Circular DNA and supercoiling

DNA that contained both closed, circular DNA and supercoiled DNA. Would you expect to see two bands in the sedimentation pattern Explain. 

With respect to circular DNAs and supercoiling, students should note the following ... 

See also Figure 4.24, Linking Number, Supercoiling, Topoisomerases, Circular DNA and Supercoiling... 

Different topologies of dsDNA on amino-terminated mica have been imaged using the tapping mode and ambient conditions [48]. Fig. 8 presents linear A-DNA, non-twisted circular DNA and circular supercoiled DNA. The measured width and height of dsDNA are 3-7 nm and around 1 nm, respectively. 

The capability of the supramolecular porphyrins to induce photodamage to calf thymus DNA and supercoiled pBR322 also was evaluated (168). The irradiation of the last one in the presence of 1.5 )J,M H2(4-TRPyP) solution led to efficient nicking and formation of the open circular species, as well as to an increase of the amount of oxidized deoxiguanosine (8-oxodGuo). Similar results were obtained for calf thymus DNA (169). 

Relaxed circular DNA with a nicked strand is less compact than supercoiled circular DNA and so has a lower effective density. As a result it will not migrate as far in the centrifuge tube as will the supercoiled DNA. 

Positive supercoiling tends to increase the twist between one nucleotide and the next, negative supercoiling reduces it. In a circular DNA, the supercoils will "cancel out" when they meet opposite the original twist. The subject of supercoiling in DNA may be referred to as DNA topology. 

Fig. 6 Photocleavage of supercoiled X174 DNA. d>X174 DNA (50 xM per base pair) was incubated with various compounds in 20% acetonitrile in Tris-HCl buffer (pH 7.5, 50 mM) at 25 °C for 2 h under a UV lamp (365 nm, 15 W) placed 10 cm from the sample, then analyzed by gel electrophoresis (0.9% agarose gel, ethidium bromide stain). Lane h DNA alone lane 2r. DNA following UV irradiation lanes 3-8-. compounds 14, 18, 8, 3, 15, and 16 (1000 p,M), respectively, following UV irradiation. Form 1 covalently closed supercoiled DNA Form II open circular DNA and Form 111 linear DNA...

Eukaryotic type IT. relax both positively and negatively supercoiled DNA during catalysis, the enzyme attaches at the 3 -end of the break via a phosphotyro-sine linkage. Rat liver type I T. catalyses the formation in vivo of chromatin-like material from relaxed circular DNA and core histones. This suggests a role in vivo for eukaryotic type 1 T. in the formation of chromatin, which correlates well with the fact that the eukaryotic enzymes are found almost entirely in... 

The length of a DNA molecule is enormously greater than its diameter, and the extended molecule is quite flexible. A DNA molecule is said to be relaxed if it has no twists other than those imposed by its secondary structure. Said another way, relaxed DNA does not have a clearly defined tertiary structure. We consider two types of tertiary structure, one type induced by perturbations in circular DNA and a second type introduced by coordination of DNA with nuclear proteins called histones. Tertiary structure, whatever the type, is referred to as supercoiling. 

Dl A. Supercoiling. Supercoiling is a topological property of closed-circular DNA molecules. Circular DNA molecules can exist in various conformations differing in the number of times one strand of the helix crosses the other. These different isomeric conformations are called topoisomers and maybe characterized in terms of the linking number, Ek. A linear DNA molecule having Nbase pairs and h base pairs per turn of the helix, if joined end to end, has the following ... 

Equation (4.68) with a = 0 applies for linear or nicked circular DNAs. When r = r, the (initially) supercoiled DNA is predicted to experience no deformational strain, as it is fully relaxed, and to bind the same amount of dye as its linear counterpart with the same concentration of free dye. Under these conditions, the supercoiled and linear DNA/chloroquine complexes are expected to exhibit identical local structures, rigidities, and deformational dynamics. This important corollary to the standard model was untested till recently.(53)... 

A related observation is that fully relaxed supercoiled DNA/dye complexes are somehow different from nicked circular DNA/dye complexes in the presence of the same concentration of free dye, where the binding ratios should be the same. This is readily seen in gel electrophoresis in the presence of sufficient dye concentration so that at least one, but not all, of the topoisomers is positively supercoiled. The slowest moving, presumably fully relaxed, topoisomer migrates significantly faster than the nicked circle, and this difference increases with the amount of dye present. This is not observed with chloroquine, perhaps because the effect is too small. However, it is readily apparent in the original gels of Keller0 61) in which ethidium was used to unwind the topoisomers. We have confirmed this effect for ethidium and have observed similar behavior for proflavine, 9-aminoacridine, and quinacrine. 

Figure 3. The stability of the nucleosome is affected by the length and the superhelicity of DNA. (a-b) The chromatin fibers were reconstituted from the purified plasmids and the histone octamers by a salt-dialysis method and observed under AFM. The 3 kb (a) or 106 kb (e) supercoiled circular plasmid was used as a template, (c) Relationship between the plasmid length and the frequency of nucleosome formation in the reconstitution process. The nucleosome frequency is represented as the number of base pairs per nucleosome and plotted against the length of the template DNA in supercoiled (filled circle) and linear (open circle) forms, (d) AFM image of the chromatin fiber reconstituted on the topoisomerase 1-treated plasmid, (e) Chromatin fiber reconstituted with Drosophila embryo extract. The chromatin fiber was reconstituted from plasmid DNA of 10kband the embryo extract of Drosophila, and was observed by AFM...

Mitochondrial DNA and the DNA of most prokaryotes are closed circular structures. These molecules may exist as relaxed circles or as supercoiled structures in which the heUx is twisted around itself in three-dimensional space. Supercoiling results from strain on the molecule caused by under- or overwinding the double helix ... 

Fig. 8 Topview of AFM images of different dsDNA topologies on amino-terminated mica a linear 2,-DNA (48.5 kbp), b non-twisted circular DNA plasmids (3.2 kbp), c circular su-percoiled DNA with twists and writhes due to internal supercoiling (supercoiled DNA ladder 2-16khp) [48]. Reprinted with permission...

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