Supercoiling of circular DNA

Fig. 38.7. Schematic depicting supercoiling of circular DNA catalyzed by DNA gyrase.

The native form of chromatin in cells assumes a higher order stmcture called the 30-nm filament, which adopts a solenoidal stmcture where the 10-nm filament is arranged in a left-handed cod (Fig. 5). The negative supercoiling of the DNA is manifested by writhing the hehcal axis around the nucleosomes. Chromatin stmcture is an example of toroidal winding whereas eukaryotic chromosomes are linear, the chromatin stmctures, attached to a nuclear matrix, define separate closed-circular topological domains. 

Supercoiling of circular duplex DNA is quantitatively considered in terms of the linking number (L), an integer that specifies the number of complete turns made by one strand around the other. The linking number can change only if a covalent linkage in the DNA backbone is broken and reformed. Enzymes called topoisomerases (see chapter... 

Figure 19-7. Yields of SSBs and DSBs induced by 0-4.2 eV electrons on supercoiled plasmid DNA films. The inset shows the dependence of the percentage of circular DNA (i.e. SSB) on irradiation time for a beam of 0.6 eV electrons of 2 nA...

Figure 5.18. Electrou Micrographs of Circular DNA from Mitochoudria. (A) Relaxed form. (B) Supercoiled form. [Courtesy of Dr. David Clayton.]...

Fig. 9. Positive supercoiling of SSVl DNA. Panels A and B show two-dimensional agarose gel electrophoresis of SSVl DNA isolated from cells of Sulfolobus shibatae, (A) before and (B) after UV induction (for methods see refs. [39,98]). The left-hand branch of the arch visible in A corresponds to negatively supercoiled DNA, the top of the arch corresponds to relaxed DNA and the right-hand branch corresponds to positively supercoiled DNA. The upper bands in A and B correspond to form II (open circular) and the middle band in B corresponds to form III (linear SSVl) (pictures courtesy of G. Mirambeau). Panel C shows a one-dimensional agarose gel electrophoresis of SSVl DNA isolated...

The first activity of DNA topoisomerases to be described was the relaxation of supercoiled closed-circular DNA, i.e., conversion to a less supercoiled form (Wang, 1971). This activity was clearly distinct from that of nucleases since the products were covalently closed and relaxation could occur in a stepwise fashion. A dependence on DNA ligase was ruled out, since no energy source was required for this reaction. All topoisomerases discovered subsequently can relax negatively supercoiled DNA the ability to relax positively supercoiled DNA is less gen-... 

Figure 4.24 illustrates three kinds of circular DNA, unstrained circle, strained circle, and supercoil. Similarly, Figure 4.18 shows an electron micrograph of a relaxed (unstrained) circle and two supercoiled circles. The unstrained circle contains the same number of twists as linear DNA. It is under no superhelical tension. To make the strained circle, one twist was removed (compared to linear DNA) and the resulting circular DNA is strained because it has the same number of base pairs (105), but fewer numbers of turns (twists). Thus, the strained circle has a higher number of base pairs per turn than the unstrained circle. To relieve the strain, the strained molecule can introduce another superhelical turn within itself, called a writhe. 

Figure 16.4 Salt-dependent buckling of circular DNA molecules. (A) Schematic representation of the preparation of the over twisted 90-base-pair circular DNA. (B) A pronounced phase transition, from circular to buckled DNA, is observed at physiological conditions (concentrations of 50-200 mM]. Q is an order parameter that quantifies the DNA circle s supercoiling. Figure reproduced with permission from Savelyev et al., 2011b.

The standard assay for reverse gyrases is the ATP-dependent positive super-coiling of circular DNA. The assay described below is applicable to all reverse gyrases, with slight modifications. For simplicity, the substrate is usually a negatively supercoiled plasmid, but it is possible to start from relaxed DNA. The reaction mixture (usually 20 p,l final volume) contains 50 mM Tris-HCl, pH 8.0, at 20° (or MOPS, pH 6.6), 1 mM ATP, 0.5 to 1 mM DTT, 0.5 mM EDTA, 30 (xg/ml... 

Fig. 3. Unlinking and melting of covalently closed DNA by topoisomerase V. (A) Electrophoresis of control relaxed plasmid DNA (1), negatively supercoiled (2), and the same DNA as in (1) but after incubation at 95°C with topo V (3). —SC and U indicate negatively supercoiled and unlinked DNA, respectively. (B) Unlinked DNA strands of pBR322 DNA by electron microscopy. The molecule shown here is magnified approximately 120,000. ssDNA circles are linked onee. (C) An illustration of incomplete melting of circular DNA at high temperature and its complete melting and unlinking by topoisomerase V. Duplex regions and melted strands are shown by thick and thin lines.

Supercoiled DNA A contortion of circular DNA into the shape of the simple figure eight. DNA supercoiling is important for DNA... 

In the much-studied E. coli cell, the closed circular DNA has a molecular weight of 2.6 x 10 with about 4 x 10 base pairs and a contour length (i.e. when stretched out) of about 1.4 mm. A considerable degree of folding and supercoiling of the DNA is implied since the cell diameter is only 0.002 mm. 

Circular DNA is a type of double-stranded DNA in which the two ends of each strand are joined by phosphodiester bonds [Figure 20.11 (a)]. This type of DNA, the most prominent form in bacteria and viruses, is also referred to as circular duplex (because it is double-stranded) DNA. One strand of circular DNA may be opened, partially unwound, and then rejoined. The unwound section introduces a strain into the molecule because the nonhelical gap is less stable than hydrogen-bonded, base-paired helical sections. The strain can be localized in the nonhelical gap. Alternatively, it may be spread uniformly over the entire circular DNA by the introduction of superhelical twists, one twist for each turn of a helix unwound. The circular DNA shown in Figure 20.11 (b) has been unwound by four complete turns of the helix. The strain introduced by this unwinding is spread uniformly over the entire molecule by the introduction of four superhelical twists [Figure 20.11 (c)]. Interconversion of relaxed and supercoiled DNA is catalyzed by groups of enzymes called topoisomerases and gyrases. 

Relaxed and supercoiled DNA. (a) Circular DNA is relaxed, (b) One strand is broken, unwound by four turns, and the ends are then rejoined.The strain of unwinding is localized in the nonhelical gap. (c) Supercoiling by four twists distributes the strain of unwinding uniformly over the entire molecule of circular DNA. 

It thus appears that many, if not all DNA s of viral or bacterial origin, even if linear In the vegetative form, circularize during replication and that the repetitive end sequences serve as locks to fuse the circles. Most of the closed circular DNA s have an additional feature they are super-coiled, i.e., they lack a certain constant number of helix turns and will therefore compensate for this strain by super-coiling. They are thus packed more tightly. The supercoil structure of circular DNA s can be relaxed spontaneously by single-stranded breaks or gradually (Fig. 5.10) by Intercalation of such aromatic dyes as ethidlum bromide (32) (see Chapter 7). 

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 ... 

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