Positive supercoiling

Bacterial as well as eukaryotic chromosomes contain too much DNA to fit easily into a cell. Therefore, the DNA must be condensed (compacted) to fit into the cell or nucleus. This is accomplished by supercoiling the DNA into a highly condensed form. When relaxed circular DNA is twisted in the direction that the helix turns, the DNA becomes positively supercoiled, if it is twisted in the opposite direction, it is called negatively supercoiled. Bacterial DNA is normally found in a negatively supercoiled state. Supercoiling reactions are catalyzed by topoisomerases. 

Several laboratories have examined whether BPDE covalently bound to DNA assumes an intercalated conformation or is externally bound. Different groups have reported different results (5,6,8,20,34,59-69). Mobility studies using relaxed circular pBR322 DNA indicate that reaction with BPDE gives rise to rapid positive supercoiling which is suggestive of a conformation in which the hydrocarbon occupies an internal site in the DNA (34). On the other hand, from results of the most recent spectroscopic studies it is concluded that the covalent adduct formed from the more carcinogenic (+) enantiomer of BPDE is in an external conformation (68,69). 

At binding ratios r > 0.27, both linear and supercoiled DNAs show evidence of a marked structural change. A component with intermediate lifetime (t 5 ns) appears in the ethidium fluorescence decay, which may represent a partially intercalated species. The apparent torsion constants become highly nonuniform and exhibit considerably altered values. The long-range torsion constant increases appreciably for the linear DNA, but decreases for the supercoiled DNAs, which are substantially positively supercoiled at that point.(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. 

Positively supercoiled DNA is formed if the DNA is wound more tightly than in Watson-Crick DNA. 

Removal of positive supercoils ahead of advancing replication forks DNA topoisomerase II (DNA gyrase) DNA topoisomerase II... 

Polymerase-induced positive supercoiling and linker positive crossing in nucleosomes... 

In the absence of conclusive data on the role of a positive supercoiling wave, static positive supercoiling elicited by nucleosome reconstitution on relaxed or slightly positively-supercoiled plasmids [51] or by ethidium bromide intercalation in the loop of mononucleosomes on DNA minicircles [52] did not succeed either in releasing dimers. Moreover, circular dichroism, histone chemical modi-flcation and H3-thiol accessibility failed to detect an even slight alteration in the structure of such torsionally-stressed nucleosomes [51]. The reason was later found to lie in the ability of nucleosome entry/exit DNAs to form a positive crossing [52]. 

A puzzling observation in the above mentioned investigation of the effect of positive supercoiling on nucleosome structure [51] was that the number of nucleosomes... 

Hamiche, A., Carol, V., Alilat, M., De Lucia, F., O Donohue, M.F., Revet, B., and Prunell, A. (1996) Interaction of the histone (H3-H4)2 tetramer of the nucleosome with positively supercoiled DNA minicircles. Potential flipping of the protein from a left- to a right-handed superhelical form. PNAS (USA) 93, 7588-7593. 

Rodriguez, A.C. and Stock, D. (2002) Crystal structure of reverse gyrase insights into the positive supercoiling of DNA. EMBO J. 21, 418 26. 

Rodriguez, A.C. (2002) Studies of a positive supercoiling machine. Nucleotide hydrolysis and a multifunctional latch in the mechatrism of reverse gyrase. J. Biol. Chem. 277, 29865-29873. 

FIGURE 24-17 Negative and positive supercoils. For the relaxed DNA molecule of Figure 24-16a, underwinding or overwinding by two helical turns (Lk = 198 or 202) will produce negative or positive supercoiling, respectively. Note that the DNA axis twists in opposite directions in the two cases. 

FIGURE 24-28 Chromatin assembly, (a) Relaxed, closed-circular DNA. (b) Binding of a histone core to form a nucleosome induces one negative supercoil, in the absence of any strand breaks, a positive supercoil must form elsewhere in the DNA (ALk = 0). (c) Relaxation of this positive supercoil by a topoisomerase leaves one net negative supercoil (ALk = -1). 

HGURE 24-35 Model for the effect of condensins on DNA super-coiling. Binding of condensins to a closed-circular DNA in the presence of topoisomerase I leads to the production of positive supercoils (+). Wrapping of the DNA about the condensin introduces positive supercoils because it wraps in the opposite sense to a solenoidal supercoil (see Fig. 24-24).The compensating negative supercoils (—) that appear elsewhere in the DNA are then relaxed by topoisomerase I. In the chromosome, it is the wrapping of the DNA about condensin that may contribute to DNA condensation. 

Solving the problem of supercoils As the two strands of the double helix are separated, a problem is encountered, namely, the appearance of positive supercoils (also called supertwists) in... 

Positive supercoiling resulting from DNA strand separation. 

Correct answer = C. Fluoroquinolones, such as ciprofloxacin, inhibit bacterial DNA gyrase—a type II DNA topoisomerase. This enzyme catalyzes the transient breaking and rejoining of the phosphodiester bonds of the DNA backbone, to allow the removal of positive supercoils during DNA replication. The other enzyme activities mentioned are not affected. Primase synthesizes RNA primers, helicase breaks hydrogen bonds in front of the replication fork, DNA polymerase I removes RNA primers, and DNA igase joins Okazaki fragments. 

Cause of positive supercoils and enzymes that can relax them... 

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