Superhelix density

The difference between the linking number of a DNA and the linking number of its relaxed form is AL AL = (L — To) - In our example with four negative supercoils, AL = 4. The superhelix density or specific linking difference... 

The effective potential governing torsional deformations could conceivably be quite anharmonic, so that overwinding is much more strongly resisted than underwinding for finite deformations. This question is addressed by examining the dependence of the torsion constant on temperature(40) and on superhelix density. 

Figure 4.10. Best-fit torsion constant a versus experimental time span for two different samples of supercoiled pUC8 dimer. The two samples are in 10 mAf Nad, 10 mAf Tris, 1 mAf EDTA, at pH 8 and T = 20°C, and differ only in their mean superhelix density (it) ,

A. S. Benight, J. Langowski, B. S. Fujimoto, and J. M. Schurr, unpublished results). It appears that the transition is not induced in samples with higher than normal superhelix densities. This suggests that the equilibrium between the secondary structure states a and b might be rather sensitive to superhelical stress. This question is addressed immediately below. 

Samples of pUC8 dimer (5434 bp) with different median superhelix densities were prepared by relaxing the native plasmid with topoisomerase I... 

Figure 4.20. Variation of the molar ellipticity [0] and torsion constant a of pUC8 dimer with superhelix density. All samples were prepared from the same stock solution as described in the text. Solution conditions were 10 mM NaCl, 10 mM Tris, 1 mM EDTA, and pH 8, and the DNA concentrations were between 40 and 50 g/ml. , Five days after preparation +, 15 days after preparation , 2 months after preparation , samples prepared from the same initial stock solution 4 months after the other samples and measured within 5 days. Top [0] versus superhelix density a. Only the a = -0.015 sample changed significantly over the first few weeks. Bottom a versus superhelix density. Torsion constants are averages for 70- and 120-ns time spans. For the FPA measurements only, ethidium was added to a concentration of 1 dye per 300 base pairs. With the exception of a - - 0.025, the samples denoted by did not change significantly over a period of 2 months, and it is the final values that are plotted. The final measurement for the a= -0.025 sample is denoted by . The error bars are less than or equal to the size of the symbols in the figure. Complete data for o= —0.048 and o= —0.031 are given in Figure 4.10, which demonstrates the ability of FPA measurements to distinguish between samples whose torsion constants differ by only 10%.

Figure 4.21. D0 and Z)pU, for pUC8 dimer versus superhelix density. Conditions are the same as in Figure 4.20. All samples were measured at 5-7 days after preparation, again at 2-3 weeks, and finally at l -2 months. , Five days after preparation +,...

Top Dplu versus superhelix density. >plat is the apparent DLS diffusion coefficient at A2 = 20 x 10 ° cm 2. The lower values at a = —0.015, —0.020, and -0.025 suggest that, in this intermediate range, DNA exhibits a different secondary structure with decreased torsional and/or bending rigidity. 

The low torsion constant at a = —0.025 is very similar to that observed in a supercoiled pBR322 that was partially relaxed by saturation binding of Escherichia coli single-strand binding (ssb) protein, and which persisted for over a month.(56) It is also similar to that recently inferred from an in vivo assay based on variation in repression efficiency with size of a putative DNA loop.(234) Indeed, it appears that anomalously low torsion constants may be universally encountered in the course of either partial or complete relaxation of supercoiled DNAs, regardless of whether the superhelix density is reduced by action of topoisomerase I, binding of ssb protein, binding of intercalated... 

An important question is whether the secondary structure of pUC8 dimer at native superhelix density is the same as that of the relaxed (o = 0) species, as the similarities in a, D and [0] would suggest. We suspect not, because their [0] values would probably be rather different after correction for the difference in superhelix density. 

Most DNA in living organisms, whether bacteria or eukaryotes, is underwound. That is, the superhelix density (Chapter 5) is about -0.05 or one supercoil per 200 base pairs. In eukaryotes this negative supercoil-ing can be accounted for by the winding of DNA around the histones within the nucleosomes (Figs. 

A cooperative transition. The transition from B-DNA to Z-DNA occurs over a small change in the superhelix density, which shows that the transition is highly cooperative. 

The superhelix density of a DNA molecule is often expressed as o = Wr/ Tw number of superhelical turns... 

The superhelix density of 0.05 observed for DNA extracted from eukaryotic cells is just equal to one negative superhelix turn per nucleosome. For example, the number of nucleosomes seen in the minichromosome of Fig. 27-3 matches the numbers of supercoils in the SV40 DNA (Fig. 5-20). If there are two negative supercoils per nucleosome, as shown in Fig. 

---