Temperature dependence of DNA persistence length

Abstract

We have determined the temperature dependence of DNA persistence length, a, using two different methods. The first approach was based on measuring the j-factors of short DNA fragments at various temperatures. Fitting the measured j-factors by the theoretical equation allowed us to obtain the values of a for temperatures between 5°C and 42°C. The second approach was based on measuring the equilibrium distribution of the linking number between the strands of circular DNA at different temperatures. The major contribution into the distribution variance comes from the fluctuations of DNA writhe in the nicked circular molecules which are specified by the value of a. The computation-based analysis of the measured variances was used to obtain the values of a for temperatures up to 60°C. We found a good agreement between the results obtained by these two methods. Our data show that DNA persistence length strongly depends on temperature and accounting for this dependence is important in quantitative comparison between experimental results obtained at different temperatures.

Figure 1.

Equilibrium between circular and linear conformations of 199-bp DNA fragments with various sticky ends. The probability of finding a fragment in circular conformation, f_c, can be estimated from its relative mobility in the polyacrylamide gel since the linear (lane M) and circular (lane 1) forms have different mobility in such a gel. The experiments were performed for sticky ends of 4 AT base pairs created by EcoRI restriction enzyme (lane 2), 2 AT and 2 GC base pairs created by HindIII (lane 3) and 4 GC base pairs created by AgeI (lane 4). The gel electrophoresis was run in TBE buffer containing additional 10 mM of MgCl₂ at 5°C (A) and 25°C (B).

Figure 2.

Dependence of the measured C/D on the concentration of T4 DNA ligase. The shown data correspond to the fragments with EcoRI (5°C, filled circle) and HindIII (42°C, filled triangle) sticky ends. The data show that the ratio does not change if the ligase concentration is below 100 U/ml.

Figure 3.

The j-factor measured for λ phage DNA fragments with EcoRI sticky ends at 5°C (top) and HindIII sticky ends at 42°C (bottom). The lines correspond to the theoretical fit of the data. The best fit shown by the solid lines, correspond to DNA persistence length of 53.2 nm and γ of 10.43 for 5°C and 42.5 nm and γ of 10.56 for 42°C; the value of C was equal to 3.10⁻¹⁹ erg·cm for both temperatures. The dotted lines correspond to the theoretical equation with a reduced/increased by 1 nm from the best fits.

Figure 4.

Equilibrium distribution of DNA molecules over the linking number difference, . To analyze the distribution, radioactively labeled DNA topoisomers were separated by agarose gel electrophoresis (A) and the gel was scanned by Phosphorimager (B). The symbols OC and L mark the bands of open circular and linear DNA molecules, correspondingly. (C) Linearized representation of the distribution. The slope of the line on the plot is specified by the distribution variance (see text for details).

Figure 5.

Temperature dependence of . The distributions of the topoisomers were obtained by applying T4 DNA ligase (open circle) or Taq DNA ligase (open triangle) to nicked samples of pUC19 plasmid.

Figure 6.

Comparison between theoretical and experimental values of the linking number variance, . Experimental data (filled circle) for 37°C were taken from reference (25). The data from the current study (open triangle) and from reference (33) (open circle) were adjusted to 37°C according to the temperature dependence of presented in Figure 5. The line represents the best theoretical fit of the data. The shown curve corresponds to C of 3.1 × 10⁻¹⁹ erg ·cm, a of 45 nm and DNA effective diameter of 5 nm (33), in very good agreement with other available data.

Figure 7.

The temperature dependence of DNA persistence length. The data obtained from the measurements of j-factors (open triangle) and the variance of (open circle) in the current study are shown together with Equation (1) (dotted gray line) and the data from reference (12). The solid line represents manual interpolation of the current results.

Figure 8.

The temperature dependence of supercoiling free energy, . The shown data represent temperature dependence of K that specifies according to Equation (6). The lines correspond to the data obtained in the current study (blue solid line), references (42) (green line), (43) (red line) and (44) (dotted blue line).