APPLICATION OF THE METHOD OF PHAGE-T4 DNA LIGASE-CATALYZED RING-CLOSURE TO THE STUDY OF DNA-STRUCTURE .2. NACL-DEPENDENCE OF DNA FLEXIBILITY AND HELICAL REPEAT

In this work, we demonstrate that it is possible to determine the molar cyclization factor jM from single ligation reactions in which both circular and linear dimer DNA species are formed concurrently from linear monomers. This approach represents a significant improvement over previous methods, in which jM is evaluated from the ratio of the rate constants for two separate processes; namely (1) the cyclization of linear DNA and (2) the association of two linear molecules to form linear dimers. Determination of jM for a 366 base-pair molecule yields 5.8 × 10-8 m, in close agreement with the value of 5.6 × 10-8m determined by Shore et al. for the same molecule. Using the current approach for the determination of jM, we have investigated the dependence on NaCl concentration (0 to 162 mm-NaCl, 1 mm-MgCl2) of both the lateral and torsional flexibilities of DNA. The principal observation is that both quantities are essentially constant over the above range of NaCl concentrations, with the persistence length P ≈ 450 (±15) A ̊, and the torsional elastic constant C ≈ 2.0 (±0.2) × 10-19 erg cm. These observations are in accord with the previous theoretical prediction that P becomes essentially independent of NaCl concentration above 10 to 20 mm. We have examined the dependence of the helical repeat of DNA on NaCl concentration over the above range, and have found the value of 10.44 base-pairs per turn to be essentially constant over that range. This last result suggests that earlier studies have overestimated the dependence of DNA helical twist on salt concentration. © 1990.