Conformational changes of the double-stranded DNA helix in response to dehydration were investigated by monitoring, by agarose gel electrophoresis, the linking number of covalently closed circular DNA generated by ligation of linear DNA in the presence of different organic solvents or at different temperatures. It was found that: (i) The Hydration and dehydration of DNA has been studied for the past two decades (see refs. 1-3 for reviews). By using the analytical ultracentrifuge, DNA has been found to be dehydrated upon increase of temperature (4-6). It has been reported that DNA denaturation occurs in a number of organic solvents due to dehydration (7-11). Extensive studies of the effect of dehydration on DNA conformation have been carried out using circular dichroism (CD). The conformation of DNA in a thin film (12) or in aqueous organic solvent mixtures (13-18) has been correlated to the spectral change of CD. The decrease of the CD band at 270 nm in the presence of organic solvent has been attributed to the presence ofC form DNA (C DNA) (12-18). On this basis, C DNA has been assumed to prevail at high salt concentrations (19)(20)(21)(22)(23)(24) or under low temperature conditions (in the premelting region) (25,26). However, the presence of C DNA is not supported by x-ray diffraction studies of the DNA in concentrated salt solutions (27) or in organic solvent/water mixtures (27, 28). DNA condensed from various ethanol/water solutions also failed to show the x-ray diffraction pattern of the C form (29).Although a considerable amount of data has been accumulated, no conclusive results have been published to relate hydration of the DNA to its helical structure in solution. Depew and Wang (30) and Pulleyblank et al. (31) showed that the thermal effect on the DNA base-pair twisting angle could be accurately determined by agarose gel electrophoresis of covalently closed circular DNA generated at different temperatures. This type of system is useful for studying DNA structure in solution. For example, using a gel electrophoresis technique, Wang (32,33) demonstrated that the DNA double helix has 10.4 ± 0.1 base pairs per helical turn in dilute solution. [It should be pointed out that Zimmerman and Pheiffer (34) found 9.9 base pairs per turn for DNA in concentrated solution. ] In the present studies, we have explored the effect of hydration on the helical structure of DNA in solution, using phage T4 DNA ligase to covalently close the linear plasmid DNA (pBR322 and pNT7) at various levels of dehydration by addition of methanol, ethanol, glycerol, ethylene glycol, dimethyl sulfoxide, and tetrahydrofuran or exposure to different temperatures. The thermodynamic properties of superhelix formation and the conformational changes of DNA resulting from the different states of dehydration are discussed. An argument is made for the interpretation of the CD spectra of DNA in organic solvent/water mixtures and those in the premelting temperature regions. The implications of DNA hydration and dehydration and their effec...

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The structures of molecules of colicin El plasmid DNA that were in the process of being replicated in vitro were examined electron microscopically. Circular molecules containing a loop of approximately 7% of the length of the molecules were the major class of replicating molecules. The location of the loop was determined by treating the molecules with restriction endonuclease EcoRl, which introduced one unique double-strand break in the colicin El plasmid DNA molecule. The loops had a specific location with two branch points at approximately 17 and 24% of the molecular length from the endonucleasesensitive site. Molecules with a larger loop were observed with a preparation labeled with 5-bromodeoxyuridine and enriched for these molecules. One of the branch points in these molecules was located at approximately 17% of the molecular length from the endonuclease-sensitive site independent of the size of the loops. These results indicate that the origin of replication of the plasmid DNA is located within the small loop and the replication proceeds unidirectionally. The molecules that had completed a round of replication had the monomeric twisted circular structure.A soluble system that is capable of replicating colicin El plasmid (Col El) DNA was described in a previous paper (1).In this system, the closed-circular Col El DNA molecules in cell extracts prepared from Escherichia coli carrying Col El can initiate semiconservative replication. The major products of Col El DNA synthesis are completely replicated molecules and a class of replicative intermediates carrying DNA fragments with an average length of approximately 7% of Col El DNA (1). The molecules carrying small DNA fragments are selectively synthesized in the presence ol" 10% (v/v) glycerol and 2 mM spermidine (2).The structures of early replicative intermediates, as well as the molecules replicated more extensively, were examined electron microscopically. A necessary internal marker for locating the replication loop in each molecule is provided by treatment of the DNA with restriction endonuclease EcoRl (3), which was found to introduce one unique double-strand break in the Col El DNA molecule. The endonuclease has been used to show bidirectional replication from a fixed origin in simian virus 40 DNA (4). MATERIALS AND METHODSMlaterials. Most of the materials used have been described in the previous papers (1, 2). Restriction endonuclease EcoRl was supplied by Drs. D. Nathans and T. J. Kelley, Jr. Cytochrome c was obtained from Calbiochem.Preparation of Replicative Intermediates and Completely Replicated Molecules. Cell extracts prepared by lysis of E. coli YS10 (Col El) (1) were incubated for 60 min in the standard reaction mixture with [a-32P]dTTP in the presence or absence of 10% (v/v) glycerol and 2 mM spermidine (2). DNA was extracted and purified by CsCl density gradient centrifugation. The DNA, which formed a single band, was dialyzed against 0.1 M Tris * HCl (pH 8.0)-l mM EDTA. With these preparations, structures of early replicative int...

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