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Nucleic Acids Research, 1991, Vol. 19, No. 11 3047-3054
© 1991

MOLECULAR BIOLOGY

Effects of temperature on excluded volume-promoted cyclization and concatemerization of cohesive-ended DNA longer than 0.04 Mb

Donna Louie and Philip Serwer

Department of Biochemistry, The University of Texas Health Science Center San Antonio, TX 78284-7760, USA

Received January 28, 1991. Revised April 19, 1991. Accepted April 19, 1991.

The 0.048502 megabase (Mb), primarily doublestranded DNA of bacteriophage {lambda} has single-stranded, complementary termini (cohesive ends) that undergo either spontaneous intramolecular joining to form open circular DNA or spontaneous intermolecular joining to form linear, end-to-end oligomeric DNAs (concatemers); concatemers also cyclize. In the present study, the effects of polyethylene glycol (PEG) on the cyclization and concatemerization of {lambda} DNA are determined at temperatures that, in the absence of PEG, favor dissociation of cohesive ends. Circular and linear {lambda} DNA, monomeric and concatemeric, are observed by use of pulsed field agarose gel (PFG) electrophoresis. During preparation of {lambda} DNA for these studies, hydrodynamic shear-induced, partial dissociation of joined cohesive ends is fortuitously observed. Although joined {lambda} cohesive ends progressively dissociate as their temperature is raised in the buffer used here (0.1 M NaCI, 0.01 M sodium phosphate, pH 7.4, 0.001 M EDTA), when PEG is added to this buffer, raising the temperature sometimes promotes joining of cohesive ends. Conditions for promotion of primarily either cyclization or concatemerization are described. Open circular DNAs as long as a 7-mer are produced and resolved. The concentration of PEG required to promote joining of cohesive ends decreases as the molecular weight of the PEG increases. The rate of cyclization is brought, the first time, to values that are high enough to be comparable to the rate observed in vivo. For doublestranded DNA bacteriophages that have a linear replicative form of DNA (bacteriophage T7, for example), a suppression, sometimes observed here, of cyclization mimics a suppression of cyclization previously observed in vivo. The PEG, temperature effects on DNA joining are explained by both the excluded volume of PEG random coils and an increase in this excluded volume that occurs when temperature increases.


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