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Nucleic Acids Research, 1977, Vol. 4, No. 6 1803-1814
© 1977

Articles

Relaxed circular SV40 DNA as cleavage intermediate of two restriction endonucleases

George Ruben, Paul Spielman, Chen-pei D. Tu, Ernest Jay, Benjamin Siegel and Ray Wu*

Section of Biochemistry, Molecular and Cell Biology, and the Department of Applied Physics, Cornell University Ithaca, NY 14853, USA

*To whom correspondence should be sent.

Received February 7, 1977. We have determined the mode of cleavage of superhelical SV40 DNA (Form I) by restriction endonucleases EcoRI and HpaII at 37°C. By analysis with agarose gel electrophoresis and direct examination with dark field electron microscopy, we found that a large amount of the single-nicked circular DNA (Form II) was produced before the linear SV40 DNA (Form III) appeared. Thus, both restriction enzymes cleave only one strand of the superhelical DNA first. The second cleavage on the complementary strand occurred after a lag period. The first order rate constant for the second cleavage by EcoRI endonuclease was determined and a kinetic reaction scheme for both enzymes is proposed.


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