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Nucleic Acids Research, 1992, Vol. 20, No. 19 5127-5130
© 1992

MOLECULAR BIOLOGY

Modified DNA fragments activate Nael cleavage of refractory DNA sites

Michael Conrad and Michael D. Topal*

Lineberger Comprehensive Cancer Center and Department of Pathology, University of North Carolina Medical School at Chapel Hill Chapel Hill, NC 27599-7295, USA

* To whom correspondence should be addressed at the Lineberger Comprehensive Cancer Center, University of North Carolina Medical School at Chapel Hill, Chapel Hill, NC 27599-7295, USA

Received June 22, 1992. Revised August 29, 1992. Accepted August 29, 1992.

Endonuclease Nael cleaves DNA using a two-site mechanism. The DNA-blnding sites are nonidentical: they recognize different families of flanking sequences. A unique Nael site that is resistant to cleavage resides in M13 double-stranded DNA. Nael can be activated to cleave this site by small DNA fragments containing one or more Nael sites. These activators are not practical for genetic engineering because unphosphorylated activators that are consumed during the cleavage of substrate give ends that may interfere with subsequent ligations. We show that a DNA fragment containing phosphorothioate linkages at the Nael scissile bonds (S-activator) is not cleaved by Nael, even though this S-activator binds to the substrate site. The S-activator activates Nael to cleave M13 DNA under conditions that completely exhaust unsubstituted activator. These results demonstrate that activation is not coupled to cleavage of activator, that Nael reverts to its inactive state soon after dissociation of the EA complex, and that S-activator makes for a nondepletable activator during prolonged incubations.


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