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Nucleic Acids Research, 1992, Vol. 20, No. 14 3567-3573
© 1992

ENZYMOLOGY

The cleavage of DNA at phoshorothioate internucleotidic linkages by DNA gyrase

Simon T. Dobbs1,2, Paul M. Cullis1 and Anthony Maxwell2,*

1Department of Chemistry Leicester LE1 7RH, UK 2Department of Biochemistry, University of Leicester Leicester LE1 7HR, UK

*To whom correspondence should be addressed

Received May 20, 1992. Accepted June 26, 1992.

We have constructed a plasmid which contains 22 copies of a 147 bp DNA fragment which contains the major DNA gyrase cleavage site from plasmid pBR322 (located at base-pair 990). We have found that this fragment is efficiently bound and cleaved by gyrase. The selectivity for the sequence corresponding to position 990 in pBR322 is maintained even when this site is located only 15 bp from one end of the 147 bp fragment. A strategy for the specific incorporation of a single thiophosphoryl linkage into the 147 bp fragment has been developed, and gyrase has been shown to catalyse efficient cleavage of fragments bearing phosphorothioate linkages at the gyrase cleavage site in one or both strands.


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