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Nucleic Acids Research, 1993, Vol. 21, No. 3 593-600
© 1993

MOLECULAR BIOLOGY

Characterization of an altered DNA catalysis of a camptothecin-resistant eukaryotic topoisomerase I

Irina I. Gromova, Eigil Kjeldsen+, Jesper Q. Svejstrup, Jan Alsner, Kent Christiansen and Ole Westergaard*

Department of Molecular Biology, Universfty of Åarhus DK-8000 Arhus C, Denmark

*To whom correspondence should be addressed

Received October 9, 1992. Revised December 22, 1992. Accepted December 22, 1992.

We investigated topoisomerase I activity at a specific camptothecin-enhanced cleavage site by use of a partly double-stranded DNA substrate. The cleavage site belongs to a group of DNA topoisomerase I sites which is only efficiently cleaved by wild-type topolsomerase I (topo i-wt) in the presence of camptothecin. With a mutated camptothecin-resistant form of topolsomerase I (topo l-K5) previous attempts to reveal cleavage activity at this site have failed. On this basis it was questioned whether the mutant enzyme has an altered DNA sequence recognition or a changed rate of catalysis at the site. Utilizing a newly developed assay system we demonstrate that topo I-K5 not only recognizes and binds to the strongly camptothecin enhanced cleavage site but also has considerable cleavage/religation activity at this particular DNA site. Thus, topo I-K5 has a 10-fold higher rate of catalysis and a 10-fold higher affinity for DNA relative to topo l-wt. Our data indicate that the higher cleavage/religation activity of topo I-K5 is a result of improved DNA binding and a concomitant shift in the equilibrium between cleavage and religatlon towards the religation step. Thus, a recently identified point mutation which characterizes the camptothecin resistant topo I-K5 has altered the enzymatic catalysis without disturbing the DNA sequence specificity of the enzyme.

+Present address: Deparuiient of Human Genetics, The Bartholin Building, University of Aarhus, DK-8000 Aarhus C, Denmark


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