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Abstract
Newly replicated duplex DNA minicircles of trypanosomal kinetoplast DNA are nicked in both their monomeric and catenated topological states, whereas mature ones are covalently sealed. The possibility that nicking may play a role during kinetoplast DNA replication by affecting the topological interconversions of monomeric DNA minicircles and catenane networks was studied here in vitro using Crithidia fasciculata DNA topoisomerase. An enzyme that catalyzes the nicking of duplex DNA circles has been purified to apparent homogeneity from C. fasciculata cell extracts. The native enzyme has a sedimentation coefficient of 6.8 S and was found to be a dimer with a protomer Mr = 60,000. Nicking of kinetoplast DNA networks by the purified enzyme inhibits their decatenation by the Crithidia DNA topoisomerase but has no effect on the catenation of monomeric DNA minicircles into networks. This differential effect on decatenation versus catenation is specific to the purified nicking enzyme. Random nicking of interlocked DNA minicircles has no detectable effect on the reversibility of the topological reaction. The potential role of Crithidia nicking enzyme in the replication of kinetoplast DNA networks in trypanosomatids is discussed.
Highlights
From the Kuvin Centrefor the Studvof Infectious and Tropical Diseases, The Hebrew University Hadassah Medical School, I
In a previousreport [14] we described a DNA topoisomerase from C. fasciculata and a crude enzyme fraction which affects the topological reaction through the introductionof nicks in the network circles
Purification of Crithidia Nicking Enzyme-Purification of Crithidia nicking enzymewas carried out monitoring both the nicking of duplexcircularDNA, and the inhibition of the decatenation of Kinetoplast DNA (kDNA) networks catalyzed by Crithidia DNA
Summary
From the Kuvin Centrefor the Studvof Infectious and Tropical Diseases, The Hebrew University Hadassah Medical School, I. In a previousreport [14] we described a DNA topoisomerase from C. fasciculata and a crude enzyme fraction which affects the topological reaction through the introductionof nicks in the network circles.
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