Replication of DNA minicircles in kinetoplasts isolated from Crithidia fasciculata: structure of nascent minicircles.

We have previously described an isolated kinetoplast system from Crithidia fasciculata capable of ATP-dependent replication of kinetoplast DNA minicircles (L. Birkenmeyer and D.S. Ray, J. Biol. Chem. 261: 2362-2368, 1986). We present here the identification of two new minicircle species observed in short pulse-labeling experiments in this system. The earliest labeled minicircle species (component A) contains both nascent H and L strands and is heterogeneous in sedimentation and electrophoretic migration. Component A has characteristics consistent with a Cairns-type structure in which the L strand is the leading strand and the H strand is the lagging strand. The other new species (component B) has a nascent 2.5-kilobase linear L strand with a single discontinuity that mapped to either of two alternative origins located 180 degrees apart on the minicircle map. Component B could be repaired to a covalently closed form by Escherichia coli polymerase I and T4 ligase but not by T4 polymerase and T4 ligase. Even though component B has a single gap in one strand, it had an electrophoretic mobility on an agarose gel (minus ethidium bromide) similar to that of a supercoiled circle with three supertwists. Treatment of component B with topoisomerase II converted it to a form that comigrated with a nicked open circular form (replicative form II). These results indicate that component B is a knotted topoisomer of a kinetoplast DNA minicircle with a single gap in the L strand.