LINEARIZED FREE MAXICIRCLE DNA IN CRITHIDIA-FASCICULATA IS A PRODUCT OF TOPOISOMERASE II-MEDIATED CLEAVAGE

Linearized free maxicircle DNA, present in detergent lysates of Crithi dia fasciculata mitochondria, was thought to be a replication intermed iate formed during rolling circle replication of maxicircle DNA. Gel e lectrophoresis of the linearized free maxicircles indicated that they were slightly larger than the maxicircle genome, raising the possibili ty of the presence of terminal repetitions (Hajduk, S.L., Klein, V.A, and Englund, P.T. (1984) Cell 36, 483-492). We recently found, however : that maxicircles replicate by a theta-mechanism, and not as rolling circles (Carpenter, L.R. and Englund, P.T. (1995) Mol. Cell Biol. 15, 6794-6803). Given that theta-replication does not easily explain the p resence of linearized free maxicircles, we investigated alternative ex planations for their existence. We present evidence that this DNA spec ies results from the double-strand cleavage of maxicircles due to dete rgent denaturation of intracellular topoisomerase II cleavable complex es. As expected for a topoisomerase II cleavage product, the linearize d free maxicircle DNA is covalently bound to protein at both 5' ends. In addition, the slightly larger apparent size of linearized free maxi circle DNA or maxicircles linearized by a restriction enzyme can be ex plained by anomalous electrophoretic migration during conventional or pulsed-field agarose gel electrophoresis. This anomalous migration is presumably due to bends or other unusual structures in the DNA.