KINETOPLAST MAXICIRCLE DNA-REPLICATION IN CRITHIDIA-FASCICULATA AND TRYPANOSOMA-BRUCEI

Kinetoplast DNA, the mitochondrial DNA of trypanosomatids, is composed of several thousand minicircles and a few dozen maxicircles, all of w hich are topologically interlocked in a giant network, We have studied the replication of maxicircle DNA, using electron microscopy to analy ze replication intermediates from both Crithidia fasciculata and Trypa nosoma brucei, Replication intermediates were stabilized against branc h migration by introducing DNA interstrand cross-links in vivo with 4, 5',8-trimethylpsoralen and UV radiation. Electron microscopy of indivi dual maxicircles resulting from a topoisomerase II decatenation of kin etoplast DNA networks revealed intact maxicircle theta structures, Ana lysis of maxicircle DNA linearized by restriction enzyme cleavage reve aled branched replication intermediates derived from theta structures. Measurements of the linearized branched molecules in both parasites i ndicate that replication initiates in the variable region (a noncoding segment characterized by repetitive sequences) and proceeds unidirect ionally, clockwise on the standard map.