CHANGES IN ORGANIZATION OF CRITHIDIA-FASCICULATA KINETOPLAST DNA-REPLICATION PROTEINS DURING THE CELL-CYCLE

Kinetoplast DNA (kDNA), the mitochondrial DNA in kinetoplastids, is a network containing several thousand topologically interlocked minicirc les. We investigated cell cycle-dependent changes in the localization of kDNA replication enzymes by combining immunofluorescence with eithe r hydroxyurea synchronization or incorporation of fluorescein-dUTP int o the endogenous gaps of newly replicated minicircles. We found that w hile both topoisomerase II and DNA polymerase beta colocalize in two a ntipodal sites flanking the kDNA during replication, they behave diffe rently at other times. Polymerase beta is not detected by immunofluore scence either during cell division or G1, but is abruptly detected in the antipodal sites at the onset of kDNA replication. In contrast, top oisomerase II is localized to sites at the network edge at all cell cy cle stages; usually it is found in two antipodal sites, but during cyt okinesis each postscission daughter network is associated with only a single site. During the subsequent G1, topoisomerase accumulates in a second localization site, forming the characteristic antipodal pattern . These data suggest that these sites at the network periphery are per manent components of the mitochondrial architecture that function in k DNA replication.