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 minicircles. We investigated cell cycle–dependent changes in the localization of kDNA replication enzymes by combining immunofluorescence with either hydroxyurea synchronization or incorporation of fluorescein–dUTP into the endogenous gaps of newly replicated minicircles. We found that while both topoisomerase II and DNA polymerase β colocalize in two antipodal sites flanking the kDNA during replication, they behave differently at other times. Polymerase β is not detected by immunofluorescence either during cell division or G1, but is abruptly detected in the antipodal sites at the onset of kDNA replication. In contrast, topoisomerase II is localized to sites at the network edge at all cell cycle stages; usually it is found in two antipodal sites, but during cytokinesis 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 permanent components of the mitochondrial architecture that function in kDNA replication.