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Changes in organization of Crithidia fasciculata kinetoplast DNA replication proteins during the cell cycle.

Johnson CE, Englund PT - J. Cell Biol. (1998)

Bottom Line: We found that while both topoisomerase II and DNA polymerase beta colocalize in two antipodal sites flanking the kDNA during replication, they behave differently at other times.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.These data suggest that these sites at the network periphery are permanent components of the mitochondrial architecture that function in kDNA replication.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, Johns Hopkins School of Medicine, Baltimore, Maryland 21205, USA.

ABSTRACT
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 beta colocalize in two antipodal sites flanking the kDNA during replication, they behave differently at other times. Polymerase beta 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.

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Analysis of pol β localization in asynchronous cells  labeled with dUTP-F. dUTP-F  was incorporated into endogenous gaps in kDNA by TdT and  cells were then processed for  immunofluorescence. Pol β was  visualized with goat anti–rabbit  antibody conjugated to Texas  red (Molecular Probes, Inc.).  Images were captured as in Fig.  4. Top row, pol β immunofluorescence. Middle row, dUTP-F  fluorescence of kDNA networks. Bottom row, DAPI fluorescence of kDNA networks. (a and b) Pre-replication networks. (c–e) Replicating networks. (f–h) Post-replication networks. (i) Postscission kDNA networks in a cell undergoing cytokinesis. Bar, 1 μm.
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Figure 5: Analysis of pol β localization in asynchronous cells labeled with dUTP-F. dUTP-F was incorporated into endogenous gaps in kDNA by TdT and cells were then processed for immunofluorescence. Pol β was visualized with goat anti–rabbit antibody conjugated to Texas red (Molecular Probes, Inc.). Images were captured as in Fig. 4. Top row, pol β immunofluorescence. Middle row, dUTP-F fluorescence of kDNA networks. Bottom row, DAPI fluorescence of kDNA networks. (a and b) Pre-replication networks. (c–e) Replicating networks. (f–h) Post-replication networks. (i) Postscission kDNA networks in a cell undergoing cytokinesis. Bar, 1 μm.

Mentions: We used immunofluorescence to determine the location of pol β in cells that had been first labeled with dUTP-F to determine the stage of kDNA replication (see Table I and Fig. 5). In cells which had not initiated kDNA replication, we detected no pol β immunofluorescence (Fig. 5, a–b). In contrast, 100% of the cells undergoing kDNA replication had pol β localized in the two antipodal sites (Fig. 5, c–e) that co-localize with the two bright spots of dUTP-F fluorescence. After replication is complete, different patterns of pol β localization were observed. In 54% of these cells, pol β was found in the two antipodal sites (Fig. 5 f). At this stage of kDNA replication, some weak pol β signal is also detected in the kDNA region (Fig. 5 f). The rest of the post-replication networks either contain no detectable pol β immunofluorescence (33%, Fig. 5 h) or the enzyme is in other locations (13% of post-replication cells, see Fig. 5 g). Finally, in dividing cells which have two daughter kDNA networks (following gap repair and network scission), none showed detectable pol β immunofluorescence (Fig. 5 i).


Changes in organization of Crithidia fasciculata kinetoplast DNA replication proteins during the cell cycle.

Johnson CE, Englund PT - J. Cell Biol. (1998)

Analysis of pol β localization in asynchronous cells  labeled with dUTP-F. dUTP-F  was incorporated into endogenous gaps in kDNA by TdT and  cells were then processed for  immunofluorescence. Pol β was  visualized with goat anti–rabbit  antibody conjugated to Texas  red (Molecular Probes, Inc.).  Images were captured as in Fig.  4. Top row, pol β immunofluorescence. Middle row, dUTP-F  fluorescence of kDNA networks. Bottom row, DAPI fluorescence of kDNA networks. (a and b) Pre-replication networks. (c–e) Replicating networks. (f–h) Post-replication networks. (i) Postscission kDNA networks in a cell undergoing cytokinesis. Bar, 1 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132953&req=5

Figure 5: Analysis of pol β localization in asynchronous cells labeled with dUTP-F. dUTP-F was incorporated into endogenous gaps in kDNA by TdT and cells were then processed for immunofluorescence. Pol β was visualized with goat anti–rabbit antibody conjugated to Texas red (Molecular Probes, Inc.). Images were captured as in Fig. 4. Top row, pol β immunofluorescence. Middle row, dUTP-F fluorescence of kDNA networks. Bottom row, DAPI fluorescence of kDNA networks. (a and b) Pre-replication networks. (c–e) Replicating networks. (f–h) Post-replication networks. (i) Postscission kDNA networks in a cell undergoing cytokinesis. Bar, 1 μm.
Mentions: We used immunofluorescence to determine the location of pol β in cells that had been first labeled with dUTP-F to determine the stage of kDNA replication (see Table I and Fig. 5). In cells which had not initiated kDNA replication, we detected no pol β immunofluorescence (Fig. 5, a–b). In contrast, 100% of the cells undergoing kDNA replication had pol β localized in the two antipodal sites (Fig. 5, c–e) that co-localize with the two bright spots of dUTP-F fluorescence. After replication is complete, different patterns of pol β localization were observed. In 54% of these cells, pol β was found in the two antipodal sites (Fig. 5 f). At this stage of kDNA replication, some weak pol β signal is also detected in the kDNA region (Fig. 5 f). The rest of the post-replication networks either contain no detectable pol β immunofluorescence (33%, Fig. 5 h) or the enzyme is in other locations (13% of post-replication cells, see Fig. 5 g). Finally, in dividing cells which have two daughter kDNA networks (following gap repair and network scission), none showed detectable pol β immunofluorescence (Fig. 5 i).

Bottom Line: We found that while both topoisomerase II and DNA polymerase beta colocalize in two antipodal sites flanking the kDNA during replication, they behave differently at other times.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.These data suggest that these sites at the network periphery are permanent components of the mitochondrial architecture that function in kDNA replication.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, Johns Hopkins School of Medicine, Baltimore, Maryland 21205, USA.

ABSTRACT
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 beta colocalize in two antipodal sites flanking the kDNA during replication, they behave differently at other times. Polymerase beta 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.

Show MeSH