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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 topo II 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. Topo II was  visualized with goat anti–mouse  antibody conjugated to Cy3  (Boehringer Mannheim Corp.).  Images were captured as in Fig.  4. Top row, topo II immunofluorescence. Middle row, dUTP-F  fluorescence of kDNA networks. Bottom row, DAPI fluorescence of kDNA networks.  (a–d) Pre-replication networks.  (e–g) Replicating kDNA networks. (h–k) Postreplication  kDNA networks. (l–n) Cells containing two kDNA networks.  Bar, 1 μm.
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Figure 6: Analysis of topo II 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. Topo II was visualized with goat anti–mouse antibody conjugated to Cy3 (Boehringer Mannheim Corp.). Images were captured as in Fig. 4. Top row, topo II immunofluorescence. Middle row, dUTP-F fluorescence of kDNA networks. Bottom row, DAPI fluorescence of kDNA networks. (a–d) Pre-replication networks. (e–g) Replicating kDNA networks. (h–k) Postreplication kDNA networks. (l–n) Cells containing two kDNA networks. Bar, 1 μm.

Mentions: At certain stages of kDNA replication the pattern of topo II localization in cells labeled with dUTP-F differed significantly from that of pol β (Table I; Fig. 6). Before the onset of kDNA replication all cells had topo II detectable in the kinetoplast region; in 22% topo II was found to be diffuse throughout the kDNA region (Fig. 6 a). In nearly half (43%) of pre-replication cells topo II was localized to both antipodal sites (Fig. 6 d), but surprisingly, 35% had topo II concentrated at only a single site at the network edge (Fig. 6, b and c).


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

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

Analysis of topo II 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. Topo II was  visualized with goat anti–mouse  antibody conjugated to Cy3  (Boehringer Mannheim Corp.).  Images were captured as in Fig.  4. Top row, topo II immunofluorescence. Middle row, dUTP-F  fluorescence of kDNA networks. Bottom row, DAPI fluorescence of kDNA networks.  (a–d) Pre-replication networks.  (e–g) Replicating kDNA networks. (h–k) Postreplication  kDNA networks. (l–n) Cells containing two kDNA networks.  Bar, 1 μm.
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Related In: Results  -  Collection

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Figure 6: Analysis of topo II 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. Topo II was visualized with goat anti–mouse antibody conjugated to Cy3 (Boehringer Mannheim Corp.). Images were captured as in Fig. 4. Top row, topo II immunofluorescence. Middle row, dUTP-F fluorescence of kDNA networks. Bottom row, DAPI fluorescence of kDNA networks. (a–d) Pre-replication networks. (e–g) Replicating kDNA networks. (h–k) Postreplication kDNA networks. (l–n) Cells containing two kDNA networks. Bar, 1 μm.
Mentions: At certain stages of kDNA replication the pattern of topo II localization in cells labeled with dUTP-F differed significantly from that of pol β (Table I; Fig. 6). Before the onset of kDNA replication all cells had topo II detectable in the kinetoplast region; in 22% topo II was found to be diffuse throughout the kDNA region (Fig. 6 a). In nearly half (43%) of pre-replication cells topo II was localized to both antipodal sites (Fig. 6 d), but surprisingly, 35% had topo II concentrated at only a single site at the network edge (Fig. 6, b and c).

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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