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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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DNA primase localization during the C. fasciculata cell  cycle. Cells from a synchronized culture were processed for immunofluorescence and stained with DAPI. (a) Percentage of cells  undergoing cell division (closed circles), and percentage of cells  with DNA primase localized above and below the kDNA disk  (open triangles). (b) Localization of DNA primase during S  phase, 60 min after release from hydroxyurea arrest. (c) Localization of DNA primase from the peak of cell division, 120 min after  release from hydroxyurea arrest. The same localization of primase was seen at other time points. Left column, DNA primase  localization detected by goat anti–mouse antibody conjugated to  fluorescein. Right column, cells stained with DAPI to visualize  the brightly stained kDNA and dimly stained nuclear DNA. Images were captured as described in the legend of Fig. 1. Bar, 3 μm.
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Figure 7: DNA primase localization during the C. fasciculata cell cycle. Cells from a synchronized culture were processed for immunofluorescence and stained with DAPI. (a) Percentage of cells undergoing cell division (closed circles), and percentage of cells with DNA primase localized above and below the kDNA disk (open triangles). (b) Localization of DNA primase during S phase, 60 min after release from hydroxyurea arrest. (c) Localization of DNA primase from the peak of cell division, 120 min after release from hydroxyurea arrest. The same localization of primase was seen at other time points. Left column, DNA primase localization detected by goat anti–mouse antibody conjugated to fluorescein. Right column, cells stained with DAPI to visualize the brightly stained kDNA and dimly stained nuclear DNA. Images were captured as described in the legend of Fig. 1. Bar, 3 μm.

Mentions: Given the cell cycle variation in localization pattern of pol β and topo II, we were interested to determine whether the localization of enzymes not detected in the antipodal sites also varied during the cell cycle. To this end, we performed immunolocalization of the DNA primase in conjunction with hydroxyurea synchronization. In asynchronous cells, primase is localized to regions adjacent to the faces of the kDNA disk (Li and Englund, 1997). Using synchronized cultures we found that the primase is detectable in virtually all cells (Fig. 7 a), and is similarly localized to the two network faces at all stages of the cell cycle (see Fig. 7 b for examples of cells in S phase; and c for examples of dividing cells).


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

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

DNA primase localization during the C. fasciculata cell  cycle. Cells from a synchronized culture were processed for immunofluorescence and stained with DAPI. (a) Percentage of cells  undergoing cell division (closed circles), and percentage of cells  with DNA primase localized above and below the kDNA disk  (open triangles). (b) Localization of DNA primase during S  phase, 60 min after release from hydroxyurea arrest. (c) Localization of DNA primase from the peak of cell division, 120 min after  release from hydroxyurea arrest. The same localization of primase was seen at other time points. Left column, DNA primase  localization detected by goat anti–mouse antibody conjugated to  fluorescein. Right column, cells stained with DAPI to visualize  the brightly stained kDNA and dimly stained nuclear DNA. Images were captured as described in the legend of Fig. 1. Bar, 3 μm.
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Related In: Results  -  Collection

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Figure 7: DNA primase localization during the C. fasciculata cell cycle. Cells from a synchronized culture were processed for immunofluorescence and stained with DAPI. (a) Percentage of cells undergoing cell division (closed circles), and percentage of cells with DNA primase localized above and below the kDNA disk (open triangles). (b) Localization of DNA primase during S phase, 60 min after release from hydroxyurea arrest. (c) Localization of DNA primase from the peak of cell division, 120 min after release from hydroxyurea arrest. The same localization of primase was seen at other time points. Left column, DNA primase localization detected by goat anti–mouse antibody conjugated to fluorescein. Right column, cells stained with DAPI to visualize the brightly stained kDNA and dimly stained nuclear DNA. Images were captured as described in the legend of Fig. 1. Bar, 3 μm.
Mentions: Given the cell cycle variation in localization pattern of pol β and topo II, we were interested to determine whether the localization of enzymes not detected in the antipodal sites also varied during the cell cycle. To this end, we performed immunolocalization of the DNA primase in conjunction with hydroxyurea synchronization. In asynchronous cells, primase is localized to regions adjacent to the faces of the kDNA disk (Li and Englund, 1997). Using synchronized cultures we found that the primase is detectable in virtually all cells (Fig. 7 a), and is similarly localized to the two network faces at all stages of the cell cycle (see Fig. 7 b for examples of cells in S phase; and c for examples of dividing cells).

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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