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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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Colocalization of pol β and topo II.  Cells from an asynchronous culture were fixed  and processed for immunofluorescence to detect  both pol β and topo II as described in Materials  and Methods. (a) DAPI visualization of the  dimly stained nucleus and brightly staining  kDNA. (b) Detection of pol β with goat anti– rabbit secondary antibody conjugated to Texas  red (Molecular Probes, Inc.). (c) Detection of  topo II with goat anti–mouse secondary antibody  conjugated to fluorescein (Boehringer Mannheim Corp.). Images were captured with a Photometrics slow-scan CCD camera (Tucson, AZ)  using IPLab software. Bar, 3 μm.
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Figure 1: Colocalization of pol β and topo II. Cells from an asynchronous culture were fixed and processed for immunofluorescence to detect both pol β and topo II as described in Materials and Methods. (a) DAPI visualization of the dimly stained nucleus and brightly staining kDNA. (b) Detection of pol β with goat anti– rabbit secondary antibody conjugated to Texas red (Molecular Probes, Inc.). (c) Detection of topo II with goat anti–mouse secondary antibody conjugated to fluorescein (Boehringer Mannheim Corp.). Images were captured with a Photometrics slow-scan CCD camera (Tucson, AZ) using IPLab software. Bar, 3 μm.

Mentions: We found, as previously reported, that in asynchronously-growing cells pol β and topo II have similar patterns of localization to two antipodal sites flanking the kDNA disk (Melendy et al., 1988; Ferguson et al., 1992). To further characterize the antipodal localizations of topo II and pol β, we used double-label immunofluorescence to probe both enzymes. We observed that pol β and topo II colocalized to the same sites at the network edge, as shown in Fig. 1. In addition, we observed that in some cells with topo II concentrated in these antipodal sites, a small amount of the enzyme is also detected either between these sites, in the region of the kDNA network (Fig. 2 d), or in a region adjacent to the flagellar face of the network (data not shown).


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

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

Colocalization of pol β and topo II.  Cells from an asynchronous culture were fixed  and processed for immunofluorescence to detect  both pol β and topo II as described in Materials  and Methods. (a) DAPI visualization of the  dimly stained nucleus and brightly staining  kDNA. (b) Detection of pol β with goat anti– rabbit secondary antibody conjugated to Texas  red (Molecular Probes, Inc.). (c) Detection of  topo II with goat anti–mouse secondary antibody  conjugated to fluorescein (Boehringer Mannheim Corp.). Images were captured with a Photometrics slow-scan CCD camera (Tucson, AZ)  using IPLab software. Bar, 3 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132953&req=5

Figure 1: Colocalization of pol β and topo II. Cells from an asynchronous culture were fixed and processed for immunofluorescence to detect both pol β and topo II as described in Materials and Methods. (a) DAPI visualization of the dimly stained nucleus and brightly staining kDNA. (b) Detection of pol β with goat anti– rabbit secondary antibody conjugated to Texas red (Molecular Probes, Inc.). (c) Detection of topo II with goat anti–mouse secondary antibody conjugated to fluorescein (Boehringer Mannheim Corp.). Images were captured with a Photometrics slow-scan CCD camera (Tucson, AZ) using IPLab software. Bar, 3 μm.
Mentions: We found, as previously reported, that in asynchronously-growing cells pol β and topo II have similar patterns of localization to two antipodal sites flanking the kDNA disk (Melendy et al., 1988; Ferguson et al., 1992). To further characterize the antipodal localizations of topo II and pol β, we used double-label immunofluorescence to probe both enzymes. We observed that pol β and topo II colocalized to the same sites at the network edge, as shown in Fig. 1. In addition, we observed that in some cells with topo II concentrated in these antipodal sites, a small amount of the enzyme is also detected either between these sites, in the region of the kDNA network (Fig. 2 d), or in a region adjacent to the flagellar face of the network (data not shown).

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