Limits...
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.

Show MeSH
Localization of pol β and topo II in hydroxyurea synchronized C. fasciculata. (a) Cells were treated with hydroxyurea and released into fresh medium at 0 min. Samples were analyzed as described in Materials and Methods for cell division (closed circles), cell  density (open triangles), and for the rate of DNA synthesis (open squares). (b) Cells from another synchronized culture were processed  for immunofluorescence to detect pol β and topo II and stained with DAPI. The percentage of cells undergoing division (closed circles),  percentage of cells with topo II detected in two antipodal sites (open squares), and percentage of cells with pol β detected in two antipodal sites (closed triangles) are shown. At least 200 randomly chosen cells per time point were scored. Results were confirmed three  times, and data shown are from a representative experiment. (c and d) Localization of pol β (c) and topo II (d) in cells synchronized  with hydroxyurea. The pol β images are from the synchronization in a, and those of topo II are from the experiment in b. Top row, cells  during interphase, 210 min after release from hydroxyurea arrest; bottom row, cells during the peak of cell division, 120 min after release  from hydroxyurea arrest. Control experiments showed that the weak signal covering the entire cell results from non-specific binding of  the secondary antibody (data not shown). Images were captured with a Zeiss 35 mm camera. Bar, 3 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132953&req=5

Figure 2: Localization of pol β and topo II in hydroxyurea synchronized C. fasciculata. (a) Cells were treated with hydroxyurea and released into fresh medium at 0 min. Samples were analyzed as described in Materials and Methods for cell division (closed circles), cell density (open triangles), and for the rate of DNA synthesis (open squares). (b) Cells from another synchronized culture were processed for immunofluorescence to detect pol β and topo II and stained with DAPI. The percentage of cells undergoing division (closed circles), percentage of cells with topo II detected in two antipodal sites (open squares), and percentage of cells with pol β detected in two antipodal sites (closed triangles) are shown. At least 200 randomly chosen cells per time point were scored. Results were confirmed three times, and data shown are from a representative experiment. (c and d) Localization of pol β (c) and topo II (d) in cells synchronized with hydroxyurea. The pol β images are from the synchronization in a, and those of topo II are from the experiment in b. Top row, cells during interphase, 210 min after release from hydroxyurea arrest; bottom row, cells during the peak of cell division, 120 min after release from hydroxyurea arrest. Control experiments showed that the weak signal covering the entire cell results from non-specific binding of the secondary antibody (data not shown). Images were captured with a Zeiss 35 mm camera. 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)

Localization of pol β and topo II in hydroxyurea synchronized C. fasciculata. (a) Cells were treated with hydroxyurea and released into fresh medium at 0 min. Samples were analyzed as described in Materials and Methods for cell division (closed circles), cell  density (open triangles), and for the rate of DNA synthesis (open squares). (b) Cells from another synchronized culture were processed  for immunofluorescence to detect pol β and topo II and stained with DAPI. The percentage of cells undergoing division (closed circles),  percentage of cells with topo II detected in two antipodal sites (open squares), and percentage of cells with pol β detected in two antipodal sites (closed triangles) are shown. At least 200 randomly chosen cells per time point were scored. Results were confirmed three  times, and data shown are from a representative experiment. (c and d) Localization of pol β (c) and topo II (d) in cells synchronized  with hydroxyurea. The pol β images are from the synchronization in a, and those of topo II are from the experiment in b. Top row, cells  during interphase, 210 min after release from hydroxyurea arrest; bottom row, cells during the peak of cell division, 120 min after release  from hydroxyurea arrest. Control experiments showed that the weak signal covering the entire cell results from non-specific binding of  the secondary antibody (data not shown). Images were captured with a Zeiss 35 mm camera. Bar, 3 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Localization of pol β and topo II in hydroxyurea synchronized C. fasciculata. (a) Cells were treated with hydroxyurea and released into fresh medium at 0 min. Samples were analyzed as described in Materials and Methods for cell division (closed circles), cell density (open triangles), and for the rate of DNA synthesis (open squares). (b) Cells from another synchronized culture were processed for immunofluorescence to detect pol β and topo II and stained with DAPI. The percentage of cells undergoing division (closed circles), percentage of cells with topo II detected in two antipodal sites (open squares), and percentage of cells with pol β detected in two antipodal sites (closed triangles) are shown. At least 200 randomly chosen cells per time point were scored. Results were confirmed three times, and data shown are from a representative experiment. (c and d) Localization of pol β (c) and topo II (d) in cells synchronized with hydroxyurea. The pol β images are from the synchronization in a, and those of topo II are from the experiment in b. Top row, cells during interphase, 210 min after release from hydroxyurea arrest; bottom row, cells during the peak of cell division, 120 min after release from hydroxyurea arrest. Control experiments showed that the weak signal covering the entire cell results from non-specific binding of the secondary antibody (data not shown). Images were captured with a Zeiss 35 mm camera. 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