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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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In situ analysis of kDNA replication in fixed cells from  an asynchronous culture. dUTP-F was incorporated into endogenous minicircle gaps by TdT as described in Materials and Methods. Top row, dUTP-F fluorescence of kDNA networks. Bottom  row, DAPI fluorescence. (a–c) Pre-replication networks. (d–i)  Replicating networks. (j–m) Postreplication networks. (n) Two  fully repaired, post scission networks, both in a cell undergoing  cytokinesis. The kDNA networks in c and i are positioned vertically. Images were captured with a Photometrics slow-scan CCD  camera using IPLab software. Networks in cells treated with proteinase K to reposition the network for a horizontal view are indicated with an asterisk. Treatment with proteinase K was modified  from a previous method (Ferguson et al., 1992). Before either immunofluorescence or dUTP-F labeling, fixed cells were treated at  room temperature for 15 min with 1 μg/ml Proteinase K in 10  mM Tris-HCl, 1 mM EDTA, 150 mM NaCl, and 0.1% SDS.  Slides were then given three 5-min washes in PBS containing 1 mM  PMSF, and one 5-min wash in PBS. Without protease treatment,  ∼8% of cells contain tipped networks. Treatment with protease  increases the frequency of horizontally positioned networks to  ∼25%. Bar, 1 μm.
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Figure 4: In situ analysis of kDNA replication in fixed cells from an asynchronous culture. dUTP-F was incorporated into endogenous minicircle gaps by TdT as described in Materials and Methods. Top row, dUTP-F fluorescence of kDNA networks. Bottom row, DAPI fluorescence. (a–c) Pre-replication networks. (d–i) Replicating networks. (j–m) Postreplication networks. (n) Two fully repaired, post scission networks, both in a cell undergoing cytokinesis. The kDNA networks in c and i are positioned vertically. Images were captured with a Photometrics slow-scan CCD camera using IPLab software. Networks in cells treated with proteinase K to reposition the network for a horizontal view are indicated with an asterisk. Treatment with proteinase K was modified from a previous method (Ferguson et al., 1992). Before either immunofluorescence or dUTP-F labeling, fixed cells were treated at room temperature for 15 min with 1 μg/ml Proteinase K in 10 mM Tris-HCl, 1 mM EDTA, 150 mM NaCl, and 0.1% SDS. Slides were then given three 5-min washes in PBS containing 1 mM PMSF, and one 5-min wash in PBS. Without protease treatment, ∼8% of cells contain tipped networks. Treatment with protease increases the frequency of horizontally positioned networks to ∼25%. Bar, 1 μm.

Mentions: Fig. 4 shows images of the kinetoplast region of dUTP-F–labeled cells from an asynchronous culture. In vivo, the kDNA network is condensed into a disk positioned perpendicularly to the flagellum, and therefore fluorescent images usually provide a view of the edge of the disk, as in Fig. 4, c and i. In fixed cells the kDNA disk is occasionally re-oriented, or “tipped,” providing a view of the face of the disk. The frequency of tipped networks can be increased by a mild protease treatment (examples in Fig. 4, e, g, and h) (Ferguson et al., 1992). Since images of a tipped disk are more informative about the degree of replication in a partially replicated network, we chose most examples in Fig. 4 with the disk tipped.


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

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

In situ analysis of kDNA replication in fixed cells from  an asynchronous culture. dUTP-F was incorporated into endogenous minicircle gaps by TdT as described in Materials and Methods. Top row, dUTP-F fluorescence of kDNA networks. Bottom  row, DAPI fluorescence. (a–c) Pre-replication networks. (d–i)  Replicating networks. (j–m) Postreplication networks. (n) Two  fully repaired, post scission networks, both in a cell undergoing  cytokinesis. The kDNA networks in c and i are positioned vertically. Images were captured with a Photometrics slow-scan CCD  camera using IPLab software. Networks in cells treated with proteinase K to reposition the network for a horizontal view are indicated with an asterisk. Treatment with proteinase K was modified  from a previous method (Ferguson et al., 1992). Before either immunofluorescence or dUTP-F labeling, fixed cells were treated at  room temperature for 15 min with 1 μg/ml Proteinase K in 10  mM Tris-HCl, 1 mM EDTA, 150 mM NaCl, and 0.1% SDS.  Slides were then given three 5-min washes in PBS containing 1 mM  PMSF, and one 5-min wash in PBS. Without protease treatment,  ∼8% of cells contain tipped networks. Treatment with protease  increases the frequency of horizontally positioned networks to  ∼25%. Bar, 1 μm.
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Related In: Results  -  Collection

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Figure 4: In situ analysis of kDNA replication in fixed cells from an asynchronous culture. dUTP-F was incorporated into endogenous minicircle gaps by TdT as described in Materials and Methods. Top row, dUTP-F fluorescence of kDNA networks. Bottom row, DAPI fluorescence. (a–c) Pre-replication networks. (d–i) Replicating networks. (j–m) Postreplication networks. (n) Two fully repaired, post scission networks, both in a cell undergoing cytokinesis. The kDNA networks in c and i are positioned vertically. Images were captured with a Photometrics slow-scan CCD camera using IPLab software. Networks in cells treated with proteinase K to reposition the network for a horizontal view are indicated with an asterisk. Treatment with proteinase K was modified from a previous method (Ferguson et al., 1992). Before either immunofluorescence or dUTP-F labeling, fixed cells were treated at room temperature for 15 min with 1 μg/ml Proteinase K in 10 mM Tris-HCl, 1 mM EDTA, 150 mM NaCl, and 0.1% SDS. Slides were then given three 5-min washes in PBS containing 1 mM PMSF, and one 5-min wash in PBS. Without protease treatment, ∼8% of cells contain tipped networks. Treatment with protease increases the frequency of horizontally positioned networks to ∼25%. Bar, 1 μm.
Mentions: Fig. 4 shows images of the kinetoplast region of dUTP-F–labeled cells from an asynchronous culture. In vivo, the kDNA network is condensed into a disk positioned perpendicularly to the flagellum, and therefore fluorescent images usually provide a view of the edge of the disk, as in Fig. 4, c and i. In fixed cells the kDNA disk is occasionally re-oriented, or “tipped,” providing a view of the face of the disk. The frequency of tipped networks can be increased by a mild protease treatment (examples in Fig. 4, e, g, and h) (Ferguson et al., 1992). Since images of a tipped disk are more informative about the degree of replication in a partially replicated network, we chose most examples in Fig. 4 with the disk tipped.

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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