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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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Steady-state level  of pol β during the C. fasciculata cell cycle. (a) Western  blot of whole cell lysates (5  μg protein per lane) made at  30-min intervals from a synchronized culture. Blots were  probed with anti–pol β serum and 125I-labeled protein  A. Control experiments indicated that PhosphorImager  signals are in the linear  range. (b) Percentage of cells undergoing cell division in the same  culture as determined by DAPI fluorescence and phase microscopy.
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Figure 3: Steady-state level of pol β during the C. fasciculata cell cycle. (a) Western blot of whole cell lysates (5 μg protein per lane) made at 30-min intervals from a synchronized culture. Blots were probed with anti–pol β serum and 125I-labeled protein A. Control experiments indicated that PhosphorImager signals are in the linear range. (b) Percentage of cells undergoing cell division in the same culture as determined by DAPI fluorescence and phase microscopy.

Mentions: Our inability to detect pol β immunofluorescence in the population of dividing cells raised the possibility that the enzyme abundance may change during the cell cycle. To address this issue, we performed Western blots on whole cell lysates taken at 30 min intervals during synchronized growth of a C. fasciculata culture. We found that the enzyme is present at similar levels in all time points (Fig. 3 a). Thus while the pol β protein is continuously present, it is detectable by immunofluorescence only at certain cell cycle stages. Possible explanations of the inability to immunolocalize this enzyme will be addressed in Discussion.


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

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

Steady-state level  of pol β during the C. fasciculata cell cycle. (a) Western  blot of whole cell lysates (5  μg protein per lane) made at  30-min intervals from a synchronized culture. Blots were  probed with anti–pol β serum and 125I-labeled protein  A. Control experiments indicated that PhosphorImager  signals are in the linear  range. (b) Percentage of cells undergoing cell division in the same  culture as determined by DAPI fluorescence and phase microscopy.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Steady-state level of pol β during the C. fasciculata cell cycle. (a) Western blot of whole cell lysates (5 μg protein per lane) made at 30-min intervals from a synchronized culture. Blots were probed with anti–pol β serum and 125I-labeled protein A. Control experiments indicated that PhosphorImager signals are in the linear range. (b) Percentage of cells undergoing cell division in the same culture as determined by DAPI fluorescence and phase microscopy.
Mentions: Our inability to detect pol β immunofluorescence in the population of dividing cells raised the possibility that the enzyme abundance may change during the cell cycle. To address this issue, we performed Western blots on whole cell lysates taken at 30 min intervals during synchronized growth of a C. fasciculata culture. We found that the enzyme is present at similar levels in all time points (Fig. 3 a). Thus while the pol β protein is continuously present, it is detectable by immunofluorescence only at certain cell cycle stages. Possible explanations of the inability to immunolocalize this enzyme will be addressed in Discussion.

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