Limits...
Chromosome association of minichromosome maintenance proteins in Drosophila mitotic cycles.

Su TT, O'Farrell PH - J. Cell Biol. (1997)

Bottom Line: Arrest of mitosis by induced expression of nondegradable forms of cyclins A and/or B showed that reassociation of MCMs to chromatin requires cyclin A destruction but not cyclin B destruction.In contrast to the earlier mitoses, mitosis 16 (M16) is followed by G1, and MCMs do not reassociate with chromatin at the end of M16. dacapo mutant embryos lack an inhibitor of cyclin E, do not enter G1 quiescence after M16, and show mitotic reassociation of MCM proteins.We propose that cyclin E, inhibited by Dacapo in M16, promotes chromosome binding of MCMs. We suggest that cyclins have both positive and negative roles in controlling MCM-chromatin association.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, University of California San Francisco 94143-0448, USA.

ABSTRACT
Minichromosome maintenance (MCM) proteins are essential DNA replication factors conserved among eukaryotes. MCMs cycle between chromatin bound and dissociated states during each cell cycle. Their absence on chromatin is thought to contribute to the inability of a G2 nucleus to replicate DNA. Passage through mitosis restores the ability of MCMs to bind chromatin and the ability to replicate DNA. In Drosophila early embryonic cell cycles, which lack a G1 phase, MCMs reassociate with condensed chromosomes toward the end of mitosis. To explore the coupling between mitosis and MCM-chromatin interaction, we tested whether this reassociation requires mitotic degradation of cyclins. Arrest of mitosis by induced expression of nondegradable forms of cyclins A and/or B showed that reassociation of MCMs to chromatin requires cyclin A destruction but not cyclin B destruction. In contrast to the earlier mitoses, mitosis 16 (M16) is followed by G1, and MCMs do not reassociate with chromatin at the end of M16. dacapo mutant embryos lack an inhibitor of cyclin E, do not enter G1 quiescence after M16, and show mitotic reassociation of MCM proteins. We propose that cyclin E, inhibited by Dacapo in M16, promotes chromosome binding of MCMs. We suggest that cyclins have both positive and negative roles in controlling MCM-chromatin association.

Show MeSH

Related in: MedlinePlus

The effect of stable cyclins on association of DmMCM2  with chromosomes in mitosis. Embryos carrying heat-inducible  transgenes for cyclin As (As), cyclin Bs (Bs), or both (As+Bs) were  fixed and stained for DmMCM2 and DNA after heat induction.  Cells indicated by arrowheads in the top two panels are magnified two times and shown in the bottom two panels. Note that regions occupied by chromosomes in panels 2 and 3 are devoid of  DmMCM2 stain. The local exclusion of the stain is more obvious  in a cyclin As arrest than in an arrest caused by coexpression of  cyclins As and Bs, perhaps because the chromosomes are more  tightly clustered in the former case. In contrast, cells arrested  with cyclin Bs (panel 1) showed no exclusion of the stain from  chromosomes, and in many cells, MCM stain on the chromosomes substantially exceeds the cytoplasmic signal (panel 1, arrowheads). Bar, 20 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139827&req=5

Figure 2: The effect of stable cyclins on association of DmMCM2 with chromosomes in mitosis. Embryos carrying heat-inducible transgenes for cyclin As (As), cyclin Bs (Bs), or both (As+Bs) were fixed and stained for DmMCM2 and DNA after heat induction. Cells indicated by arrowheads in the top two panels are magnified two times and shown in the bottom two panels. Note that regions occupied by chromosomes in panels 2 and 3 are devoid of DmMCM2 stain. The local exclusion of the stain is more obvious in a cyclin As arrest than in an arrest caused by coexpression of cyclins As and Bs, perhaps because the chromosomes are more tightly clustered in the former case. In contrast, cells arrested with cyclin Bs (panel 1) showed no exclusion of the stain from chromosomes, and in many cells, MCM stain on the chromosomes substantially exceeds the cytoplasmic signal (panel 1, arrowheads). Bar, 20 μm.

Mentions: To test if MCM–chromosome association requires cyclin degradation, we analyzed three Drosophila MCMs after induction of stable cyclins in cell cycles 14 and 15. All three MCMs dispersed from the nucleus upon entry to mitosis in the presence of stable cyclins, as they do in normal mitoses (not shown). During arrests in M14 and M15, reassociation with chromosomes occurred in the presence of cyclin Bs but not cyclin As (Fig. 2; data not shown). In the presence of cyclin As, DmMCM2 staining was uniform throughout the cell, except for exclusion from the region occupied by chromosomes (Fig. 2, panel 2, arrowheads). This is similar to what is seen in metaphases of wild-type embryos and heat-shocked w67 controls lacking the transgenes (not shown). In contrast, in the presence of cyclin Bs, no such clearing of the DmMCM2 stain from the chromosomal region was seen. In most cells, colocalization of DmMCM2 stain and DNA stain is observed, suggesting concentration of DmMCM2 on chromosomes; this is similar to chromosomes in late anaphase of wild-type mitoses (e.g., Fig. 1, F–H, white arrows). The intensity of DmMCM2 stain on the chromosomes varied from cell to cell; this may be because cells of a Drosophila embryo undergo M14 and M15 asynchronously (Foe, 1989) and had been arrested in mitosis for various lengths of time at fixation.


Chromosome association of minichromosome maintenance proteins in Drosophila mitotic cycles.

Su TT, O'Farrell PH - J. Cell Biol. (1997)

The effect of stable cyclins on association of DmMCM2  with chromosomes in mitosis. Embryos carrying heat-inducible  transgenes for cyclin As (As), cyclin Bs (Bs), or both (As+Bs) were  fixed and stained for DmMCM2 and DNA after heat induction.  Cells indicated by arrowheads in the top two panels are magnified two times and shown in the bottom two panels. Note that regions occupied by chromosomes in panels 2 and 3 are devoid of  DmMCM2 stain. The local exclusion of the stain is more obvious  in a cyclin As arrest than in an arrest caused by coexpression of  cyclins As and Bs, perhaps because the chromosomes are more  tightly clustered in the former case. In contrast, cells arrested  with cyclin Bs (panel 1) showed no exclusion of the stain from  chromosomes, and in many cells, MCM stain on the chromosomes substantially exceeds the cytoplasmic signal (panel 1, arrowheads). Bar, 20 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139827&req=5

Figure 2: The effect of stable cyclins on association of DmMCM2 with chromosomes in mitosis. Embryos carrying heat-inducible transgenes for cyclin As (As), cyclin Bs (Bs), or both (As+Bs) were fixed and stained for DmMCM2 and DNA after heat induction. Cells indicated by arrowheads in the top two panels are magnified two times and shown in the bottom two panels. Note that regions occupied by chromosomes in panels 2 and 3 are devoid of DmMCM2 stain. The local exclusion of the stain is more obvious in a cyclin As arrest than in an arrest caused by coexpression of cyclins As and Bs, perhaps because the chromosomes are more tightly clustered in the former case. In contrast, cells arrested with cyclin Bs (panel 1) showed no exclusion of the stain from chromosomes, and in many cells, MCM stain on the chromosomes substantially exceeds the cytoplasmic signal (panel 1, arrowheads). Bar, 20 μm.
Mentions: To test if MCM–chromosome association requires cyclin degradation, we analyzed three Drosophila MCMs after induction of stable cyclins in cell cycles 14 and 15. All three MCMs dispersed from the nucleus upon entry to mitosis in the presence of stable cyclins, as they do in normal mitoses (not shown). During arrests in M14 and M15, reassociation with chromosomes occurred in the presence of cyclin Bs but not cyclin As (Fig. 2; data not shown). In the presence of cyclin As, DmMCM2 staining was uniform throughout the cell, except for exclusion from the region occupied by chromosomes (Fig. 2, panel 2, arrowheads). This is similar to what is seen in metaphases of wild-type embryos and heat-shocked w67 controls lacking the transgenes (not shown). In contrast, in the presence of cyclin Bs, no such clearing of the DmMCM2 stain from the chromosomal region was seen. In most cells, colocalization of DmMCM2 stain and DNA stain is observed, suggesting concentration of DmMCM2 on chromosomes; this is similar to chromosomes in late anaphase of wild-type mitoses (e.g., Fig. 1, F–H, white arrows). The intensity of DmMCM2 stain on the chromosomes varied from cell to cell; this may be because cells of a Drosophila embryo undergo M14 and M15 asynchronously (Foe, 1989) and had been arrested in mitosis for various lengths of time at fixation.

Bottom Line: Arrest of mitosis by induced expression of nondegradable forms of cyclins A and/or B showed that reassociation of MCMs to chromatin requires cyclin A destruction but not cyclin B destruction.In contrast to the earlier mitoses, mitosis 16 (M16) is followed by G1, and MCMs do not reassociate with chromatin at the end of M16. dacapo mutant embryos lack an inhibitor of cyclin E, do not enter G1 quiescence after M16, and show mitotic reassociation of MCM proteins.We propose that cyclin E, inhibited by Dacapo in M16, promotes chromosome binding of MCMs. We suggest that cyclins have both positive and negative roles in controlling MCM-chromatin association.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, University of California San Francisco 94143-0448, USA.

ABSTRACT
Minichromosome maintenance (MCM) proteins are essential DNA replication factors conserved among eukaryotes. MCMs cycle between chromatin bound and dissociated states during each cell cycle. Their absence on chromatin is thought to contribute to the inability of a G2 nucleus to replicate DNA. Passage through mitosis restores the ability of MCMs to bind chromatin and the ability to replicate DNA. In Drosophila early embryonic cell cycles, which lack a G1 phase, MCMs reassociate with condensed chromosomes toward the end of mitosis. To explore the coupling between mitosis and MCM-chromatin interaction, we tested whether this reassociation requires mitotic degradation of cyclins. Arrest of mitosis by induced expression of nondegradable forms of cyclins A and/or B showed that reassociation of MCMs to chromatin requires cyclin A destruction but not cyclin B destruction. In contrast to the earlier mitoses, mitosis 16 (M16) is followed by G1, and MCMs do not reassociate with chromatin at the end of M16. dacapo mutant embryos lack an inhibitor of cyclin E, do not enter G1 quiescence after M16, and show mitotic reassociation of MCM proteins. We propose that cyclin E, inhibited by Dacapo in M16, promotes chromosome binding of MCMs. We suggest that cyclins have both positive and negative roles in controlling MCM-chromatin association.

Show MeSH
Related in: MedlinePlus