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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.

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MCM–chromosome  association in M16 in dacapo  mutants is detected by histochemistry. Homozygous dap  mutant embryos and their  wild-type or heterozygous siblings were fixed and stained  for DmMCM2 and DNA.  Histochemical staining used  for detection of DmMCM2  results in a dark-colored deposit that quenches the DNA  stain when DmMCM2 and  DNA are colocalized. Lateral views of the posterior  third of stage 11 embryos are  shown in A and B. Many of  the epidermal cells in this  view are completing M16  (dorsolateral epidermis). (A)  In wild type or heterozygotes, identified by β-galactosidase stain in the wingless  pattern (arrowheads), many pairs of anaphase/telophase chromosomes lack the DmMCM2 stain and are seen as brightly fluorescent  (boxes show examples magnified in panels 1–3). This is similar to what is seen for DmMCM5 in wild-type embryos undergoing M16  (Fig. 4 B). (B) In contrast, in dap homozygous mutants, identified by the absence of β-galactosidase stain, many anaphase/telophase  chromosomes acquire DmMCM2 stain, which quenches the DNA signal. Consequently, chromosome pairs (boxes) are not readily visible, except when magnified as in panels 4–6. Bar, 20 μm in A and B; 7 μm in panels 1–6.
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Figure 5: MCM–chromosome association in M16 in dacapo mutants is detected by histochemistry. Homozygous dap mutant embryos and their wild-type or heterozygous siblings were fixed and stained for DmMCM2 and DNA. Histochemical staining used for detection of DmMCM2 results in a dark-colored deposit that quenches the DNA stain when DmMCM2 and DNA are colocalized. Lateral views of the posterior third of stage 11 embryos are shown in A and B. Many of the epidermal cells in this view are completing M16 (dorsolateral epidermis). (A) In wild type or heterozygotes, identified by β-galactosidase stain in the wingless pattern (arrowheads), many pairs of anaphase/telophase chromosomes lack the DmMCM2 stain and are seen as brightly fluorescent (boxes show examples magnified in panels 1–3). This is similar to what is seen for DmMCM5 in wild-type embryos undergoing M16 (Fig. 4 B). (B) In contrast, in dap homozygous mutants, identified by the absence of β-galactosidase stain, many anaphase/telophase chromosomes acquire DmMCM2 stain, which quenches the DNA signal. Consequently, chromosome pairs (boxes) are not readily visible, except when magnified as in panels 4–6. Bar, 20 μm in A and B; 7 μm in panels 1–6.

Mentions: Analysis of dacapo (dap) mutants suggests that this gene has a role in MCM–chromosome association in M16. dap encodes a p21/p27 type cdk inhibitor (Dap) whose developmentally regulated expression just before M16 contributes to the withdrawal of epidermal cells from the cell cycle (de Nooij et al., 1997; Lane et al., 1997). In homozygous dap mutants, many epidermal cells enter an additional S phase (S17) immediately after M16. Analysis of DmMCM2 in dap homozygotes showed that many late anaphase/telophase chromosomes acquire DmMCM2 in M16, unlike those in M16 of wild-type or heterozygous sibling embryos (Fig. 5). This is illustrated most clearly in embryos in which the DNA has been stained with the fluorescent dye Hoechst 33258 and DmMCM2 has been stained immunohistochemically. When the MCMs colocalize with DNA, the histochemical stain efficiently quenches the fluorescent staining (e.g., see Fig. 4). Focusing on the bright cells, one can see that the later stages of mitosis are not quenched in cell cycle 16 of wild-type or heterozygous dap embryos, but they are quenched in homozygous dap mutant embryos (Fig. 5). For example, late anaphase/telophase nuclei in wild type/heterozygotes are devoid of histochemical DmMCM2 stain and are clearly visible when visualized for DNA (Fig. 5 A, boxes, and panels 1–3). In contrast, late anaphase/telophase nuclei in homozygous dap mutants have acquired the DmMCM2 stain and are barely visible when visualized for DNA (Fig. 5 B and panels 4–6). Not all anaphase/telophase figures acquire MCM stain in dap mutants (e.g., Fig. 5 B, arrowhead). Likewise, not all cells of the epidermis undergo S17 in a homozygous dap mutant (de Nooij et al., 1997; Lane et al., 1997). Direct analysis of DmMCM2 immunofluorescent staining also revealed association of MCMs with late anaphase/telophase nuclei of homozygous dap mutants (not shown). These data suggest that expression of Dap in cycle 16 normally inhibits chromosome association of DmMCM2.


Chromosome association of minichromosome maintenance proteins in Drosophila mitotic cycles.

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

MCM–chromosome  association in M16 in dacapo  mutants is detected by histochemistry. Homozygous dap  mutant embryos and their  wild-type or heterozygous siblings were fixed and stained  for DmMCM2 and DNA.  Histochemical staining used  for detection of DmMCM2  results in a dark-colored deposit that quenches the DNA  stain when DmMCM2 and  DNA are colocalized. Lateral views of the posterior  third of stage 11 embryos are  shown in A and B. Many of  the epidermal cells in this  view are completing M16  (dorsolateral epidermis). (A)  In wild type or heterozygotes, identified by β-galactosidase stain in the wingless  pattern (arrowheads), many pairs of anaphase/telophase chromosomes lack the DmMCM2 stain and are seen as brightly fluorescent  (boxes show examples magnified in panels 1–3). This is similar to what is seen for DmMCM5 in wild-type embryos undergoing M16  (Fig. 4 B). (B) In contrast, in dap homozygous mutants, identified by the absence of β-galactosidase stain, many anaphase/telophase  chromosomes acquire DmMCM2 stain, which quenches the DNA signal. Consequently, chromosome pairs (boxes) are not readily visible, except when magnified as in panels 4–6. Bar, 20 μm in A and B; 7 μm in panels 1–6.
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Figure 5: MCM–chromosome association in M16 in dacapo mutants is detected by histochemistry. Homozygous dap mutant embryos and their wild-type or heterozygous siblings were fixed and stained for DmMCM2 and DNA. Histochemical staining used for detection of DmMCM2 results in a dark-colored deposit that quenches the DNA stain when DmMCM2 and DNA are colocalized. Lateral views of the posterior third of stage 11 embryos are shown in A and B. Many of the epidermal cells in this view are completing M16 (dorsolateral epidermis). (A) In wild type or heterozygotes, identified by β-galactosidase stain in the wingless pattern (arrowheads), many pairs of anaphase/telophase chromosomes lack the DmMCM2 stain and are seen as brightly fluorescent (boxes show examples magnified in panels 1–3). This is similar to what is seen for DmMCM5 in wild-type embryos undergoing M16 (Fig. 4 B). (B) In contrast, in dap homozygous mutants, identified by the absence of β-galactosidase stain, many anaphase/telophase chromosomes acquire DmMCM2 stain, which quenches the DNA signal. Consequently, chromosome pairs (boxes) are not readily visible, except when magnified as in panels 4–6. Bar, 20 μm in A and B; 7 μm in panels 1–6.
Mentions: Analysis of dacapo (dap) mutants suggests that this gene has a role in MCM–chromosome association in M16. dap encodes a p21/p27 type cdk inhibitor (Dap) whose developmentally regulated expression just before M16 contributes to the withdrawal of epidermal cells from the cell cycle (de Nooij et al., 1997; Lane et al., 1997). In homozygous dap mutants, many epidermal cells enter an additional S phase (S17) immediately after M16. Analysis of DmMCM2 in dap homozygotes showed that many late anaphase/telophase chromosomes acquire DmMCM2 in M16, unlike those in M16 of wild-type or heterozygous sibling embryos (Fig. 5). This is illustrated most clearly in embryos in which the DNA has been stained with the fluorescent dye Hoechst 33258 and DmMCM2 has been stained immunohistochemically. When the MCMs colocalize with DNA, the histochemical stain efficiently quenches the fluorescent staining (e.g., see Fig. 4). Focusing on the bright cells, one can see that the later stages of mitosis are not quenched in cell cycle 16 of wild-type or heterozygous dap embryos, but they are quenched in homozygous dap mutant embryos (Fig. 5). For example, late anaphase/telophase nuclei in wild type/heterozygotes are devoid of histochemical DmMCM2 stain and are clearly visible when visualized for DNA (Fig. 5 A, boxes, and panels 1–3). In contrast, late anaphase/telophase nuclei in homozygous dap mutants have acquired the DmMCM2 stain and are barely visible when visualized for DNA (Fig. 5 B and panels 4–6). Not all anaphase/telophase figures acquire MCM stain in dap mutants (e.g., Fig. 5 B, arrowhead). Likewise, not all cells of the epidermis undergo S17 in a homozygous dap mutant (de Nooij et al., 1997; Lane et al., 1997). Direct analysis of DmMCM2 immunofluorescent staining also revealed association of MCMs with late anaphase/telophase nuclei of homozygous dap mutants (not shown). These data suggest that expression of Dap in cycle 16 normally inhibits chromosome association of DmMCM2.

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