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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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Subcellular localization of  DmMCMs during embryonic divisions. Drosophila embryos were  fixed and stained with DmMCM2  antibody (A, E, and H), WGA (to  detect NPCs; D and G) and Hoechst  33258 (to visualize DNA; B, C, and  F). (A and B) The head region of an  embryo in transition from postblastoderm division cycle 14 to cycle 15  is shown. DmMCM2 antigen is  present in the nuclei of G2 cells in  cycle 14 (white, open arrows). As  cells enter mitosis, the chromatin  condenses, and DmMCM2 antigen  disperses from the nuclei into the  cytoplasm (white arrowheads).  DmMCM2 staining is again nuclear  in anaphase/telophase cells (black  arrows, see also C–H). (C–H) Reaccumulation of DmMCM2 to the nucleus at exit from mitosis during  precellular nuclear division cycles  (C–E) and postblastoderm cellular  divisions (cycles 15; F–H). The embryo in C–E was undergoing a wave  of mitoses such that nuclei toward  the bottom of the figure are at a more  advanced state of mitosis. A progression from anaphase (top) to telophase  (bottom) is shown. In F–H, asynchronously dividing cells of a mitotic  domain in M15 are shown. Chromosomes early in anaphase do not accumulate detectable levels of DmMCM2  (C–E, top, F–H, arrowheads). Reaccumulation of DmMCM2 (E and  H) to the nuclear region begins in  late anaphase (white, open arrows)  and precedes the reappearance of  WGA staining (D and G). Nuclear  MCM stain increased as cells progressed into interphase (S phase of  cycle 16) and completely reformed  a nuclear envelope (F–H, black arrows). Bar, 20 μm.
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Figure 1: Subcellular localization of DmMCMs during embryonic divisions. Drosophila embryos were fixed and stained with DmMCM2 antibody (A, E, and H), WGA (to detect NPCs; D and G) and Hoechst 33258 (to visualize DNA; B, C, and F). (A and B) The head region of an embryo in transition from postblastoderm division cycle 14 to cycle 15 is shown. DmMCM2 antigen is present in the nuclei of G2 cells in cycle 14 (white, open arrows). As cells enter mitosis, the chromatin condenses, and DmMCM2 antigen disperses from the nuclei into the cytoplasm (white arrowheads). DmMCM2 staining is again nuclear in anaphase/telophase cells (black arrows, see also C–H). (C–H) Reaccumulation of DmMCM2 to the nucleus at exit from mitosis during precellular nuclear division cycles (C–E) and postblastoderm cellular divisions (cycles 15; F–H). The embryo in C–E was undergoing a wave of mitoses such that nuclei toward the bottom of the figure are at a more advanced state of mitosis. A progression from anaphase (top) to telophase (bottom) is shown. In F–H, asynchronously dividing cells of a mitotic domain in M15 are shown. Chromosomes early in anaphase do not accumulate detectable levels of DmMCM2 (C–E, top, F–H, arrowheads). Reaccumulation of DmMCM2 (E and H) to the nuclear region begins in late anaphase (white, open arrows) and precedes the reappearance of WGA staining (D and G). Nuclear MCM stain increased as cells progressed into interphase (S phase of cycle 16) and completely reformed a nuclear envelope (F–H, black arrows). Bar, 20 μm.

Mentions: Identification and characterization of three Drosophila MCMs, MCM2, MCM4, and MCM5, have been described (Treisman et al., 1995; Feger et al., 1995; Su et al., 1996, 1997). Here, we first describe their localization during embryonic divisions as detected by immunostaining of fixed Drosophila embryos. Fig. 1, A and B, shows nuclear staining for DmMCM2 during G2 of cycle 14, dispersal of staining during mitosis 14, and reaccummulation in telophase 14 (DmMCM4 and DmMCM5 staining was indistinguishable from DmMCM2; not shown). Although all interphase cells exhibited nuclear MCM staining (see below), these early cell cycles lack a G1 phase. Fig. 4 documents that DmMCM5 is nuclear during G1 of cell cycle 17 (similar observations were made for DmMCM2 and DMCM4; not shown). We conclude that these proteins are nuclear in G1, S, and G2, and that they are dispersed from chromatin upon entry into mitosis. This cell cycle pattern of localization is similar to the behavior of vertebrate MCMs (reviewed in Chong et al., 1996).


Chromosome association of minichromosome maintenance proteins in Drosophila mitotic cycles.

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

Subcellular localization of  DmMCMs during embryonic divisions. Drosophila embryos were  fixed and stained with DmMCM2  antibody (A, E, and H), WGA (to  detect NPCs; D and G) and Hoechst  33258 (to visualize DNA; B, C, and  F). (A and B) The head region of an  embryo in transition from postblastoderm division cycle 14 to cycle 15  is shown. DmMCM2 antigen is  present in the nuclei of G2 cells in  cycle 14 (white, open arrows). As  cells enter mitosis, the chromatin  condenses, and DmMCM2 antigen  disperses from the nuclei into the  cytoplasm (white arrowheads).  DmMCM2 staining is again nuclear  in anaphase/telophase cells (black  arrows, see also C–H). (C–H) Reaccumulation of DmMCM2 to the nucleus at exit from mitosis during  precellular nuclear division cycles  (C–E) and postblastoderm cellular  divisions (cycles 15; F–H). The embryo in C–E was undergoing a wave  of mitoses such that nuclei toward  the bottom of the figure are at a more  advanced state of mitosis. A progression from anaphase (top) to telophase  (bottom) is shown. In F–H, asynchronously dividing cells of a mitotic  domain in M15 are shown. Chromosomes early in anaphase do not accumulate detectable levels of DmMCM2  (C–E, top, F–H, arrowheads). Reaccumulation of DmMCM2 (E and  H) to the nuclear region begins in  late anaphase (white, open arrows)  and precedes the reappearance of  WGA staining (D and G). Nuclear  MCM stain increased as cells progressed into interphase (S phase of  cycle 16) and completely reformed  a nuclear envelope (F–H, black arrows). Bar, 20 μm.
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Figure 1: Subcellular localization of DmMCMs during embryonic divisions. Drosophila embryos were fixed and stained with DmMCM2 antibody (A, E, and H), WGA (to detect NPCs; D and G) and Hoechst 33258 (to visualize DNA; B, C, and F). (A and B) The head region of an embryo in transition from postblastoderm division cycle 14 to cycle 15 is shown. DmMCM2 antigen is present in the nuclei of G2 cells in cycle 14 (white, open arrows). As cells enter mitosis, the chromatin condenses, and DmMCM2 antigen disperses from the nuclei into the cytoplasm (white arrowheads). DmMCM2 staining is again nuclear in anaphase/telophase cells (black arrows, see also C–H). (C–H) Reaccumulation of DmMCM2 to the nucleus at exit from mitosis during precellular nuclear division cycles (C–E) and postblastoderm cellular divisions (cycles 15; F–H). The embryo in C–E was undergoing a wave of mitoses such that nuclei toward the bottom of the figure are at a more advanced state of mitosis. A progression from anaphase (top) to telophase (bottom) is shown. In F–H, asynchronously dividing cells of a mitotic domain in M15 are shown. Chromosomes early in anaphase do not accumulate detectable levels of DmMCM2 (C–E, top, F–H, arrowheads). Reaccumulation of DmMCM2 (E and H) to the nuclear region begins in late anaphase (white, open arrows) and precedes the reappearance of WGA staining (D and G). Nuclear MCM stain increased as cells progressed into interphase (S phase of cycle 16) and completely reformed a nuclear envelope (F–H, black arrows). Bar, 20 μm.
Mentions: Identification and characterization of three Drosophila MCMs, MCM2, MCM4, and MCM5, have been described (Treisman et al., 1995; Feger et al., 1995; Su et al., 1996, 1997). Here, we first describe their localization during embryonic divisions as detected by immunostaining of fixed Drosophila embryos. Fig. 1, A and B, shows nuclear staining for DmMCM2 during G2 of cycle 14, dispersal of staining during mitosis 14, and reaccummulation in telophase 14 (DmMCM4 and DmMCM5 staining was indistinguishable from DmMCM2; not shown). Although all interphase cells exhibited nuclear MCM staining (see below), these early cell cycles lack a G1 phase. Fig. 4 documents that DmMCM5 is nuclear during G1 of cell cycle 17 (similar observations were made for DmMCM2 and DMCM4; not shown). We conclude that these proteins are nuclear in G1, S, and G2, and that they are dispersed from chromatin upon entry into mitosis. This cell cycle pattern of localization is similar to the behavior of vertebrate MCMs (reviewed in Chong et al., 1996).

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