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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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Chromosomes do not associate with DmMCM5 in M16  of wild-type embryos. Embryos were fixed and stained with purified antibodies against DmMCM5 and Hoechst 33258 to visualize  DNA. The primary antibody was detected immunohistochemically for increased sensitivity (see Materials and Methods). Embryos were visualized simultaneously for DmMCM5 (in bright  field) and DNA (fluorescence). DmMCM5 signal appears as a  dark stain and, when it colocalizes with DNA, quenches the  DNA fluorescence. All interphase nuclei are dark reflecting nuclear localization of MCMs in interphase (white arrows). (A and  B) During cycle 15, DmMCM5 disperses from the nucleus in mitosis and reaccumulates on pairs of late anaphase/telophase chromosomes (A, bracket). Further reaccumulation is seen as nuclei  progress into interphase (A, arrow). This pattern of DmMCM5  relocalization is identical to the pattern detected by immunofluoresence in Fig. 1, F–H. In contrast, cells of the dorsal epidermis  that are going through M16 in a stage 11 embryo fail to acquire  DmMCM5 stain at a similar stage in mitosis (B, bracket). Note,  however, that when these cells have completed M16 and are in  G117, DmMCM5 stain is present in the nuclei (B and C, arrows),  presumably because of nuclear import (see Fig. 1 legend). A–C  are magnified from areas indicated by brackets in A′, B′, and C′.  Embryos are shown anterior to the right and dorsal facing forward (A′) or down (B′ and C′). Bar, 10 μm in A–C.
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Figure 4: Chromosomes do not associate with DmMCM5 in M16 of wild-type embryos. Embryos were fixed and stained with purified antibodies against DmMCM5 and Hoechst 33258 to visualize DNA. The primary antibody was detected immunohistochemically for increased sensitivity (see Materials and Methods). Embryos were visualized simultaneously for DmMCM5 (in bright field) and DNA (fluorescence). DmMCM5 signal appears as a dark stain and, when it colocalizes with DNA, quenches the DNA fluorescence. All interphase nuclei are dark reflecting nuclear localization of MCMs in interphase (white arrows). (A and B) During cycle 15, DmMCM5 disperses from the nucleus in mitosis and reaccumulates on pairs of late anaphase/telophase chromosomes (A, bracket). Further reaccumulation is seen as nuclei progress into interphase (A, arrow). This pattern of DmMCM5 relocalization is identical to the pattern detected by immunofluoresence in Fig. 1, F–H. In contrast, cells of the dorsal epidermis that are going through M16 in a stage 11 embryo fail to acquire DmMCM5 stain at a similar stage in mitosis (B, bracket). Note, however, that when these cells have completed M16 and are in G117, DmMCM5 stain is present in the nuclei (B and C, arrows), presumably because of nuclear import (see Fig. 1 legend). A–C are magnified from areas indicated by brackets in A′, B′, and C′. Embryos are shown anterior to the right and dorsal facing forward (A′) or down (B′ and C′). Bar, 10 μm in A–C.

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)

Chromosomes do not associate with DmMCM5 in M16  of wild-type embryos. Embryos were fixed and stained with purified antibodies against DmMCM5 and Hoechst 33258 to visualize  DNA. The primary antibody was detected immunohistochemically for increased sensitivity (see Materials and Methods). Embryos were visualized simultaneously for DmMCM5 (in bright  field) and DNA (fluorescence). DmMCM5 signal appears as a  dark stain and, when it colocalizes with DNA, quenches the  DNA fluorescence. All interphase nuclei are dark reflecting nuclear localization of MCMs in interphase (white arrows). (A and  B) During cycle 15, DmMCM5 disperses from the nucleus in mitosis and reaccumulates on pairs of late anaphase/telophase chromosomes (A, bracket). Further reaccumulation is seen as nuclei  progress into interphase (A, arrow). This pattern of DmMCM5  relocalization is identical to the pattern detected by immunofluoresence in Fig. 1, F–H. In contrast, cells of the dorsal epidermis  that are going through M16 in a stage 11 embryo fail to acquire  DmMCM5 stain at a similar stage in mitosis (B, bracket). Note,  however, that when these cells have completed M16 and are in  G117, DmMCM5 stain is present in the nuclei (B and C, arrows),  presumably because of nuclear import (see Fig. 1 legend). A–C  are magnified from areas indicated by brackets in A′, B′, and C′.  Embryos are shown anterior to the right and dorsal facing forward (A′) or down (B′ and C′). Bar, 10 μm in A–C.
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Figure 4: Chromosomes do not associate with DmMCM5 in M16 of wild-type embryos. Embryos were fixed and stained with purified antibodies against DmMCM5 and Hoechst 33258 to visualize DNA. The primary antibody was detected immunohistochemically for increased sensitivity (see Materials and Methods). Embryos were visualized simultaneously for DmMCM5 (in bright field) and DNA (fluorescence). DmMCM5 signal appears as a dark stain and, when it colocalizes with DNA, quenches the DNA fluorescence. All interphase nuclei are dark reflecting nuclear localization of MCMs in interphase (white arrows). (A and B) During cycle 15, DmMCM5 disperses from the nucleus in mitosis and reaccumulates on pairs of late anaphase/telophase chromosomes (A, bracket). Further reaccumulation is seen as nuclei progress into interphase (A, arrow). This pattern of DmMCM5 relocalization is identical to the pattern detected by immunofluoresence in Fig. 1, F–H. In contrast, cells of the dorsal epidermis that are going through M16 in a stage 11 embryo fail to acquire DmMCM5 stain at a similar stage in mitosis (B, bracket). Note, however, that when these cells have completed M16 and are in G117, DmMCM5 stain is present in the nuclei (B and C, arrows), presumably because of nuclear import (see Fig. 1 legend). A–C are magnified from areas indicated by brackets in A′, B′, and C′. Embryos are shown anterior to the right and dorsal facing forward (A′) or down (B′ and C′). Bar, 10 μm in A–C.
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