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

Su TT, O'Farrell PH - J. Cell Biol. (1998)

Bottom Line: We found that MCMs in polytene nuclei exist in two states: associated with or dissociated from chromosomes.We demonstrate that cyclin E can drive chromosome association of DmMCM2 and that DNA synthesis erases this association.These results are discussed in a model in which the cycle of MCM-chromosome associations is uncoupled from mitosis because of the distinctive program of cyclin expression in endocycles.

View Article: PubMed Central - PubMed

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

ABSTRACT
Minichromosome maintenance (MCM) proteins are essential eukaryotic DNA replication factors. The binding of MCMs to chromatin oscillates in conjunction with progress through the mitotic cell cycle. This oscillation is thought to play an important role in coupling DNA replication to mitosis and limiting chromosome duplication to once per cell cycle. The coupling of DNA replication to mitosis is absent in Drosophila endoreplication cycles (endocycles), during which discrete rounds of chromosome duplication occur without intervening mitoses. We examined the behavior of MCM proteins in endoreplicating larval salivary glands, to determine whether oscillation of MCM-chromosome localization occurs in conjunction with passage through an endocycle S phase. We found that MCMs in polytene nuclei exist in two states: associated with or dissociated from chromosomes. We demonstrate that cyclin E can drive chromosome association of DmMCM2 and that DNA synthesis erases this association. We conclude that mitosis is not required for oscillations in chromosome binding of MCMs and propose that cycles of MCM-chromosome association normally occur in endocycles. These results are discussed in a model in which the cycle of MCM-chromosome associations is uncoupled from mitosis because of the distinctive program of cyclin expression in endocycles.

Show MeSH
DmMCM2 is retained on chromosomes  when DNA synthesis is  inhibited. Larval salivary  glands from control larvae  (−aphi) and larvae on aphidicolin-containing diet (+aphi)  were dissected, fixed, and  stained for DmMCM2 and  DNA as indicated. The nucleus indicated with an arrowhead in A and B is magnified and shown in C–E.  The nucleus indicated with  an arrowhead in F and G is  magnified and shown in H–J. In F and H, colocalization of DmMCM2 and DNA results in uniform DmMCM2 stain in the nonnucleolar  region of nuclei when viewed in cross-section (similar to Fig. 1, 4 and schematized in Fig. 4 as chromosomal). In contrast, in most nuclei  in A and in the nucleus in C, DmMCM2 did not colocalize with DNA and appears netlike (as in Fig. 1, 5 and schematized in Fig. 4 as nucleoplasmic).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2140170&req=5

Figure 5: DmMCM2 is retained on chromosomes when DNA synthesis is inhibited. Larval salivary glands from control larvae (−aphi) and larvae on aphidicolin-containing diet (+aphi) were dissected, fixed, and stained for DmMCM2 and DNA as indicated. The nucleus indicated with an arrowhead in A and B is magnified and shown in C–E. The nucleus indicated with an arrowhead in F and G is magnified and shown in H–J. In F and H, colocalization of DmMCM2 and DNA results in uniform DmMCM2 stain in the nonnucleolar region of nuclei when viewed in cross-section (similar to Fig. 1, 4 and schematized in Fig. 4 as chromosomal). In contrast, in most nuclei in A and in the nucleus in C, DmMCM2 did not colocalize with DNA and appears netlike (as in Fig. 1, 5 and schematized in Fig. 4 as nucleoplasmic).

Mentions: Although the aphidicolin treatment clearly stabilized chromosomal DmMCM2, the proportion of nuclei showing chromosomal staining varied from gland to gland (not shown), suggesting that inhibition of DNA synthesis may not have been efficient. This may be due to a substantial time, required for dissection, between the beginning of heat induction of cyclin E and the delivery of aphidicolin to the samples. As an alternative way to inhibit DNA synthesis, we added aphidicolin to the food of feeding third-instar larvae for 15 h. During this time, we expect that a large fraction of polytene nuclei would attempt endoreplication (Rudkin, 1972). We reasoned that the MCMs would bind to chromosomes as cells prepare to enter S phase (e.g., Fig. 4 B), but that the presence of aphidicolin would then block DNA synthesis. If DNA synthesis was required for dissociation of MCMs from chromosomes, we would essentially “trap” MCMs on polytene chromosomes of larvae that have been fed aphidicolin, but not in control larvae. After aphidicolin feeding, salivary glands were dissected, fixed, and stained for DmMCM2. Efficient retention of DmMCM2 on chromosomes was seen using this procedure (Fig. 5): 65% of nuclei in salivary glands of aphidicolin-fed larvae showed chromosomal DmMCM2 (data from four glands), compared to <5% in controls. Note that we do not expect to see retention of chromosomal MCMs in all the nuclei. This is because at the beginning of the experiment some of the nuclei would have been in S phase with MCMs dissociated from chromosomes and may have remained arrested in this state in the presence of aphidicolin. In addition, because of asynchrony in endocycles, some of the nuclei may not have gone through the period of loading MCMs onto DNA during the course of aphidicolin feeding. On the basis of these data, we conclude that DNA replication is required for dissociation of DmMCM2 from the chromosomes.


Chromosome association of minichromosome maintenance proteins in Drosophila endoreplication cycles.

Su TT, O'Farrell PH - J. Cell Biol. (1998)

DmMCM2 is retained on chromosomes  when DNA synthesis is  inhibited. Larval salivary  glands from control larvae  (−aphi) and larvae on aphidicolin-containing diet (+aphi)  were dissected, fixed, and  stained for DmMCM2 and  DNA as indicated. The nucleus indicated with an arrowhead in A and B is magnified and shown in C–E.  The nucleus indicated with  an arrowhead in F and G is  magnified and shown in H–J. In F and H, colocalization of DmMCM2 and DNA results in uniform DmMCM2 stain in the nonnucleolar  region of nuclei when viewed in cross-section (similar to Fig. 1, 4 and schematized in Fig. 4 as chromosomal). In contrast, in most nuclei  in A and in the nucleus in C, DmMCM2 did not colocalize with DNA and appears netlike (as in Fig. 1, 5 and schematized in Fig. 4 as nucleoplasmic).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2140170&req=5

Figure 5: DmMCM2 is retained on chromosomes when DNA synthesis is inhibited. Larval salivary glands from control larvae (−aphi) and larvae on aphidicolin-containing diet (+aphi) were dissected, fixed, and stained for DmMCM2 and DNA as indicated. The nucleus indicated with an arrowhead in A and B is magnified and shown in C–E. The nucleus indicated with an arrowhead in F and G is magnified and shown in H–J. In F and H, colocalization of DmMCM2 and DNA results in uniform DmMCM2 stain in the nonnucleolar region of nuclei when viewed in cross-section (similar to Fig. 1, 4 and schematized in Fig. 4 as chromosomal). In contrast, in most nuclei in A and in the nucleus in C, DmMCM2 did not colocalize with DNA and appears netlike (as in Fig. 1, 5 and schematized in Fig. 4 as nucleoplasmic).
Mentions: Although the aphidicolin treatment clearly stabilized chromosomal DmMCM2, the proportion of nuclei showing chromosomal staining varied from gland to gland (not shown), suggesting that inhibition of DNA synthesis may not have been efficient. This may be due to a substantial time, required for dissection, between the beginning of heat induction of cyclin E and the delivery of aphidicolin to the samples. As an alternative way to inhibit DNA synthesis, we added aphidicolin to the food of feeding third-instar larvae for 15 h. During this time, we expect that a large fraction of polytene nuclei would attempt endoreplication (Rudkin, 1972). We reasoned that the MCMs would bind to chromosomes as cells prepare to enter S phase (e.g., Fig. 4 B), but that the presence of aphidicolin would then block DNA synthesis. If DNA synthesis was required for dissociation of MCMs from chromosomes, we would essentially “trap” MCMs on polytene chromosomes of larvae that have been fed aphidicolin, but not in control larvae. After aphidicolin feeding, salivary glands were dissected, fixed, and stained for DmMCM2. Efficient retention of DmMCM2 on chromosomes was seen using this procedure (Fig. 5): 65% of nuclei in salivary glands of aphidicolin-fed larvae showed chromosomal DmMCM2 (data from four glands), compared to <5% in controls. Note that we do not expect to see retention of chromosomal MCMs in all the nuclei. This is because at the beginning of the experiment some of the nuclei would have been in S phase with MCMs dissociated from chromosomes and may have remained arrested in this state in the presence of aphidicolin. In addition, because of asynchrony in endocycles, some of the nuclei may not have gone through the period of loading MCMs onto DNA during the course of aphidicolin feeding. On the basis of these data, we conclude that DNA replication is required for dissociation of DmMCM2 from the chromosomes.

Bottom Line: We found that MCMs in polytene nuclei exist in two states: associated with or dissociated from chromosomes.We demonstrate that cyclin E can drive chromosome association of DmMCM2 and that DNA synthesis erases this association.These results are discussed in a model in which the cycle of MCM-chromosome associations is uncoupled from mitosis because of the distinctive program of cyclin expression in endocycles.

View Article: PubMed Central - PubMed

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

ABSTRACT
Minichromosome maintenance (MCM) proteins are essential eukaryotic DNA replication factors. The binding of MCMs to chromatin oscillates in conjunction with progress through the mitotic cell cycle. This oscillation is thought to play an important role in coupling DNA replication to mitosis and limiting chromosome duplication to once per cell cycle. The coupling of DNA replication to mitosis is absent in Drosophila endoreplication cycles (endocycles), during which discrete rounds of chromosome duplication occur without intervening mitoses. We examined the behavior of MCM proteins in endoreplicating larval salivary glands, to determine whether oscillation of MCM-chromosome localization occurs in conjunction with passage through an endocycle S phase. We found that MCMs in polytene nuclei exist in two states: associated with or dissociated from chromosomes. We demonstrate that cyclin E can drive chromosome association of DmMCM2 and that DNA synthesis erases this association. We conclude that mitosis is not required for oscillations in chromosome binding of MCMs and propose that cycles of MCM-chromosome association normally occur in endocycles. These results are discussed in a model in which the cycle of MCM-chromosome associations is uncoupled from mitosis because of the distinctive program of cyclin expression in endocycles.

Show MeSH