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. 1998 Feb 9;140(3):451-60.
doi: 10.1083/jcb.140.3.451.

Chromosome association of minichromosome maintenance proteins in Drosophila endoreplication cycles

T T Su  1 , P H O'Farrell
Affiliations

Chromosome association of minichromosome maintenance proteins in Drosophila endoreplication cycles

T T Su et al. J Cell Biol. .

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.

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Figures

Figure 1
Figure 1
Chromosomal and nucleoplasmic localization of DmMCM2 polytene nuclei. Salivary glands from third-instar larvae were fixed and stained for DmMCM2 and DNA. Note that all nuclei contain DmMCM2 (1) despite differences in the pattern of staining (for example, nuclei indicated by arrows). 2–5 show, at higher magnification, representative nuclei showing two distinct types of nuclear DmMCM2 staining. 2 and 3 are grazing optical sections and 4 and 5 are optical sections across the nucleus. The “space” in the center of each nucleus is the nucleolus. Note the colocalization of DmMCM2 and DNA signals in 2 and 4, such that distinct green or red regions are not seen. For example, regions indicated by arrowheads in 4 lack the DNA stain as well as the DmMCM2 signal. In contrast, DmMCM2 stain is excluded from the chromosomes in 3 and 5, such that distinct green or red regions are visible. For example, regions indicated by arrowheads in 3 have the DNA stain but lack the DmMCM2 signal, and regions indicated by arrowheads in 5 lack the DNA stain but have the DmMCM2 signal.
Figure 3
Figure 3
Induction of DmMCM2–chromosome association by cyclin E. Salivary glands were dissected from transgenic larvae before heat shock (E-hs) and at 25 min after heat shock to induce cyclin E (E +hs), or from heat-shocked controls lacking the transgene (W67 +hs), fixed, and stained for DmMCM2 and DNA. Nuclei indicated by arrowheads are magnified and shown in a (from A and A′), b (from B and B′) and c (from C and C′). In A and B, DmMCM2 did not colocalize with DNA in most nuclei and appears netlike when viewed in cross-section (similar to Fig. 1, lane 5 and schematized in Fig. 4 as nucleoplasmic). In contrast, in C, colocalization of DmMCM2 and DNA results in uniform DmMCM2 stain in the nonnucleolar region of many nuclei (similar to Fig. 1, 4 and schematized in Fig. 4 as chromosomal).
Figure 4
Figure 4
Quantification of DmMCM2–chromosome association. A shows the time course of changes in DmMCM2 localization after heat shock of larvae with the inducible cyclin E transgene (filled circles) or of w67 controls (open circles). DmMCM2 was detected immunologically in parallel samples from experiments such as those in Fig. 2. Salivary glands were not analyzed for BrdU and DmMCM2 simultaneously for technical reasons outlined in Materials and Methods. The percentage of nuclei showing chromosomal-associated DmMCM2 staining in each salivary gland was plotted against time after heat shock. Data from three of four experiments performed are shown. The value for the no– heat shock control for cyclin E transgenic larvae is shown at t = 0 (half-filled circle). Dashed lines represent extrapolation to the no–heat shock value. At least three and up to eight glands of ∼120 nuclei each are counted for each time point. Induced DNA synthesis was detectable at about 1 h after heat shock (Fig. 2; arrowhead). Slight variations in the time of onset of MCM–chromosome association is seen, as in the examples shown here. But in all cases, maximal chromosome association occurred before induction of BrdU incorporation. B shows the inferred pattern of DmMCM2 localization in a polytene nucleus during a cyclin E–induced S phase.
Figure 2
Figure 2
Induction of DNA replication by cyclin E. (A) The detection of cyclin E in salivary gland extracts by immunoblotting. cyclin E was produced by heat-shocking feeding-stage third-instar larvae carrying the appropriate transgene. Salivary glands were dissected from control larvae (−hs lanes) or from heat-shocked larvae at various times after heat shock (in h, indicated above each lane). Extracts were separated on denaturing gels and immunoblotted using a previously characterized antiserum against cyclin E (Sauer et al., 1995). “−hs” lanes contain extract from either 20 pairs or one pair of salivary glands as indicated. Each of the other lanes contain extract from one pair of salivary glands. The positions of molecular mass markers, in kD, are indicated on the side. (B) Induction of DNA synthesis by cyclin E. cyclin E was produced by heat-shocking feeding-stage third-instar larvae carrying the appropriate transgene. Salivary glands were dissected and labeled with a nucleotide analog, BrdU, at various times after heat shock as indicated above the lanes (min or h:min). Incorporated BrdU was detected immunologically and DNA was stained with Hoechst 33258. −hs: no heat shock control.
Figure 2
Figure 2
Induction of DNA replication by cyclin E. (A) The detection of cyclin E in salivary gland extracts by immunoblotting. cyclin E was produced by heat-shocking feeding-stage third-instar larvae carrying the appropriate transgene. Salivary glands were dissected from control larvae (−hs lanes) or from heat-shocked larvae at various times after heat shock (in h, indicated above each lane). Extracts were separated on denaturing gels and immunoblotted using a previously characterized antiserum against cyclin E (Sauer et al., 1995). “−hs” lanes contain extract from either 20 pairs or one pair of salivary glands as indicated. Each of the other lanes contain extract from one pair of salivary glands. The positions of molecular mass markers, in kD, are indicated on the side. (B) Induction of DNA synthesis by cyclin E. cyclin E was produced by heat-shocking feeding-stage third-instar larvae carrying the appropriate transgene. Salivary glands were dissected and labeled with a nucleotide analog, BrdU, at various times after heat shock as indicated above the lanes (min or h:min). Incorporated BrdU was detected immunologically and DNA was stained with Hoechst 33258. −hs: no heat shock control.
Figure 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).
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