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First published online 1 September 2005
doi: 10.1242/jcs.02534

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Levels of the origin-binding protein Double parked and its inhibitor Geminin increase in response to replication stress

Noah R. May^*, Marguerite Thomer^*, Katherine F. Murnen and Brian R. Calvi

Department of Genetics, University of Pennsylvania School of Medicine, 415 Curie Blvd, Philadelphia, PA 19104-6145, USA

View larger version (91K): [in a new window]   Fig. 1. Dup persists past the G1-S transition and accumulates to high levels in Mcm63 mutant brains, (A,C,E) Wild type. (B,D,F) Mcm63. (A) One lobe of a wild-type third-instar brain labeled with anti-Dup antibody (red) highlights dividing lamina-precursor cells (LPCs) in the optic lobe and neuroblast stem cells (NB) in the mid-brain (arrow). (B) A brain from an Mcm63 mutant third-instar larva has many cells with high levels of Dup and is smaller than the wild type owing to defects in cell proliferation. The Dup increase is especially pronounced in the large neuroblast stem cells. (C,D) Double labeling for Dup (red) and cyclin B (green). (C) Most neuroblasts in G2 (top arrow) have Dup in the nucleus and cyclin B in the cytoplasm, whereas those in mitosis (bottom arrow) have Dup and cyclin B in the cytoplasm and nucleus. By contrast, the smaller, cyclin-B-positive daughter cells (DC, extent of one shown with bracket) surrounding the neuroblast have no detectable Dup during S, G2 or M phase. Cells in G1 phase have Dup in the nucleus but no cyclin B (arrowhead). Asterisk indicates the nucleolus in the stem cell. (D) An Mcm63 mutant brain has many cells with high levels of Dup (red) in the nucleus or nucleus and cytoplasm. Many of these cells are in late S, G2 or early M, as evidenced by cyclin-B expression (green; overlap is yellow). (E,F) Labeling for Dup (red) and PH3 (green). (E) Wild-type neuroblast-stem-cell division in anaphase shows that there is a low but detectable level of Dup during mitosis. (F) Some Mcm63 mutant cells with elevated Dup levels enter M phase and have PH3-positive chromosomes that are abnormal in number and structure. The wild-type and mutant images are matched for exposure, resulting in pixel saturation for some mutant cells owing to intense Dup labeling. Images are composites of confocal sections. Scale bar, 50 µm (A,B), 10 µm (C-F).

View larger version (89K): [in a new window]   Fig. 2. Dup mRNA levels and E2F1/DP-dependent transcription are not increased during replication stress. (A,B) In-situ hybridization to Dup mRNA in a third-instar brain from wild-type (A) and Mcm63 mutant (B) larvae. (C-F) E2F1/DP transcription-factor activity is not increased in brain cells with elevated Dup levels. Dup labeling (red) and anti-Myc labeling (green) to detect expression of the E2F1/DP-sensitive reporter ORC1p:ftz-GFP-Myc in control (C,D) or HU-treated (E,F) brain cells. (E) In HU-treated cells, Dup was often abundant in the nucleus and cytoplasm but was excluded from the nucleolus, which appears as an absence of staining in a spherical area in the nucleus. HU-treated cells did not have significantly elevated E2F/DP reporter activity (F) despite having high levels of Dup in the same cells (E). Images in A,B are bright field and those in C-F are composites of confocal sections. Scale bar, 50 µm (A,B), 10 µm (C-F).

View larger version (152K): [in a new window]   Fig. 3. The increase in Dup levels in response to replication stress is not sensitive to ATM/ATR-checkpoint-kinase activity. Dup labeling in a single third-instar brain lobe from HU-fed wild-type (A), HU-fed mei-4129D mutant (B) and HU-plus caffeine-fed wild-type (C) larvae. (D) High-power image of cells from an HU-treated mei-4129D brain labeled for Dup (red) and PH3 (green). Some mei-4129D cells proceeded into mitosis in the presence of HU and had chromosomes that were highly aberrant in morphology. Arrow indicates a neuroblast stem cell (NB). Scale bars, 10 µm.

View larger version (136K): [in a new window]   Fig. 4. Replication defects delocalize and increase Dup levels in follicle cells. (A,B) Immunolabeling for Orc2 (red) indicates that it is localized to amplification foci in wild-type (A) and Mcm6K1214 mutant (B) early-stage 10B follicle cell nuclei (TOTO-3, blue). (C-H) Immunolabeling for Dup (red). Dup is localized to chorion foci in wild-type stage 10B follicle cells (C), whereas in Mcm6K1214 (D) Dup protein is largely dispersed and more abundant, although some nuclei had detectable concentrations of Dup protein at chorion loci (arrow). Because late-stage 10B follicle cells are shown in C and D, one focus of Dup staining at the chorion locus on the third chromosome locus predominates. The bright blue foci are heterochromatin. (E-H) Dup localization and abundance are altered in the other amplification mutant strains mus101K451 (E), chiffon0233 (F), Orc2fs293 (G) and dupPA77 (H). Images are composites of confocal sections. Scale bar, 5 µm.

View larger version (103K): [in a new window]   Fig. 5. Gem levels increase during replication stress. (A) Cyclin B (green) and Gem (red) labeling in third-instar brain lobe. Gem is abundant in late-S-, G2- and early M-phase nuclei, whose cytoplasm labels for cyclin B. This is most evident in the lamina-precursor cells (LPCs), which undergo synchronized cell cycles as they migrate (left to right) towards the lamina furrow (LF, arrow). The cells immediately to the left of the LF are in G2 phase and have high levels of Gem in the nucleus and of cyclin B in the cytoplasm. Gem is also expressed during S and G2 phase in the nuclei of neuroblast stem cells (NB) (e.g. arrowhead). (B) Dup (red) and Gem (green) are both present in the nucleus in mid-brain neuroblast stem cells during G2 phase (yellow overlap). During mitosis, stem cells have Dup and Gem distributed throughout the cell (arrow). By contrast, the smaller daughter cells have nuclear Gem (green) during S and G2 phase but little Dup (arrowhead). Cells in G1 are positive for Dup (red) but not Gem. (C) One lobe of an Mcm63 mutant brain. Most cells with high levels of Dup (red) also have elevated levels of Gem (green; overlap is yellow), whereas other cells have only high levels of Gem (green). (D,E) HU increases levels of Dup and Gem in neuroblasts and LPCs. (D) A lower-power micrograph shows that the mid-brain NBs have greater increases in Dup and Gem than other cells in response to replication stress. (E) Higher magnification of mid-brain stem cells from D. Neuroblast stem cells (arrows) have very high levels of Dup and Gem (bright yellow), whereas surrounding cells have high levels of Gem only (green) or normal levels of either protein. Scale bar, 50 µm (A,D), 10 µm (B,C,E).

View larger version (27K): [in a new window]   Fig. 6. A model for Dup degradation during S phase. (A) Summary of proteins and activities at origins and forks that were tested and found to be required for normal Dup degradation. Only the origin and replication-fork proteins that were tested are shown, except for the pre-RC protein CDC6, which was not tested. HU inhibits polymerase indirectly by inhibiting ribonucleotide reductase, which results in depletion of dNTPs. (B) Dup degradation during a normal S phase. We had previously shown that cyclin-E/CDK2 is indirectly required for Dup degradation (Thomer et al., 2004). Cyclin-E/CDK2 phosphorylates Dup but the relative contribution of this modification to the instability of Dup appears to be relatively minor (dotted arrow). Based on current evidence, we propose that DNA replication is the CDK2-dependent activity that is required for Dup degradation. This might be required to promote ubiquitylation of Dup by an unknown ubiquitin (Ub) ligase, leading to the rapid destruction of Dup at the proteasome. (C) When CDK2 activity is inhibited (Thomer et al., 2004) or other problems with DNA replication are encountered (this study), Dup is not degraded, perhaps because of reduced ubiquitylation. Because Dup mRNA continues to be translated, Dup accumulates to high levels. Replication stress also results in an increase in Gem levels, which probably plays a prominent role in preventing the relicensing of origins when Dup degradation fails

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