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. 2002 Mar;22(6):1868-80.
doi: 10.1128/MCB.22.6.1868-1880.2002.

Control of DNA replication and chromosome ploidy by geminin and cyclin A

Ivailo S Mihaylov  1 , Takeshi Kondo, Lynn Jones, Sophia Ryzhikov, Junko Tanaka, Jianyu Zheng, Leigh Ann Higa, Naoto Minamino, Lynn Cooley, Hui Zhang
Affiliations

Control of DNA replication and chromosome ploidy by geminin and cyclin A

Ivailo S Mihaylov et al. Mol Cell Biol. 2002 Mar.

Abstract

Alteration of the control of DNA replication and mitosis is considered to be a major cause of genome instability. To investigate the mechanism that controls DNA replication and genome stability, we used the RNA silencing-interference technique (RNAi) to eliminate the Drosophila geminin homologue from Schneider D2 (SD2) cells. Silencing of geminin by RNAi in SD2 cells leads to the cessation of mitosis and asynchronous overreplication of the genome, with cells containing single giant nuclei and partial ploidy between 4N and 8N DNA content. The effect of geminin deficiency is completely suppressed by cosilencing of Double parked (Dup), the Drosophila homologue of Cdt1, a replication factor to which geminin binds. The geminin deficiency-induced phenotype is also partially suppressed by coablation of Chk1/Grapes, indicating the involvement of Chk1/Grapes in the checkpoint control in response to overreplication. We found that the silencing of cyclin A, but not of cyclin B, also promotes the formation of a giant nucleus and overreplication. However, in contrast to the effect of geminin knockout, cyclin A deficiency leads to the complete duplication of the genome from 4N to 8N. We observed that the silencing of geminin causes rapid downregulation of Cdt1/Dup, which may contribute to the observed partial overreplication in geminin-deficient cells. Analysis of cyclin A and geminin double knockout suggests that the effect of cyclin A deficiency is dominant over that of geminin deficiency for cell cycle arrest and overreplication. Together, our studies indicate that both cyclin A and geminin are required for the suppression of overreplication and for genome stability in Drosophila cells.

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Figures

FIG. 1.
FIG. 1.
Identification of a Drosophila geminin homologue. (A) Homology between human (Homo sapiens), Xenopus (Xenopus laevis), and Drosophila (Drosophila melanogaster) geminin proteins. Xenopus has two cDNAs encoding geminin H and L. Only geminin H is shown. (B) Association between geminin and Dup. The geminin and Cdt1/Dup proteins were immunoprecipitated from SD2 cell lysates with antigeminin, preimmune serum, and anti-Cdt1/Dup antibodies. Western blotting was performed with anti-Cdt1/Dup (top) and antigeminin (bottom) antibodies. The molecular mass standards are indicated on the left (in kilodaltons). Cdt1/Dup migrates at about 98 kDa, and the faster migrating band is likely a proteolytic fragment.
FIG. 2.
FIG. 2.
Drosophila geminin accumulates in S phase and disappears in mitosis during embryonic divisions. Drosophila embryos were collected and stained with antigeminin antibody and then with fluorescein isothiocyanate-conjugated secondary antibody (green). The nuclei were counterstained with propidium iodide (red). (A) Mitotic embryos in late prophase-metaphase; (B) S-phase embryos; (C) anaphase-telophase embryos; (D) an embryo undergoing S phase (upper right) and mitosis (lower left).
FIG. 3.
FIG. 3.
Effects of silencing geminin or Cdt1/Dup and cosilencing geminin and Cdt1/Dup or Cdc6, measured by flow cytometry analysis. (A to C) Geminin knockout induces asynchronous overreplication and partial polyploidy. 2N and 4N represent the cells containing 2N and 4N DNA content, respectively. (D to F) Cdt1/Dup silencing promotes accumulation of the sub-G1 population. (G to I) Cdt1/Dup knockout suppresses geminin deficiency-induced polyploidy and overreplication. For all profiles, the control (Neo) is indicated by a thin line while the geminin, Cdt1/Dup, and geminin plus Cdt1/Dup knockouts are indicated by thick lines. (J and K) Cdc6 knockout partially suppresses geminin deficiency-induced polyploidy and overreplication. The geminin single knockout is indicated by a thin line, while the geminin plus Cdc6 double knockout is indicated by a thick line.
FIG. 3.
FIG. 3.
Effects of silencing geminin or Cdt1/Dup and cosilencing geminin and Cdt1/Dup or Cdc6, measured by flow cytometry analysis. (A to C) Geminin knockout induces asynchronous overreplication and partial polyploidy. 2N and 4N represent the cells containing 2N and 4N DNA content, respectively. (D to F) Cdt1/Dup silencing promotes accumulation of the sub-G1 population. (G to I) Cdt1/Dup knockout suppresses geminin deficiency-induced polyploidy and overreplication. For all profiles, the control (Neo) is indicated by a thin line while the geminin, Cdt1/Dup, and geminin plus Cdt1/Dup knockouts are indicated by thick lines. (J and K) Cdc6 knockout partially suppresses geminin deficiency-induced polyploidy and overreplication. The geminin single knockout is indicated by a thin line, while the geminin plus Cdc6 double knockout is indicated by a thick line.
FIG. 3.
FIG. 3.
Effects of silencing geminin or Cdt1/Dup and cosilencing geminin and Cdt1/Dup or Cdc6, measured by flow cytometry analysis. (A to C) Geminin knockout induces asynchronous overreplication and partial polyploidy. 2N and 4N represent the cells containing 2N and 4N DNA content, respectively. (D to F) Cdt1/Dup silencing promotes accumulation of the sub-G1 population. (G to I) Cdt1/Dup knockout suppresses geminin deficiency-induced polyploidy and overreplication. For all profiles, the control (Neo) is indicated by a thin line while the geminin, Cdt1/Dup, and geminin plus Cdt1/Dup knockouts are indicated by thick lines. (J and K) Cdc6 knockout partially suppresses geminin deficiency-induced polyploidy and overreplication. The geminin single knockout is indicated by a thin line, while the geminin plus Cdc6 double knockout is indicated by a thick line.
FIG. 3.
FIG. 3.
Effects of silencing geminin or Cdt1/Dup and cosilencing geminin and Cdt1/Dup or Cdc6, measured by flow cytometry analysis. (A to C) Geminin knockout induces asynchronous overreplication and partial polyploidy. 2N and 4N represent the cells containing 2N and 4N DNA content, respectively. (D to F) Cdt1/Dup silencing promotes accumulation of the sub-G1 population. (G to I) Cdt1/Dup knockout suppresses geminin deficiency-induced polyploidy and overreplication. For all profiles, the control (Neo) is indicated by a thin line while the geminin, Cdt1/Dup, and geminin plus Cdt1/Dup knockouts are indicated by thick lines. (J and K) Cdc6 knockout partially suppresses geminin deficiency-induced polyploidy and overreplication. The geminin single knockout is indicated by a thin line, while the geminin plus Cdc6 double knockout is indicated by a thick line.
FIG. 4.
FIG. 4.
Morphological analysis of silencing geminin or Cdt1/Dup and cosilencing geminin and Cdt1/Dup. The cells were incubated with dsRNAs. At various times, the cells were fixed and the nuclei were stained with DAPI. The nuclear stainings were examined under the same magnification. (A to C) The control (Neo) knockout. (D to F) The geminin knockout. Note the induction of giant nuclei containing densely and diffusely stained regions in geminin-deficient cells. (G to I) The Cdt1/Dup knockout. Note the accumulation of unusual, small cells after the silencing of Cdt1/Dup. (J to L) The geminin and Cdt1/Dup double knockout. The morphological suppression of geminin deficiency by the cosilencing of Cdt1/Dup is shown. (M) Western blot analysis of knockout samples for control (Neo), geminin (Gem), Cdt1/Dup (DUP), and geminin plus Cdt1/Dup (GEM + DUP) knockouts, as shown in panels A to L and in Fig. 3.
FIG. 4.
FIG. 4.
Morphological analysis of silencing geminin or Cdt1/Dup and cosilencing geminin and Cdt1/Dup. The cells were incubated with dsRNAs. At various times, the cells were fixed and the nuclei were stained with DAPI. The nuclear stainings were examined under the same magnification. (A to C) The control (Neo) knockout. (D to F) The geminin knockout. Note the induction of giant nuclei containing densely and diffusely stained regions in geminin-deficient cells. (G to I) The Cdt1/Dup knockout. Note the accumulation of unusual, small cells after the silencing of Cdt1/Dup. (J to L) The geminin and Cdt1/Dup double knockout. The morphological suppression of geminin deficiency by the cosilencing of Cdt1/Dup is shown. (M) Western blot analysis of knockout samples for control (Neo), geminin (Gem), Cdt1/Dup (DUP), and geminin plus Cdt1/Dup (GEM + DUP) knockouts, as shown in panels A to L and in Fig. 3.
FIG. 5.
FIG. 5.
Silencing cyclin A induces the accumulation of 8N cells and the formation of a single giant nucleus within a cell. (A to D) Flow cytometry analysis of cyclin A silencing (A and B) and cyclin B knockout (C and D) compared with the control knockout (Neo). The cyclin A and B knockouts are indicated by thick lines, and the control is indicated by a thin line. (E to J) Nuclear morphology analysis of control (Neo) (E and F), cyclin A silencing (G and H), and cyclin B knockout (I and J). The same magnification was used for all panels. (K) Cyclin A knockout suppresses cyclin B expression. Western blot analysis of equal amounts of cell lysates from cyclin A (upper) and cyclin B (lower) levels in the control (Neo), cyclin A (A), cyclin B (B), and cyclin A plus cyclin B (A + B) knockout samples is shown. (L) Northern blot analysis of cyclin B RNA levels in the knockout samples. Abbreviations are the same as those for panel K.
FIG. 6.
FIG. 6.
Drosophila Chk1/Grapes silencing suppresses geminin deficiency-induced polyploidy and giant nucleus formation. (A and B) Flow cytometry analysis. Control (Neo) is indicated by a thin line; geminin silencing is indicated by a thick line; Chk1/Grapes and geminin double knockout is indicated by a dotted thin line. (C to J) Nuclear morphological analysis of control (Neo) knockout (C and D), geminin knockout (E and F), Chk1/Grapes knockout (G and H), and geminin and Chk1/Grapes double knockout (I and J). The same magnification was used for all panels. (K) Western blot of geminin (top) and Northern blot of Chk1/Grapes (bottom) in the knockout samples. Abbeviations are the same as those for Fig. 4M.
FIG. 6.
FIG. 6.
Drosophila Chk1/Grapes silencing suppresses geminin deficiency-induced polyploidy and giant nucleus formation. (A and B) Flow cytometry analysis. Control (Neo) is indicated by a thin line; geminin silencing is indicated by a thick line; Chk1/Grapes and geminin double knockout is indicated by a dotted thin line. (C to J) Nuclear morphological analysis of control (Neo) knockout (C and D), geminin knockout (E and F), Chk1/Grapes knockout (G and H), and geminin and Chk1/Grapes double knockout (I and J). The same magnification was used for all panels. (K) Western blot of geminin (top) and Northern blot of Chk1/Grapes (bottom) in the knockout samples. Abbeviations are the same as those for Fig. 4M.
FIG. 7.
FIG. 7.
Drosophila geminin silencing induces rapid downregulation of Cdt1/Dup. (A) Time course of Cdt1/Dup in control and geminin knockout samples. Proteins from equal amounts of cell lysates were detected by anti-Cdt1/Dup (top) and antigeminin (bottom) Western blotting. (B) The kinetics of loss of Cdt1/Dup in control, geminin, Cdt1/Dup, and geminin plus Cdt1/Dup knockout cells. The levels of geminin are also shown. The bottom row shows a Ponceau S staining of the protein gel to demonstrate equal protein loading. (C) Northern blots of Cdt1/Dup and Cdc6 in control and geminin knockout samples. (D) The effect of Chk1 cosilencing on the geminin deficiency-induced Cdt1/Dup downregulation. Neo, control; Gem, geminin; cdt1, Cdt1/Dup; Gem + cdt1, geminin plus Cdt1/Dup.
FIG. 8.
FIG. 8.
Cyclin A deficiency is dominant over geminin deficiency for cell cycle arrest and overreplication. (A and B) Flow cytometry analysis of control, cyclin A, geminin, and cyclin A plus geminin knockouts for 1 (A) or 2 (B) days. (C) Protein analysis of equal amounts of cell lysates from geminin, cyclin A, and cyclin B knockout cells. The top panel shows an examination of Cdt1/Dup, cyclin A, cyclin B, and geminin levels in the control, geminin, cyclin A, and cyclin A plus geminin knockout cells by direct Western blotting of equal amounts of cell lysates with respective antibodies. The lower left panel shows cyclin A, Cdt1/Dup, and geminin levels in cyclin B knockout cells. The lower right panel shows an analysis of the Cdt1/geminin complex in geminin and cyclin A knockout cells. The Cdt1/Dup complex was immunoprecipitated from control, geminin, and cyclin A knockout cells and then analyzed by Western blotting with Cdt1/Dup and geminin antibodies. (D) Examination of cyclin B-associated histone H1 kinase activity in geminin knockout cells. The left panel shows an analysis of Cdt1/Dup, cyclin A, cyclin B, and geminin protein levels in control and geminin knockout cells by direct Western blotting of equal amounts of cell lysates with respective antibodies. The right panel shows the results of cyclin B being immunoprecipitated from the same set of cell lysates as shown in the left panel and assayed for kinase activity towards histone H1 (H). The cyclin A antibody could not immunoprecipitate the cyclin A protein and therefore has no associated protein kinase activity. Neo, control; CycA, cyclin A; CycB, cyclin B; Gem, geminin; Gem + cycA, geminin plus cyclin A; IP, immunoprecipitate.
FIG. 8.
FIG. 8.
Cyclin A deficiency is dominant over geminin deficiency for cell cycle arrest and overreplication. (A and B) Flow cytometry analysis of control, cyclin A, geminin, and cyclin A plus geminin knockouts for 1 (A) or 2 (B) days. (C) Protein analysis of equal amounts of cell lysates from geminin, cyclin A, and cyclin B knockout cells. The top panel shows an examination of Cdt1/Dup, cyclin A, cyclin B, and geminin levels in the control, geminin, cyclin A, and cyclin A plus geminin knockout cells by direct Western blotting of equal amounts of cell lysates with respective antibodies. The lower left panel shows cyclin A, Cdt1/Dup, and geminin levels in cyclin B knockout cells. The lower right panel shows an analysis of the Cdt1/geminin complex in geminin and cyclin A knockout cells. The Cdt1/Dup complex was immunoprecipitated from control, geminin, and cyclin A knockout cells and then analyzed by Western blotting with Cdt1/Dup and geminin antibodies. (D) Examination of cyclin B-associated histone H1 kinase activity in geminin knockout cells. The left panel shows an analysis of Cdt1/Dup, cyclin A, cyclin B, and geminin protein levels in control and geminin knockout cells by direct Western blotting of equal amounts of cell lysates with respective antibodies. The right panel shows the results of cyclin B being immunoprecipitated from the same set of cell lysates as shown in the left panel and assayed for kinase activity towards histone H1 (H). The cyclin A antibody could not immunoprecipitate the cyclin A protein and therefore has no associated protein kinase activity. Neo, control; CycA, cyclin A; CycB, cyclin B; Gem, geminin; Gem + cycA, geminin plus cyclin A; IP, immunoprecipitate.
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