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. 2005 Jan 1;19(1):114-26.
doi: 10.1101/gad.1255805. Epub 2004 Dec 14.

Replication-dependent destruction of Cdt1 limits DNA replication to a single round per cell cycle in Xenopus egg extracts

Emily E Arias  1 , Johannes C Walter
Affiliations

Replication-dependent destruction of Cdt1 limits DNA replication to a single round per cell cycle in Xenopus egg extracts

Emily E Arias et al. Genes Dev. .

Abstract

In eukaryotes, prereplication complexes (pre-RCs) containing ORC, Cdc6, Cdt1, and MCM2-7 are assembled on chromatin in the G1 phase. In S phase, when DNA replication initiates, pre-RCs are disassembled, and new pre-RC assembly is restricted until the following G1 period. As a result, DNA replication is limited to a single round per cell cycle. One inhibitor of pre-RC assembly, geminin, was discovered in Xenopus, and it binds and inactivates Cdt1 in S phase. However, removal of geminin from Xenopus egg extracts is insufficient to cause rereplication, suggesting that other safeguards against rereplication exist. Here, we show that Cdt1 is completely degraded by ubiquitin-mediated proteolysis during the course of the first round of DNA replication in Xenopus egg extracts. Degradation depends on Cdk2/Cyclin E, Cdc45, RPA, and polymerase alpha, demonstrating a requirement for replication initiation. Cdt1 is ubiquitinated on chromatin, and this process also requires replication initiation. Once replication has initiated, Cdk2/Cyclin E is dispensable for Cdt1 degradation. When fresh Cdt1 is supplied after the first round of DNA replication, significant rereplication results, and rereplication is enhanced in the absence of geminin. Our results identify a replication-dependent proteolytic pathway that targets Cdt1 and that acts redundantly with geminin to inactivate Cdt1 in S phase.

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Figures

Figure 1.
Figure 1.
Geminin is the principal inhibitor of MCM2-7 loading in NPE. (A) A total of 1 μL of mock-, Cdk2-, or geminin-depleted NPE was analyzed by Western blotting using antibodies to Cdk2 and geminin. (B) Sperm chromatin (4000/μL) was incubated in HSS or a 1:1 mixture of HSS and mock-, Cdk2-, or geminin-depleted NPE that contained 50 μg/mL aphidicolin. The DNA was isolated after 30 min, and chromatin-bound proteins were analyzed by Western blotting using a mixture of ORC2 and Cdc45 antibodies, as well as MCM3 antibodies. (C) HSS, NPE, or NPE supplemented with 200 nM rCdt1 was incubated for 30 min before addition of sperm chromatin (3000/μL). Chromatin was isolated after 30 min, and chromatin-bound proteins were analyzed using antibodies against MCM3, ORC2, and RPA. In lanes 2 and 3, the NPE contained 50 μg/mL aphidicolin. (D) Sperm chromatin was incubated in 4 μL HSS, followed by addition of 8 μL mock- or geminin-depleted NPE to yield 3000 sperm/μL final concentration (left and middle columns). Sperm chromatin was incubated in 12 μL NPE (3000/μL final concentration) supplemented with 200 nM rCdt1 (right column). The percentage of input DNA replicated after 90 min is plotted. (E) Sperm chromatin was incubated in HSS, followed by addition of geminin-depleted NPE to yield 3000 sperm/μL final concentration in the presence of [α-32P]dATP and BrdU. Replication products were fractionated on a CsCl gradient and radioactivity across the gradient was measured. (HL) Heavy–Light DNA, 1.75 g/mL, (HH) Heavy–Heavy DNA, 1.80 g/mL. (F) NPE was supplemented with 200 nM rCdt1 and incubated for 15 min before addition of sperm chromatin (1500/μL) in the presence of [α-32P]dATP and BrdU. Replication products were analyzed as in E.
Figure 2.
Figure 2.
Cdt1 is degraded by ubiquitin-mediated proteolysis in Xenopus egg extracts. (A) Sperm chromatin (10,000/μL) was incubated in HSS for 30 min, followed by addition of NPE. A total of 1 μL of extract was withdrawn at the indicated times and blotted for Cdt1. Asterisk denotes a cross-reacting protein that served as a loading control. This protein is more abundant in NPE than in LSS. (B) Sperm chromatin was incubated in LSS and analyzed as in A. In all experiments using LSS, sperm chromatin was present at 3000/μL, unless otherwise stated. (C) Sperm chromatin was incubated in LSS in the presence of DMSO or 1 mM MG132 and analyzed as in A.(D) Sperm chromatin was incubated in LSS and isolated at various times after sperm addition. The indicated chromatin-bound proteins were analyzed by Western blotting. (E) As in D, except that lanes 2 and 4 contained 2 mg/mL methylated ubiquitin.
Figure 3.
Figure 3.
Cdt1 degradation requires entry into S phase. (A) LSS was incubated in the presence or absence of sperm chromatin. Where indicated, 0.2 mg/mL WGA was included. Reactions were stopped at the indicated times, and 1 μL of the total extract was blotted for Cdt1. (B) Sperm chromatin was incubated in LSS in the presence or absence of 1 μM p27Kip and analyzed as in A.
Figure 4.
Figure 4.
Cdt1 degradation requires initiation of DNA replication. LSS was immunodepleted of Cdc45, RPA, or pol α, as indicated. Where indicated, extracts contained 1.6 μM recombinant 6xhis-Cdc45 (+rCdc45) or 50nM recombinant human pol α complex (+rpol α). (A,D,G) Sperm chromatin was incubated in the indicated LSS in the presence of [α-32P]dATP. The percentage of input DNA replicated after 90 min is plotted. (B,E,H) Sperm chromatin was incubated in the indicated LSS, and at various times, reactions were stopped and 1 μL of extract was blotted for Cdt1. (C,F,I) Sperm chromatin was incubated in the indicated LSS supplemented with 2 mg/mL methylated ubiquitin. Chromatin was isolated after 45 min, and the indicated proteins were visualized by Western blotting. Note, the Xenopus pol α antibody does not react with recombinant human pol α.
Figure 5.
Figure 5.
Degradation of Cdt1 does not require extensive DNA synthesis. (A) Sperm chromatin was incubated in LSS containing the indicated concentrations of aphidicolin. At various times, reactions were stopped and 1 μL of extract was blotted for Cdt1 and MCM3. Parallel reactions were carried out in the presence of [α-32P]dATP and the percentage of input DNA replicated after 120 min is indicated. (B) Sperm chromatin was incubated in LSS containing 2 mg/mL methylated ubiquitin and the indicated concentrations of aphidicolin or 1 μM p27Kip. Chromatin was isolated after 45 min and blotted for various replication proteins and RCC1 as a loading control.
Figure 6.
Figure 6.
Cdk2 activity is not required for Cdt1 degradation following initiation of replication. (Left) Sperm chromatin (10,000/μL) was incubated in HSS followed by addition of NPE supplemented with p27Kip or buffer, and 60 nM rCdt1 was added immediately. Reactions were incubated at 19°C for 20 min, and then transferred to 22°C. (Right) Sperm chromatin was incubated in HSS followed by addition of NPE. Reactions were incubated at 19°C for 20 min, followed by addition of p27Kip or buffer and transfer to 22°C. A total of 60 nM rCdt1 was added after an additional 10 min. In both panels, reactions were stopped at the indicated times after addition of rCdt1 and 0.5 μL of extract was blotted for Cdt1.
Figure 7.
Figure 7.
Addition of Cdt1 to replicated nuclei results in reloading of MCM2-7 and rereplication. (A) Sperm chromatin was incubated in LSS for 70 min, and chromatin was isolated (lanes 1,2) or samples were supplemented with control buffer (lanes 3,4), or 0.2 mg/mL WGA (lanes 5,6) and incubated further. After 20 min, reactions were supplemented with 100 nM rCdt1. DNA was isolated after a further 20 or 30 min, and chromatin-bound proteins were analyzed. Reaction 1 contained 50 μg/mL aphidicolin to retain MCM3 and Cdc45 on chromatin. (B,C,D) Sperm chromatin (2000/μL) was incubated in mock- or geminin-depleted LSS in the presence of [α-32P]dATP and BrdU. Reactions were supplemented with 80 nM rCdt1 immediately or 60 min after addition of sperm or with control buffer at the time of sperm addition. (B) The percentage of input DNA replicated after 180 min is plotted. Parallel reactions were stopped 180 min after sperm addition and replication products in mock (C) or geminin-depleted extracts (D) were separated on a CsCl gradient. The radioactivity in the peak Heavy–Light fractions among the six conditions did not differ by >10%, and was adjusted to a value of 100.
Figure 8.
Figure 8.
Replication-dependent Cdt1 degradation and geminin-mediated inhibition of Cdt1 occur independently of one another. (A) Sperm chromatin was incubated in mock- or geminin-depleted LSS. At the indicated times, reactions were stopped and 1 μL of extract was blotted for Cdt1. (B) (Chromatin) Sperm chromatin was incubated in mock-depleted LSS, Cdt1-depleted LSS, or Cdt1-depleted LSS containing 50 nM rCdt1. All samples contained 50 μg/mL aphidicolin. Chromatin was isolated after 30 min and blotted for the indicated factors. (Extract) One microliter of the indicated extract was blotted for Cdt1. (C) Sperm chromatin was incubated in LSS containing 0.2 mg/mL WGA (lane 1), 1 μM p27Kip (lane 2), or no additions (lanes 3–5). Chromatin was isolated at the indicated times (lanes 3–5) or after 40 min (lanes 1,2) and blotted for the indicated factors.
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