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PMID: 15598982 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

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

Genes & development ·Vol. 19 ·No. 1 ·2005-01-01 ·Pages 114-26

Arias EE, Walter JC

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.

MeSH Terms
Animals CDC2-CDC28 Kinases/physiology Cell Cycle/genetics Cell Cycle Proteins/metabolism,physiology Cell Extracts Chromatin Cyclin-Dependent Kinase 2 DNA Polymerase I DNA Replication DNA-Binding Proteins/metabolism Ovum/cytology Peptide Hydrolases/metabolism S Phase Ubiquitin Xenopus Xenopus Proteins
Chemicals
Cdt1 protein, Xenopus Cell Cycle Proteins Cell Extracts Chromatin DNA-Binding Proteins Ubiquitin Xenopus Proteins CDC2-CDC28 Kinases Cdk2 protein, Xenopus Cyclin-Dependent Kinase 2 DNA Polymerase I Peptide Hydrolases
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Arias Emily E
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Walter Johannes C
References (72)
72 references, click to expand
  1. Radiation-mediated proteolysis of CDT1 by CUL4-ROC1 and CSN complexes constitutes a new checkpoint.
    Nat Cell Biol. 2003 Nov;5(11):1008-15 PMID: 14578910
  2. Cell cycle arrest and DNA endoreduplication following p21Waf1/Cip1 expression.
    Oncogene. 1998 Oct 1;17(13):1691-703 PMID: 9796698
  3. The role of Cdc6 in ensuring complete genome licensing and S phase checkpoint activation.
    J Cell Biol. 2004 Apr 26;165(2):181-90 PMID: 15096526
  4. Cdt1 phosphorylation by cyclin A-dependent kinases negatively regulates its function without affecting geminin binding.
    J Biol Chem. 2004 May 7;279(19):19691-7 PMID: 14993212
  5. Loss of Geminin induces rereplication in the presence of functional p53.
    J Cell Biol. 2004 May 24;165(4):473-82 PMID: 15159417
  6. Rapid degradation of Cdt1 upon UV-induced DNA damage is mediated by SCFSkp2 complex.
    J Biol Chem. 2004 Jun 25;279(26):27315-9 PMID: 15102855
  7. Deregulation of cyclin E in human cells interferes with prereplication complex assembly.
    J Cell Biol. 2004 Jun 21;165(6):789-800 PMID: 15197178
  8. Proteolysis of DNA replication licensing factor Cdt1 in S-phase is performed independently of geminin through its N-terminal region.
    J Biol Chem. 2004 Jul 16;279(29):30807-16 PMID: 15138268
  9. Clb/Cdc28 kinases promote nuclear export of the replication initiator proteins Mcm2-7.
    Curr Biol. 2000 Feb 24;10(4):195-205 PMID: 10704410
  10. DNA replication of mitotic chromatin in Xenopus egg extracts.
    Proc Natl Acad Sci U S A. 2003 Nov 11;100(23):13241-6 PMID: 14597706
  11. Non-proteolytic inactivation of geminin requires CDK-dependent ubiquitination.
    Nat Cell Biol. 2004 Mar;6(3):260-7 PMID: 14767479
  12. Cell cycle regulation of the licensing activity of Cdt1 in Xenopus laevis.
    Exp Cell Res. 2004 Apr 15;295(1):138-49 PMID: 15051497
  13. Cyclin-dependent kinases phosphorylate human Cdt1 and induce its degradation.
    J Biol Chem. 2004 Apr 23;279(17):17283-8 PMID: 15004027
  14. Rereplication by depletion of geminin is seen regardless of p53 status and activates a G2/M checkpoint.
    Mol Cell Biol. 2004 Aug;24(16):7140-50 PMID: 15282313
  15. Drosophila double-parked is sufficient to induce re-replication during development and is regulated by cyclin E/CDK2.
    Development. 2004 Oct;131(19):4807-18 PMID: 15342466
  16. A requirement for MCM7 and Cdc45 in chromosome unwinding during eukaryotic DNA replication.
    EMBO J. 2004 Sep 15;23(18):3667-76 PMID: 15329670
  17. A role for Ran-GTP and Crm1 in blocking re-replication.
    Cell. 2004 Oct 1;119(1):145 PMID: 15478275
  18. DNA unwinding is an Mcm complex-dependent and ATP hydrolysis-dependent process.
    J Biol Chem. 2004 Oct 29;279(44):45586-93 PMID: 15326181
  19. Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components.
    Science. 1983 May 13;220(4598):719-21 PMID: 6601299
  20. Occurrence of a polyubiquitin structure in ubiquitin-protein conjugates.
    Biochem Biophys Res Commun. 1985 May 16;128(3):1079-86 PMID: 2988526
  21. Initiation of DNA replication in nuclei and purified DNA by a cell-free extract of Xenopus eggs.
    Cell. 1986 Nov 21;47(4):577-87 PMID: 3779837
  22. Nuclear reconstitution in vitro: stages of assembly around protein-free DNA.
    Cell. 1987 Jan 30;48(2):205-17 PMID: 3026635
  23. Inhibition of in vitro nuclear transport by a lectin that binds to nuclear pores.
    J Cell Biol. 1987 Feb;104(2):189-200 PMID: 3805121
  24. A role for the nuclear envelope in controlling DNA replication within the cell cycle.
    Nature. 1988 Apr 7;332(6164):546-8 PMID: 3357511
  25. Involvement of p34cdc2 in establishing the dependency of S phase on mitosis.
    Nature. 1991 Jan 31;349(6308):388-93 PMID: 1992340
  26. Preventing re-replication of DNA in a single cell cycle: evidence for a replication licensing factor.
    J Cell Biol. 1993 Sep;122(5):993-1002 PMID: 8354699
  27. p65cdc18 plays a major role controlling the initiation of DNA replication in fission yeast.
    Cell. 1995 Nov 3;83(3):397-405 PMID: 8521469
  28. S-phase-promoting cyclin-dependent kinases prevent re-replication by inhibiting the transition of replication origins to a pre-replicative state.
    Curr Biol. 1995 Nov 1;5(11):1257-69 PMID: 8574583
  29. Once and only once upon a time: specifying and regulating origins of DNA replication in eukaryotic cells.
    Genes Dev. 1996 Nov 15;10(22):2819-30 PMID: 8918884
  30. Regulation of replicon size in Xenopus egg extracts.
    Science. 1997 Feb 14;275(5302):993-5 PMID: 9020085
  31. Construction by gene targeting in human cells of a "conditional' CDC2 mutant that rereplicates its DNA.
    Nat Genet. 1997 Mar;15(3):258-65 PMID: 9054937
  32. A role for Cdk2 kinase in negatively regulating DNA replication during S phase of the cell cycle.
    J Cell Biol. 1997 Apr 7;137(1):183-92 PMID: 9105046
  33. Proteolysis and DNA replication: the CDC34 requirement in the Xenopus egg cell cycle.
    Science. 1997 Sep 12;277(5332):1672-6 PMID: 9287222
  34. The Cdc4/34/53 pathway targets Cdc6p for proteolysis in budding yeast.
    EMBO J. 1997 Oct 1;16(19):5966-76 PMID: 9312054
  35. Chromosome association of minichromosome maintenance proteins in Drosophila mitotic cycles.
    J Cell Biol. 1997 Oct 6;139(1):13-21 PMID: 9314525
  36. Fluctuations in cyclin E levels are required for multiple rounds of endocycle S phase in Drosophila.
    Curr Biol. 1998 Feb 12;8(4):235-8 PMID: 9501987
  37. Continuous Cyclin E expression inhibits progression through endoreduplication cycles in Drosophila.
    Curr Biol. 1998 Feb 12;8(4):239-42 PMID: 9501988
  38. Geminin, an inhibitor of DNA replication, is degraded during mitosis.
    Cell. 1998 Jun 12;93(6):1043-53 PMID: 9635433
  39. Regulated chromosomal DNA replication in the absence of a nucleus.
    Mol Cell. 1998 Mar;1(4):519-29 PMID: 9660936
  40. Xenopus Cdc45-dependent loading of DNA polymerase alpha onto chromatin under the control of S-phase Cdk.
    EMBO J. 1998 Oct 1;17(19):5699-707 PMID: 9755170
  41. XCDT1 is required for the assembly of pre-replicative complexes in Xenopus laevis.
    Nature. 2000 Apr 6;404(6778):622-5 PMID: 10766247
  42. The Cdt1 protein is required to license DNA for replication in fission yeast.
    Nature. 2000 Apr 6;404(6778):625-8 PMID: 10766248
  43. Initiation of eukaryotic DNA replication: origin unwinding and sequential chromatin association of Cdc45, RPA, and DNA polymerase alpha.
    Mol Cell. 2000 Apr;5(4):617-27 PMID: 10882098
  44. Central role for cdc45 in establishing an initiation complex of DNA replication in Xenopus egg extracts.
    Genes Cells. 2000 Jun;5(6):439-52 PMID: 10886370
  45. Requirement for Atr in phosphorylation of Chk1 and cell cycle regulation in response to DNA replication blocks and UV-damaged DNA in Xenopus egg extracts.
    Genes Dev. 2000 Nov 1;14(21):2745-56 PMID: 11069891
  46. Evidence for sequential action of cdc7 and cdk2 protein kinases during initiation of DNA replication in Xenopus egg extracts.
    J Biol Chem. 2000 Dec 15;275(50):39773-8 PMID: 11005825
  47. Inhibition of eukaryotic DNA replication by geminin binding to Cdt1.
    Science. 2000 Dec 22;290(5500):2309-12 PMID: 11125146
  48. G1-phase and B-type cyclins exclude the DNA-replication factor Mcm4 from the nucleus.
    Nat Cell Biol. 1999 Nov;1(7):415-22 PMID: 10559985
  49. Is the MCM2-7 complex the eukaryotic DNA replication fork helicase?
    Curr Opin Genet Dev. 2001 Feb;11(1):64-70 PMID: 11163153
  50. Repression of origin assembly in metaphase depends on inhibition of RLF-B/Cdt1 by geminin.
    Nat Cell Biol. 2001 Feb;3(2):107-13 PMID: 11175741
  51. Use of peptides from p21 (Waf1/Cip1) to investigate PCNA function in Xenopus egg extracts.
    Exp Cell Res. 2001 May 1;265(2):242-51 PMID: 11302689
  52. DNA replication: building the perfect switch.
    Curr Biol. 2001 May 1;11(9):R367-70 PMID: 11369250
  53. Cyclin-dependent kinases prevent DNA re-replication through multiple mechanisms.
    Nature. 2001 Jun 28;411(6841):1068-73 PMID: 11429609
  54. Triggering ubiquitination of a CDK inhibitor at origins of DNA replication.
    Nat Cell Biol. 2001 Aug;3(8):715-22 PMID: 11483956
  55. Control of DNA rereplication via Cdc2 phosphorylation sites in the origin recognition complex.
    Mol Cell Biol. 2001 Sep;21(17):5767-77 PMID: 11486016
  56. Cell cycle checkpoint signaling through the ATM and ATR kinases.
    Genes Dev. 2001 Sep 1;15(17):2177-96 PMID: 11544175
  57. The Drosophila Geminin homolog: roles for Geminin in limiting DNA replication, in anaphase and in neurogenesis.
    Genes Dev. 2001 Oct 15;15(20):2741-54 PMID: 11641279
  58. The human licensing factor for DNA replication Cdt1 accumulates in G1 and is destabilized after initiation of S-phase.
    J Biol Chem. 2001 Nov 30;276(48):44905-11 PMID: 11555648
  59. Control of DNA replication and chromosome ploidy by geminin and cyclin A.
    Mol Cell Biol. 2002 Mar;22(6):1868-80 PMID: 11865064
  60. Interdependent nuclear accumulation of budding yeast Cdt1 and Mcm2-7 during G1 phase.
    Nat Cell Biol. 2002 Mar;4(3):198-207 PMID: 11836525
  61. Human origin recognition complex large subunit is degraded by ubiquitin-mediated proteolysis after initiation of DNA replication.
    Mol Cell. 2002 Mar;9(3):481-91 PMID: 11931757
  62. Geminin becomes activated as an inhibitor of Cdt1/RLF-B following nuclear import.
    Curr Biol. 2002 Apr 16;12(8):678-83 PMID: 11967157
  63. Xic1 degradation in Xenopus egg extracts is coupled to initiation of DNA replication.
    Genes Dev. 2002 May 15;16(10):1182-94 PMID: 12023298
  64. Eukaryotic DNA polymerases.
    Annu Rev Biochem. 2002;71:133-63 PMID: 12045093
  65. DNA replication in eukaryotic cells.
    Annu Rev Biochem. 2002;71:333-74 PMID: 12045100
  66. Geminin deficiency causes a Chk1-dependent G2 arrest in Xenopus.
    Mol Biol Cell. 2002 Oct;13(10):3662-71 PMID: 12388764
  67. Reconstitution of licensed replication origins on Xenopus sperm nuclei using purified proteins.
    BMC Biochem. 2001;2:15 PMID: 11737877
  68. A role for Ran-GTP and Crm1 in blocking re-replication.
    Cell. 2003 Apr 4;113(1):115-25 PMID: 12679039
  69. A p53-dependent checkpoint pathway prevents rereplication.
    Mol Cell. 2003 Apr;11(4):997-1008 PMID: 12718885
  70. CUL-4 ubiquitin ligase maintains genome stability by restraining DNA-replication licensing.
    Nature. 2003 Jun 19;423(6942):885-9 PMID: 12815436
  71. The SCF(Skp2) ubiquitin ligase complex interacts with the human replication licensing factor Cdt1 and regulates Cdt1 degradation.
    J Biol Chem. 2003 Aug 15;278(33):30854-8 PMID: 12840033
  72. Cyclin E ablation in the mouse.
    Cell. 2003 Aug 22;114(4):431-43 PMID: 12941272
Article Info
Journal
Genes & development
Abbr.
Genes Dev
ISSN
0890-9369
DOI
10.1101/gad.1255805
Published
2005-01-01
Epub
2004-00-14
Pages
114-26
Language
English
Region
United States
NLM ID
8711660
PMCID
PMC540230
Subset
IM
External Links
PubMed source DOI: 10.1101/gad.1255805 PMC Full Text View journal details
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