Abstract
We exploit an improved mammalian cell-free DNA replication system to analyse quiescence and Cdc6 function. Quiescent 3T3 nuclei cannot initiate replication in S phase cytosol from HeLa or 3T3 cells. Following release from quiescence, nuclei become competent to initiate semiconservative DNA replication in S phase cytosol, but not in G0 phase cytosol. Immunoblots show that quiescent cells lack Cdc6 and that minichromosome maintenance (MCM) proteins are not associated with chromatin. Competence of G1 phase nuclei to replicate in vitro coincides with maximum Cdc6 accumulation and MCM protein binding to chromatin in vivo. Addition of recombinant Cdc6 to permeabilized, but not intact, G1 nuclei causes up to 82% of the nuclei to initiate and accelerates G1 progression, making nuclei competent to replicate prematurely.
Full Text
The Full Text of this article is available as a PDF (530.3 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Aparicio O. M., Weinstein D. M., Bell S. P. Components and dynamics of DNA replication complexes in S. cerevisiae: redistribution of MCM proteins and Cdc45p during S phase. Cell. 1997 Oct 3;91(1):59–69. doi: 10.1016/s0092-8674(01)80009-x. [DOI] [PubMed] [Google Scholar]
- Bell S. P., Stillman B. ATP-dependent recognition of eukaryotic origins of DNA replication by a multiprotein complex. Nature. 1992 May 14;357(6374):128–134. doi: 10.1038/357128a0. [DOI] [PubMed] [Google Scholar]
- Blow J. J., Laskey R. A. A role for the nuclear envelope in controlling DNA replication within the cell cycle. Nature. 1988 Apr 7;332(6164):546–548. doi: 10.1038/332546a0. [DOI] [PubMed] [Google Scholar]
- Blow J. J., Laskey R. A. Initiation of DNA replication in nuclei and purified DNA by a cell-free extract of Xenopus eggs. Cell. 1986 Nov 21;47(4):577–587. doi: 10.1016/0092-8674(86)90622-7. [DOI] [PubMed] [Google Scholar]
- Blow J. J. 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. doi: 10.1083/jcb.122.5.993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Botchan M. Coordinating DNA replication with cell division: current status of the licensing concept. Proc Natl Acad Sci U S A. 1996 Sep 17;93(19):9997–10000. doi: 10.1073/pnas.93.19.9997. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bueno A., Russell P. Dual functions of CDC6: a yeast protein required for DNA replication also inhibits nuclear division. EMBO J. 1992 Jun;11(6):2167–2176. doi: 10.1002/j.1460-2075.1992.tb05276.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Carpenter P. B., Mueller P. R., Dunphy W. G. Role for a Xenopus Orc2-related protein in controlling DNA replication. Nature. 1996 Jan 25;379(6563):357–360. doi: 10.1038/379357a0. [DOI] [PubMed] [Google Scholar]
- Chong J. P., Mahbubani H. M., Khoo C. Y., Blow J. J. Purification of an MCM-containing complex as a component of the DNA replication licensing system. Nature. 1995 Jun 1;375(6530):418–421. doi: 10.1038/375418a0. [DOI] [PubMed] [Google Scholar]
- Cocker J. H., Piatti S., Santocanale C., Nasmyth K., Diffley J. F. An essential role for the Cdc6 protein in forming the pre-replicative complexes of budding yeast. Nature. 1996 Jan 11;379(6561):180–182. doi: 10.1038/379180a0. [DOI] [PubMed] [Google Scholar]
- Coleman T. R., Carpenter P. B., Dunphy W. G. The Xenopus Cdc6 protein is essential for the initiation of a single round of DNA replication in cell-free extracts. Cell. 1996 Oct 4;87(1):53–63. doi: 10.1016/s0092-8674(00)81322-7. [DOI] [PubMed] [Google Scholar]
- Connell-Crowley L., Elledge S. J., Harper J. W. G1 cyclin-dependent kinases are sufficient to initiate DNA synthesis in quiescent human fibroblasts. Curr Biol. 1998 Jan 1;8(1):65–68. doi: 10.1016/s0960-9822(98)70021-1. [DOI] [PubMed] [Google Scholar]
- Correa-Bordes J., Nurse P. p25rum1 orders S phase and mitosis by acting as an inhibitor of the p34cdc2 mitotic kinase. Cell. 1995 Dec 15;83(6):1001–1009. doi: 10.1016/0092-8674(95)90215-5. [DOI] [PubMed] [Google Scholar]
- Coué M., Kearsey S. E., Méchali M. Chromotin binding, nuclear localization and phosphorylation of Xenopus cdc21 are cell-cycle dependent and associated with the control of initiation of DNA replication. EMBO J. 1996 Mar 1;15(5):1085–1097. [PMC free article] [PubMed] [Google Scholar]
- Coverley D., Downes C. S., Romanowski P., Laskey R. A. Reversible effects of nuclear membrane permeabilization on DNA replication: evidence for a positive licensing factor. J Cell Biol. 1993 Sep;122(5):985–992. doi: 10.1083/jcb.122.5.985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Coverley D., Wilkinson H. R., Madine M. A., Mills A. D., Laskey R. A. Protein kinase inhibition in G2 causes mammalian Mcm proteins to reassociate with chromatin and restores ability to replicate. Exp Cell Res. 1998 Jan 10;238(1):63–69. doi: 10.1006/excr.1997.3829. [DOI] [PubMed] [Google Scholar]
- Dahmann C., Diffley J. F., Nasmyth K. A. 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):1257–1269. doi: 10.1016/s0960-9822(95)00252-1. [DOI] [PubMed] [Google Scholar]
- Dalton S., Whitbread L. Cell cycle-regulated nuclear import and export of Cdc47, a protein essential for initiation of DNA replication in budding yeast. Proc Natl Acad Sci U S A. 1995 Mar 28;92(7):2514–2518. doi: 10.1073/pnas.92.7.2514. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Detweiler C. S., Li J. J. Cdc6p establishes and maintains a state of replication competence during G1 phase. J Cell Sci. 1997 Mar;110(Pt 6):753–763. doi: 10.1242/jcs.110.6.753. [DOI] [PubMed] [Google Scholar]
- Detweiler C. S., Li J. J. Ectopic induction of Clb2 in early G1 phase is sufficient to block prereplicative complex formation in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1998 Mar 3;95(5):2384–2389. doi: 10.1073/pnas.95.5.2384. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Diffley J. F., Cocker J. H., Dowell S. J., Rowley A. Two steps in the assembly of complexes at yeast replication origins in vivo. Cell. 1994 Jul 29;78(2):303–316. doi: 10.1016/0092-8674(94)90299-2. [DOI] [PubMed] [Google Scholar]
- Diffley J. F., Cocker J. H. Protein-DNA interactions at a yeast replication origin. Nature. 1992 May 14;357(6374):169–172. doi: 10.1038/357169a0. [DOI] [PubMed] [Google Scholar]
- Donovan S., Diffley J. F. Replication origins in eukaroytes. Curr Opin Genet Dev. 1996 Apr;6(2):203–207. doi: 10.1016/s0959-437x(96)80051-7. [DOI] [PubMed] [Google Scholar]
- Donovan S., Harwood J., Drury L. S., Diffley J. F. Cdc6p-dependent loading of Mcm proteins onto pre-replicative chromatin in budding yeast. Proc Natl Acad Sci U S A. 1997 May 27;94(11):5611–5616. doi: 10.1073/pnas.94.11.5611. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Drury L. S., Perkins G., Diffley J. F. The Cdc4/34/53 pathway targets Cdc6p for proteolysis in budding yeast. EMBO J. 1997 Oct 1;16(19):5966–5976. doi: 10.1093/emboj/16.19.5966. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dutta A., Bell S. P. Initiation of DNA replication in eukaryotic cells. Annu Rev Cell Dev Biol. 1997;13:293–332. doi: 10.1146/annurev.cellbio.13.1.293. [DOI] [PubMed] [Google Scholar]
- Gavin K. A., Hidaka M., Stillman B. Conserved initiator proteins in eukaryotes. Science. 1995 Dec 8;270(5242):1667–1671. doi: 10.1126/science.270.5242.1667. [DOI] [PubMed] [Google Scholar]
- Hayles J., Fisher D., Woollard A., Nurse P. Temporal order of S phase and mitosis in fission yeast is determined by the state of the p34cdc2-mitotic B cyclin complex. Cell. 1994 Sep 9;78(5):813–822. doi: 10.1016/s0092-8674(94)90542-8. [DOI] [PubMed] [Google Scholar]
- Heintz N. H., Stillman B. W. Nuclear DNA synthesis in vitro is mediated via stable replication forks assembled in a temporally specific fashion in vivo. Mol Cell Biol. 1988 May;8(5):1923–1931. doi: 10.1128/mcb.8.5.1923. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hendrickson M., Madine M., Dalton S., Gautier J. Phosphorylation of MCM4 by cdc2 protein kinase inhibits the activity of the minichromosome maintenance complex. Proc Natl Acad Sci U S A. 1996 Oct 29;93(22):12223–12228. doi: 10.1073/pnas.93.22.12223. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jallepalli P. V., Kelly T. J. Rum1 and Cdc18 link inhibition of cyclin-dependent kinase to the initiation of DNA replication in Schizosaccharomyces pombe. Genes Dev. 1996 Mar 1;10(5):541–552. doi: 10.1101/gad.10.5.541. [DOI] [PubMed] [Google Scholar]
- Kelly T. J., Martin G. S., Forsburg S. L., Stephen R. J., Russo A., Nurse P. The fission yeast cdc18+ gene product couples S phase to START and mitosis. Cell. 1993 Jul 30;74(2):371–382. doi: 10.1016/0092-8674(93)90427-r. [DOI] [PubMed] [Google Scholar]
- Kimura H., Nozaki N., Sugimoto K. DNA polymerase alpha associated protein P1, a murine homolog of yeast MCM3, changes its intranuclear distribution during the DNA synthetic period. EMBO J. 1994 Sep 15;13(18):4311–4320. doi: 10.1002/j.1460-2075.1994.tb06751.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Krude T., Jackman M., Pines J., Laskey R. A. Cyclin/Cdk-dependent initiation of DNA replication in a human cell-free system. Cell. 1997 Jan 10;88(1):109–119. doi: 10.1016/s0092-8674(00)81863-2. [DOI] [PubMed] [Google Scholar]
- Krude T., Musahl C., Laskey R. A., Knippers R. Human replication proteins hCdc21, hCdc46 and P1Mcm3 bind chromatin uniformly before S-phase and are displaced locally during DNA replication. J Cell Sci. 1996 Feb;109(Pt 2):309–318. doi: 10.1242/jcs.109.2.309. [DOI] [PubMed] [Google Scholar]
- Kubota Y., Mimura S., Nishimoto S., Takisawa H., Nojima H. Identification of the yeast MCM3-related protein as a component of Xenopus DNA replication licensing factor. Cell. 1995 May 19;81(4):601–609. doi: 10.1016/0092-8674(95)90081-0. [DOI] [PubMed] [Google Scholar]
- Kubota Y., Takisawa H. Determination of initiation of DNA replication before and after nuclear formation in Xenopus egg cell free extracts. J Cell Biol. 1993 Dec;123(6 Pt 1):1321–1331. doi: 10.1083/jcb.123.6.1321. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Labib K., Craven R. A., Crawford K., Nurse P. Dominant mutants identify new roles for p34cdc2 in mitosis. EMBO J. 1995 May 15;14(10):2155–2165. doi: 10.1002/j.1460-2075.1995.tb07209.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Laskey R. A., Görlich D., Madine M. A., Makkerh J. P., Romanowski P. Regulatory roles of the nuclear envelope. Exp Cell Res. 1996 Dec 15;229(2):204–211. doi: 10.1006/excr.1996.0361. [DOI] [PubMed] [Google Scholar]
- Leno G. H., Downes C. S., Laskey R. A. The nuclear membrane prevents replication of human G2 nuclei but not G1 nuclei in Xenopus egg extract. Cell. 1992 Apr 3;69(1):151–158. doi: 10.1016/0092-8674(92)90126-w. [DOI] [PubMed] [Google Scholar]
- Leno G. H., Munshi R. Initiation of DNA replication in nuclei from quiescent cells requires permeabilization of the nuclear membrane. J Cell Biol. 1994 Oct;127(1):5–14. doi: 10.1083/jcb.127.1.5. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Liang C., Stillman B. Persistent initiation of DNA replication and chromatin-bound MCM proteins during the cell cycle in cdc6 mutants. Genes Dev. 1997 Dec 15;11(24):3375–3386. doi: 10.1101/gad.11.24.3375. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Liang C., Weinreich M., Stillman B. ORC and Cdc6p interact and determine the frequency of initiation of DNA replication in the genome. Cell. 1995 Jun 2;81(5):667–676. doi: 10.1016/0092-8674(95)90528-6. [DOI] [PubMed] [Google Scholar]
- Madine M. A., Khoo C. Y., Mills A. D., Laskey R. A. MCM3 complex required for cell cycle regulation of DNA replication in vertebrate cells. Nature. 1995 Jun 1;375(6530):421–424. doi: 10.1038/375421a0. [DOI] [PubMed] [Google Scholar]
- Madine M. A., Khoo C. Y., Mills A. D., Musahl C., Laskey R. A. The nuclear envelope prevents reinitiation of replication by regulating the binding of MCM3 to chromatin in Xenopus egg extracts. Curr Biol. 1995 Nov 1;5(11):1270–1279. doi: 10.1016/s0960-9822(95)00253-3. [DOI] [PubMed] [Google Scholar]
- Meijer L. Chemical inhibitors of cyclin-dependent kinases. Trends Cell Biol. 1996 Oct;6(10):393–397. doi: 10.1016/0962-8924(96)10034-9. [DOI] [PubMed] [Google Scholar]
- Miroux B., Walker J. E. Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J Mol Biol. 1996 Jul 19;260(3):289–298. doi: 10.1006/jmbi.1996.0399. [DOI] [PubMed] [Google Scholar]
- Moreno S., Nurse P. Regulation of progression through the G1 phase of the cell cycle by the rum1+ gene. Nature. 1994 Jan 20;367(6460):236–242. doi: 10.1038/367236a0. [DOI] [PubMed] [Google Scholar]
- Muzi Falconi M., Brown G. W., Kelly T. J. cdc18+ regulates initiation of DNA replication in Schizosaccharomyces pombe. Proc Natl Acad Sci U S A. 1996 Feb 20;93(4):1566–1570. doi: 10.1073/pnas.93.4.1566. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Newlon C. S. Putting it all together: building a prereplicative complex. Cell. 1997 Dec 12;91(6):717–720. doi: 10.1016/s0092-8674(00)80459-6. [DOI] [PubMed] [Google Scholar]
- Nishitani H., Nurse P. p65cdc18 plays a major role controlling the initiation of DNA replication in fission yeast. Cell. 1995 Nov 3;83(3):397–405. doi: 10.1016/0092-8674(95)90117-5. [DOI] [PubMed] [Google Scholar]
- Ohtsubo M., Theodoras A. M., Schumacher J., Roberts J. M., Pagano M. Human cyclin E, a nuclear protein essential for the G1-to-S phase transition. Mol Cell Biol. 1995 May;15(5):2612–2624. doi: 10.1128/mcb.15.5.2612. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Piatti S., Böhm T., Cocker J. H., Diffley J. F., Nasmyth K. Activation of S-phase-promoting CDKs in late G1 defines a "point of no return" after which Cdc6 synthesis cannot promote DNA replication in yeast. Genes Dev. 1996 Jun 15;10(12):1516–1531. doi: 10.1101/gad.10.12.1516. [DOI] [PubMed] [Google Scholar]
- Piatti S., Lengauer C., Nasmyth K. Cdc6 is an unstable protein whose de novo synthesis in G1 is important for the onset of S phase and for preventing a 'reductional' anaphase in the budding yeast Saccharomyces cerevisiae. EMBO J. 1995 Aug 1;14(15):3788–3799. doi: 10.1002/j.1460-2075.1995.tb00048.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rao P. N., Johnson R. T. Mammalian cell fusion: studies on the regulation of DNA synthesis and mitosis. Nature. 1970 Jan 10;225(5228):159–164. doi: 10.1038/225159a0. [DOI] [PubMed] [Google Scholar]
- Resnitzky D., Gossen M., Bujard H., Reed S. I. Acceleration of the G1/S phase transition by expression of cyclins D1 and E with an inducible system. Mol Cell Biol. 1994 Mar;14(3):1669–1679. doi: 10.1128/mcb.14.3.1669. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Resnitzky D., Reed S. I. Different roles for cyclins D1 and E in regulation of the G1-to-S transition. Mol Cell Biol. 1995 Jul;15(7):3463–3469. doi: 10.1128/mcb.15.7.3463. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Romanowski P., Madine M. A., Laskey R. A. XMCM7, a novel member of the Xenopus MCM family, interacts with XMCM3 and colocalizes with it throughout replication. Proc Natl Acad Sci U S A. 1996 Sep 17;93(19):10189–10194. doi: 10.1073/pnas.93.19.10189. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Romanowski P., Madine M. A. Mechanisms restricting DNA replication to once per cell cycle: MCMS, pre-replicative complexes and kinases. Trends Cell Biol. 1996 May;6(5):184–188. doi: 10.1016/0962-8924(96)10015-5. [DOI] [PubMed] [Google Scholar]
- Romanowski P., Madine M. A. Mechanisms restricting DNA replication to once per cell cycle: the role of Cdc6p and ORC. Trends Cell Biol. 1997 Jan;7(1):9–10. doi: 10.1016/S0962-8924(97)30077-4. [DOI] [PubMed] [Google Scholar]
- Romanowski P., Madine M. A., Rowles A., Blow J. J., Laskey R. A. The Xenopus origin recognition complex is essential for DNA replication and MCM binding to chromatin. Curr Biol. 1996 Nov 1;6(11):1416–1425. doi: 10.1016/s0960-9822(96)00746-4. [DOI] [PubMed] [Google Scholar]
- Rowles A., Chong J. P., Brown L., Howell M., Evan G. I., Blow J. J. Interaction between the origin recognition complex and the replication licensing system in Xenopus. Cell. 1996 Oct 18;87(2):287–296. doi: 10.1016/s0092-8674(00)81346-x. [DOI] [PubMed] [Google Scholar]
- Stillman B. Cell cycle control of DNA replication. Science. 1996 Dec 6;274(5293):1659–1664. doi: 10.1126/science.274.5293.1659. [DOI] [PubMed] [Google Scholar]
- Tanaka T., Knapp D., Nasmyth K. Loading of an Mcm protein onto DNA replication origins is regulated by Cdc6p and CDKs. Cell. 1997 Aug 22;90(4):649–660. doi: 10.1016/s0092-8674(00)80526-7. [DOI] [PubMed] [Google Scholar]
- Todorov I. T., Attaran A., Kearsey S. E. BM28, a human member of the MCM2-3-5 family, is displaced from chromatin during DNA replication. J Cell Biol. 1995 Jun;129(6):1433–1445. doi: 10.1083/jcb.129.6.1433. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Veselý J., Havlicek L., Strnad M., Blow J. J., Donella-Deana A., Pinna L., Letham D. S., Kato J., Detivaud L., Leclerc S. Inhibition of cyclin-dependent kinases by purine analogues. Eur J Biochem. 1994 Sep 1;224(2):771–786. doi: 10.1111/j.1432-1033.1994.00771.x. [DOI] [PubMed] [Google Scholar]
- Walter J., Sun L., Newport J. Regulated chromosomal DNA replication in the absence of a nucleus. Mol Cell. 1998 Mar;1(4):519–529. doi: 10.1016/s1097-2765(00)80052-0. [DOI] [PubMed] [Google Scholar]
- Williams R. S., Shohet R. V., Stillman B. A human protein related to yeast Cdc6p. Proc Natl Acad Sci U S A. 1997 Jan 7;94(1):142–147. doi: 10.1073/pnas.94.1.142. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yan Z., DeGregori J., Shohet R., Leone G., Stillman B., Nevins J. R., Williams R. S. Cdc6 is regulated by E2F and is essential for DNA replication in mammalian cells. Proc Natl Acad Sci U S A. 1998 Mar 31;95(7):3603–3608. doi: 10.1073/pnas.95.7.3603. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zwerschke W., Rottjakob H. W., Küntzel H. The Saccharomyces cerevisiae CDC6 gene is transcribed at late mitosis and encodes a ATP/GTPase controlling S phase initiation. J Biol Chem. 1994 Sep 16;269(37):23351–23356. [PubMed] [Google Scholar]