Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

Reducing MCM levels in human primary T cells during the G0→G1 transition causes genomic instability during the first cell cycle

Abstract

DNA replication is tightly regulated, but paradoxically there is reported to be an excess of MCM DNA replication proteins over the number of replication origins. Here, we show that MCM levels in primary human T cells are induced during the G0→G1 transition and are not in excess in proliferating cells. The level of induction is critical as we show that a 50% reduction leads to increased centromere separation, premature chromatid separation (PCS) and gross chromosomal abnormalities typical of genomic instability syndromes. We investigated the mechanisms involved and show that a reduction in MCM levels causes dose-dependent DNA damage involving activation of ATR & ATM and Chk1 & Chk2. There is increased DNA mis-repair by non-homologous end joining (NHEJ) and both NHEJ and homologous recombination are necessary for Mcm7-depleted cells to progress to metaphase. Therefore, a simple reduction in MCM loading onto DNA, which occurs in cancers as a result of aberrant cell cycle control, is sufficient to cause PCS and gross genomic instability within one cell cycle.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6

Similar content being viewed by others

References

  • Aggarwal P, Lessie MD, Lin DI, Pontano L, Gladden AB, Nuskey B et al. (2007). Nuclear accumulation of cyclin D1 during S phase inhibits Cul4-dependent Cdt1 proteolysis and triggers p53-dependent DNA rereplication. Genes Dev 21: 2908–2922.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Arnaudeau C, Lundin C, Helleday T . (2001). DNA double-strand breaks associated with replication forks are predominantly repaired by homologous recombination involving an exchange mechanism in mammalian cells. J Mol Biol 307: 1235–1245.

    Article  CAS  PubMed  Google Scholar 

  • Bailis JM, Luche DD, Hunter T, Forsburg SL . (2008). Minichromosome maintenance proteins interact with checkpoint and recombination proteins to promote s-phase genome stability. Mol Cell Biol 28: 1724–1738.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bell SP, Dutta A . (2002). DNA replication in eukaryotic cells. Annu Rev Biochem 71: 333–374.

    Article  CAS  PubMed  Google Scholar 

  • Blow JJ, Dutta A . (2005). Preventing re-replication of chromosomal DNA. Nat Rev Mol Cell Biol 6: 476–486.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Blow JJ, Hodgson B . (2002). Replication licensing—defining the proliferative state? Trends Cell Biol 12: 72–78.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bruemmer D, Yin F, Liu J, Kiyono T, Fleck E, Van Herle AJ et al. (2003). Rapamycin inhibits E2F-dependent expression of minichromosome maintenance proteins in vascular smooth muscle cells. Biochem Biophys Res Commun 303: 251–258.

    Article  CAS  PubMed  Google Scholar 

  • Budman J, Kim SA, Chu G . (2007). Processing of DNA for nonhomologous end-joining is controlled by kinase activity and XRCC4/LigaseIV. J Biol Chem 282: 11950–11959.

    Article  CAS  PubMed  Google Scholar 

  • Chong JP, Blow JJ . (1996). DNA replication licensing factor. Prog Cell Cycle Res 2: 83–90.

    Article  CAS  PubMed  Google Scholar 

  • Cortez D, Glick G, Elledge SJ . (2004). Minichromosome maintenance proteins are direct targets of the ATM and ATR checkpoint kinases. Proc Natl Acad Sci USA 101: 10078–10083.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Coxon A, Maundrell K, Kearsey SE . (1992). Fission yeast cdc21+ belongs to a family of proteins involved in an early step of chromosome replication. Nucleic Acids Res 20: 5571–5577.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Crevel G, Hashimoto R, Vass S, Sherkow J, Yamaguchi M, Heck MM et al. (2007). Differential requirements for MCM proteins in DNA replication in Drosophila S2 cells. PLoS One 2: e833.

    Article  PubMed  PubMed Central  Google Scholar 

  • Ekholm-Reed S, Mendez J, Tedesco D, Zetterberg A, Stillman B, Reed SI . (2004). Deregulation of cyclin E in human cells interferes with prereplication complex assembly. J Cell Biol 165: 789–800.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Feng D, Tu Z, Wu W, Liang C . (2003). Inhibiting the expression of DNA replication-initiation proteins induces apoptosis in human cancer cells. Cancer Res 63: 7356–7364.

    CAS  PubMed  Google Scholar 

  • Flores-Rozas H, Kolodner RD . (2000). Links between replication, recombination and genome instability in eukaryotes. Trends Biochem Sci 25: 196–200.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Françon P, Mechali M . (2006). DNA replication origins. Encyclopedia of Life Sciences, www3.interscience.wiley.com, John Wiley & Sons, Ltd.

    Google Scholar 

  • Gaymes TJ, Mufti GJ, Rassool FV . (2002a). Myeloid leukemias have increased activity of the nonhomologous end-joining pathway and concomitant DNA misrepair that is dependent on the Ku70/86 heterodimer. Cancer Res 62: 2791–2797.

    CAS  PubMed  Google Scholar 

  • Gaymes TJ, North PS, Brady N, Hickson ID, Mufti GJ, Rassool FV . (2002b). Increased error-prone non homologous DNA end-joining—a proposed mechanism of chromosomal instability in Bloom's syndrome. Oncogene 21: 2525–2533.

    Article  CAS  PubMed  Google Scholar 

  • Ge XQ, Jackson DA, Blow JJ . (2007). Dormant origins licensed by excess Mcm2-7 are required for human cells to survive replicative stress. Genes Dev 21: 3331–3341.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gillespie PJ, Hirano T . (2004). Scc2 couples replication licensing to sister chromatid cohesion in Xenopus egg extracts. Curr Biol 14: 1598–1603.

    Article  CAS  PubMed  Google Scholar 

  • Hans F, Dimitrov S . (2001). Histone H3 phosphorylation and cell division. Oncogene 20: 3021–3027.

    Article  CAS  PubMed  Google Scholar 

  • Howell RT, Taylor AMR . (1992). Chromosomal instability syndromes. In: Rooney DE, Czepulkowski, BH (eds). Human Cytogenetics. A Practical Approach. Oxford University Press: Oxford. pp 209–234.

    Google Scholar 

  • Hua XH, Yan H, Newport J . (1997). A role for Cdk2 kinase in negatively regulating DNA replication during S phase of the cell cycle. J Cell Biol 137: 183–192.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hyrien O, Marheineke K, Goldar A . (2003). Paradoxes of eukaryotic DNA replication: MCM proteins and the random completion problem. Bioessays 25: 116–125.

    Article  CAS  PubMed  Google Scholar 

  • Ibarra A, Schwob E, Mendez J . (2008). Excess MCM proteins protect human cells from replicative stress by licensing backup origins of replication. Proc Natl Acad Sci USA 105: 8956–8961.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kajii T, Ikeuchi T, Yang ZQ, Nakamura Y, Tsuji Y, Yokomori K et al. (2001). Cancer-prone syndrome of mosaic variegated aneuploidy and total premature chromatid separation: report of five infants. Am J Med Genet 104: 57–64.

    Article  CAS  PubMed  Google Scholar 

  • Kajii T, Kawai T, Takumi T, Misu H, Mabuchi O, Takahashi Y et al. (1998). Mosaic variegated aneuploidy with multiple congenital abnormalities: homozygosity for total premature chromatid separation trait. Am J Med Genet 78: 245–249.

    Article  CAS  PubMed  Google Scholar 

  • Kolodner RD, Putnam CD, Myung K . (2002). Maintenance of genome stability in Saccharomyces cerevisiae. Science 297: 552–557.

    Article  CAS  PubMed  Google Scholar 

  • Lea NC, Orr SJ, Stoeber K, Williams GH, Lam EW, Ibrahim MA et al. (2003). Commitment point during G0→G1 that controls entry into the cell cycle. Mol Cell Biol 23: 2351–2361.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lei M, Kawasaki Y, Tye BK . (1996). Physical interactions among Mcm proteins and effects of Mcm dosage on DNA replication in Saccharomyces cerevisiae. Mol Cell Biol 16: 5081–5090.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lengronne A, McIntyre J, Katou Y, Kanoh Y, Hopfner KP, Shirahige K et al. (2006). Establishment of sister chromatid cohesion at the S. cerevisiae replication fork. Mol Cell 23: 787–799.

    Article  CAS  PubMed  Google Scholar 

  • Liang DT, Hodson JA, Forsburg SL . (1999). Reduced dosage of a single fission yeast MCM protein causes genetic instability and S phase delay. J Cell Sci 112 (Part 4): 559–567.

    CAS  PubMed  Google Scholar 

  • Lundin C, Erixon K, Arnaudeau C, Schultz N, Jenssen D, Meuth M et al. (2002). Different roles for nonhomologous end joining and homologous recombination following replication arrest in mammalian cells. Mol Cell Biol 22: 5869–5878.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Mailand N, Diffley JF . (2005). CDKs promote DNA replication origin licensing in human cells by protecting Cdc6 from APC/C-dependent proteolysis. Cell 122: 915–926.

    Article  CAS  PubMed  Google Scholar 

  • Moldovan GL, Pfander B, Jentsch S . (2006). PCNA controls establishment of sister chromatid cohesion during S phase. Mol Cell 23: 723–732.

    Article  CAS  PubMed  Google Scholar 

  • Nakanishi M, Shimada M, Niida H . (2006). Genetic instability in cancer cells by impaired cell cycle checkpoints. Cancer Sci 97: 984–989.

    Article  CAS  PubMed  Google Scholar 

  • Nasmyth K, Haering CH . (2005). The structure and function of SMC and kleisin complexes. Annu Rev Biochem 74: 595–648.

    Article  CAS  PubMed  Google Scholar 

  • Niida H, Nakanishi M . (2006). DNA damage checkpoints in mammals. Mutagenesis 21: 3–9.

    Article  CAS  PubMed  Google Scholar 

  • Ohtani K, Iwanaga R, Nakamura M, Ikeda M, Yabuta N, Tsuruga H et al. (1999). Cell growth-regulated expression of mammalian MCM5 and MCM6 genes mediated by the transcription factor E2F. Oncogene 18: 2299–2309.

    Article  CAS  PubMed  Google Scholar 

  • Pruitt SC, Bailey KJ, Freeland A . (2007). Reduced Mcm2 expression results in severe stem/progenitor cell deficiency and cancer. Stem Cells 12: 3121–3132.

    Article  Google Scholar 

  • Shima N, Alcaraz A, Liachko I, Buske TR, Andrews CA, Munroe RJ et al. (2007). A viable allele of Mcm4 causes chromosome instability and mammary adenocarcinomas in mice. Nat Genet 39: 93–98.

    Article  CAS  PubMed  Google Scholar 

  • Shimada K, Gasser SM . (2007). The origin recognition complex functions in sister-chromatid cohesion in Saccharomyces cerevisiae. Cell 128: 85–99.

    Article  CAS  PubMed  Google Scholar 

  • Shreeram S, Sparks A, Lane DP, Blow JJ . (2002). Cell type-specific responses of human cells to inhibition of replication licensing. Oncogene 21: 6624–6632.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Shukla A, Navadgi VM, Mallikarjuna K, Rao BJ . (2005). Interaction of hRad51 and hRad52 with MCM complex: a cross-talk between recombination and replication proteins. Biochem Biophys Res Commun 329: 1240–1245.

    Article  CAS  PubMed  Google Scholar 

  • Smith AE, Chronis C, Christodoulakis M, Orr SJ, Lea NC, Twine NA et al. (2009). Epigenetics of human T cells during the G0->G1 transition. Genome Res 19: 1325–1337.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Stoeber K, Tlsty TD, Happerfield L, Thomas GA, Romanov S, Bobrow L et al. (2001). DNA replication licensing and human cell proliferation. J Cell Sci 114: 2027–2041.

    CAS  PubMed  Google Scholar 

  • Suzuki S, Adachi A, Hiraiwa A, Ohashi M, Ishibashi M, Kiyono T . (1998). Cloning and characterization of human MCM7 promoter. Gene 216: 85–91.

    Article  CAS  PubMed  Google Scholar 

  • Takahashi TS, Yiu P, Chou MF, Gygi S, Walter JC . (2004). Recruitment of Xenopus Scc2 and cohesin to chromatin requires the pre-replication complex. Nat Cell Biol 6: 991–996.

    Article  CAS  PubMed  Google Scholar 

  • Tanaka S, Diffley JF . (2002). Deregulated G1-cyclin expression induces genomic instability by preventing efficient pre-RC formation. Genes Dev 16: 2639–2649.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Taubert S, Gorrini C, Frank SR, Parisi T, Fuchs M, Chan HM et al. (2004). E2F-dependent histone acetylation and recruitment of the Tip60 acetyltransferase complex to chromatin in late G1. Mol Cell Biol 24: 4546–4556.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tsao CC, Geisen C, Abraham RT . (2004). Interaction between human MCM7 and Rad17 proteins is required for replication checkpoint signaling. EMBO J 23: 4660–4669.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Williams GH, Stoeber K . (2007). Cell cycle markers in clinical oncology. Curr Opin Cell Biol 19: 672–679.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We sincerely thank Hye Kyung Hong, Kai Stoeber and Gareth Williams, UCL for the Mcm2 plasmid and advice on expressing recombinant Mcm2. We also thank our colleagues for helpful comments. This work was supported by grants from the Leukaemia Research Fund (LRF, now Leukaemia and Lymphoma Research (LLR)), Department of Trade and Industry (dti), the British Society for Haematology, the Welch and Packard Foundations and the National Institutes of Health (NIH).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to N S B Thomas.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies the paper on the Oncogene website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Orr, S., Gaymes, T., Ladon, D. et al. Reducing MCM levels in human primary T cells during the G0→G1 transition causes genomic instability during the first cell cycle. Oncogene 29, 3803–3814 (2010). https://doi.org/10.1038/onc.2010.138

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/onc.2010.138

Keywords

This article is cited by

Search

Quick links