Animal cells and systems

The Korean Society for Integrative Biology (한국통합생물학회)
권호 표지

Depletion of the Pre-RC Proteins Induces Chk1/Chk2 Independent Checkpoint Responses and Apoptotic Cell Death in HeLa Cells

  • Im, Jun-Sub (Department of Biology Education, Seoul National University) ;
  • Lee, Joon-Kyu (Department of Biology Education, Seoul National University)
  • Published : 2007.12.31
Download PDF
⟨ Previous Next ⟩

Abstract

The initiation of eukaryotic DNA replication requires assembly of the pre-replicative complex (Pre-RC) through the concerted action of Orc, Cdc6, Cdt1 and Mcm2-7 complex during G1 phase. The pre-RC assembly licenses individual replication origins for the initiation of DNA replication and sufficient number of the pre-RC is essential for proper progression of S phase. However, it is not well known how cells recognize the completion of the pre-RC assembly before G1-S transition. In order to understand the cellular responses to the defects in pre-RC assembly, we depleted the known components of pre-RC proteins using the small interference RNAs in HeLa cells. Although the defects of pre-RC assembly by the depletion of the pre-RC proteins such as Orc2, Cdt1, Mcm2 & Mcm10 did not elicit the activation of Chk1- or Chk2-dependent checkpoint pathways, these cells still showed significant decrease in the cellular level of Cdc25A proteins. These results suggests that a novel checkpoint pathway exist in HeLa cells, which is not dependent upon Chk1 or Chk2 proteins and play essential roles in the cellular responses to the defects in the pre-RC assembly. Also, among those four proteins tested in this study, the depletion of Mcm10 and Cdt1 proteins significantly increased the apoptotic cell death in HeLa cells, suggesting that these proteins not only play roles in the pre-RC assembly, but also are involved in the checkpoint responses to the defects in the pre-RC assembly.

Keywords

References

  1. Abraham RT (2001) Cell cycle checkpoint signaling through the ATM and ATR kinases. Genes Dev 15: 2177-96 https://doi.org/10.1101/gad.914401
  2. Bailis JM and Forsburg SL (2004) MCM proteins: DNA damage, mutagenesis and repair. Curr Opin Genet Dev 14: 17-21 https://doi.org/10.1016/j.gde.2003.11.002
  3. Bartek J, Lukas C and Lukas J (2004) Checking on DNA damage in S phase. Nat Rev Mol Cell Biol 5: 792-804 https://doi.org/10.1038/nrm1493
  4. Bell SP and Dutta A (2002) DNA replication in eukaryotic cells. Annu Rev Biochem 71: 333-374 https://doi.org/10.1146/annurev.biochem.71.110601.135425
  5. Cho WH, Lee YJ, Kong SI, Hurwitz J and Lee JK (2006) CDC7 kinase phosphory1ates serine residues adjacent to acidic amino acids in the mini chromosome maintenance 2 protein. Proc Natl Acad Sci USA 103: 11521-11526
  6. Cortez D, Glick G and Elledge SJ (2004) Minichromosome maintenance proteins are direct targets of the ATM and ATR checkpoint kinases. Proc Natl Acad Sci USA 101: 10078-10083
  7. Falck J, Mailand N, Syljuasen RG, Bartek J and Lukas J (2001) The ATM-Chk2-Cdc25A checkpoint pathway guards against radioresistant DNA synthesis. Nature 410: 842-847 https://doi.org/10.1038/35071124
  8. Forsburg SL (2004) Eukaryotic MCM proteins: beyond replication initiation. Microbiol Mol Biol Rev 68: 109-131 https://doi.org/10.1128/MMBR.68.1.109-131.2004
  9. Fujita M (2006) Cdtl revisited: complex and tight regulation during the cell cycle and consequences of deregulation in mammalian cells. Cell Div 1: 22 https://doi.org/10.1186/1747-1028-1-1
  10. Gambus A, Jones RC, Sanchez-Diaz A, Kanemaki M, van Deursen F, Edmondson RD and Labib K (2006) GINS maintains association of Cdc45 with MCM in replisome progression complexes at eukaryotic DNA replication forks. Nat Cell Biol 8: 358-366 https://doi.org/10.1038/ncb1382
  11. Gibson DG, Bell SP and Aparicio OM (2006) Cell cycle execution point analysis of ORC function and characterization of the checkpoint response to ORC inactivation in Saccharomyces cerevisiae. Genes Cells 11: 557-573 https://doi.org/10.1111/j.1365-2443.2006.00967.x
  12. Gillespie PJ, Li A and Blow JJ (2001) Reconstitution oflicensed replication origins on Xenopus sperm nuclei using purified proteins. BMC Biochem 2: 15 https://doi.org/10.1186/1471-2091-2-15
  13. Heffernan TP, Simpson DA, Frank AR, Heinloth AN, Paules RS, Cordeiro-Stone M and Kaufmann WK (2002) An ATR- and Chk1-dependent S checkpoint inhibits replicon initiation following UVC-induced DNA damage. Mol Cell Biol 22: 8552-8561 https://doi.org/10.1128/MCB.22.24.8552-8561.2002
  14. Higa LA, Mihaylov IS, Banks DP, Zheng J and Zhang H (2003) Radiation-mediated proteolysis of CDn by CUL4-ROC1 and CSN complexes constitutes a new checkpoint. Nat Cell Biol 5: 1008-1015 https://doi.org/10.1038/ncb1061
  15. Hu J, McCall CM, Ohta T and Xiong Y (2004) Targeted ubiquitination of CDT1 by the DDBI-CUL4A-ROC1 ligase in response to DNA damage. Nat Cell Biol 6: 1003-1009 https://doi.org/10.1038/ncb1172
  16. Kaufmann WK and Paules RS (1996) DNA damage and cell cycle checkpoints. Faseb J 10: 238-247 https://doi.org/10.1096/fasebj.10.2.8641557
  17. Kumar S (2007) Caspase function in programmed cell death. Cell Death Differ 14: 32-43 https://doi.org/10.1038/sj.cdd.4402060
  18. Lau E, Zhu C, Abraham RT and Jiang W (2006) The functional role of Cdc6 in S-G2/M in mammalian cells. EMBO Rep 7: 425-430
  19. Li A and Blow JJ (2005) Cdtl downregulation by proteolysis and geminin inhibition prevents DNA re-replication in Xenopus. EMBO J 24: 395-404 https://doi.org/10.1038/sj.emboj.7600520
  20. Maiorano D, Krasinska L, Lutzmann M and Mechali M (2005) Recombinant Cdt1 induces rereplication of G2 nuclei in Xenopus egg extracts. Curr Biol 15: 146-153 https://doi.org/10.1016/j.cub.2004.12.040
  21. Melixetian M, Ballabeni A, Masiero L, Gasparini P, Zamponi R, Bartek J, Lukas J and Helin K (2004) Loss of Geminin induces rereplication in the presence of functional p53. J Cell Biol 165: 473-482 https://doi.org/10.1083/jcb.200403106
  22. Montagnoli A, Tenca P, Sola F, Carpani D, Brotherton D, Albanese C and Santocanale C (2004) Cdc7 inhibition reveals a p53-dependent replication checkpoint that is defective in cancer cells. Cancer Res 64: 7110-7116 https://doi.org/10.1158/0008-5472.CAN-04-1547
  23. Okada H and Mak TW (2004) Pathways of apoptotic and nonapoptotic death in tumour cells. Nat Rev Cancer 4: 592-603 https://doi.org/10.1038/nrc1412
  24. Park JH, Bang SW, Kim SH and Hwang DS (2007) Knockdown of human MCM10 activates G2 checkpoint pathway. Biochem Biophys Res Commun Nov 13 [Epub ahead of print]
  25. Pflumm MF and Botchan MR (2001) Orc mutants arrest in metaphase with abnormally condensed chromosomes. Development 128: 1697-1707
  26. Prasanth SG, Prasanth KY, Siddiqui K, Spector DL and Stillman B (2004) Human Orc2 localizes to centrosomes, centromeres and heterochromatin during chromosome inheritance. Embo J 23: 2651-2563 https://doi.org/10.1038/sj.emboj.7600255
  27. Randell JC, Bowers JL, Rodriguez HK and Bell SP (2006) Sequential ATP hydrolysis by Cdc6 and ORC directs loading of the Mcm2-7 helicase. Mol Cell 21: 29-39 https://doi.org/10.1016/j.molcel.2005.11.023
  28. Ricke RM and Bielinsky AK (2004) Mcm10 regulates the stability and chromatin association of DNA polymerase-alpha. Mol Cell 16: 173-185 https://doi.org/10.1016/j.molcel.2004.09.017
  29. Sawyer SL, Cheng IH, Chai Wand Tye BK (2004) Mcm10 and Cdc45 cooperate in origin activation in Saccharomyces cerevisiae. J Mol Biol 340: 195-202 https://doi.org/10.1016/j.jmb.2004.04.066
  30. Stillman B, Bell SP, Dutta A and Marahrens Y (1992) DNA replication and the cell cycle. Ciba Found Symp 170: 147-160
  31. Tada S (2007) Cdtl and geminin: role during cell cycle progression and DNA damage in higher eukaryotes. Front Biosci 12: 1629-1641 https://doi.org/10.2741/2175
  32. Teer JK, Machida YJ, Labit H, Novac O, Hyrien O, Marheineke K, Zannis-Hadjopoulos M and Dutta A (2006) Proliferating human cells hypomorphic for origin recognition complex 2 and pre-replicative complex formation have a defect in p53 activation and Cdk2 kinase activation. J Biol Chem 281: 6253-6260 https://doi.org/10.1074/jbc.M507150200
  33. Tercero JA, Longhese MP and Diffley JF (2003) A central role for DNA replication forks in checkpoint activation and response. Mol Cell 11: 1323-1336 https://doi.org/10.1016/S1097-2765(03)00169-2
  34. WohlscWegel JA, Dwyer BT, Dhar SK, Cvetic C, Walter JC and Dutta A (2000) Inhibition of eukaryotic DNA replication by geminin binding to Cdt1. Science 290: 2309-2312 https://doi.org/10.1126/science.290.5500.2309
  35. Xiao Z, Chen Z, Gunasekera AH, Sowin TJ, Rosenberg SH, Fesik S and Zhang H (2003) Chkl mediates Sand G2 arrests through Cdc25A degradation in response to DNA-damaging agents. J Biol Chem 278: 21767-21773 https://doi.org/10.1074/jbc.M300229200