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Asymmetric bidirectional replication at the human DBF4 origin

Abstract

Faithful replication of the entire genome once per cell cycle is essential for maintaining genetic integrity, and the origin of DNA replication is key in this regulation. Unlike that in unicellular organisms, the replication initiation mechanism in mammalian cells is not well understood. We have identified a strong origin of replication at the DBF4 promoter locus, which contains two initiation zones, two origin recognition complex (ORC) binding sites and two DNase I–hypersensitive regions within 1.5 kb. Notably, similar to the Escherichia coli oriC, replication at the DBF4 locus starts from initiation zone I, which contains an ORC-binding site, and progresses in the direction of transcription toward initiation zone II, located 0.4 kb downstream. Replication on the opposite strand from zone II, which contains another ORC-binding site, may be activated or facilitated by replication from zone I. We term this new mammalian replication mode 'asymmetric bidirectional replication'.

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Figure 1: The DBF4 promoter locus contains a strong ori.
Figure 2: Replication initiation at the DBF4 locus is largely confined to around 7 h after mitosis or 1 h after G1/S.
Figure 3: Determining RIPs at the DBF4 locus in asynchronous HeLa cells.
Figure 4: DBF4 ori contains two initiation zones within 1.1 kb DNA segment.
Figure 5: Replication initiation occurs from replication zone I before zone II.
Figure 6: Mapping of the ORC-binding and DNase I–hypersensitive sites.
Figure 7: An ABR initiation model.

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References

  1. Aladjem, M.I. & Fanning, E. The replicon revisited: an old model learns new tricks in metazoan chromosomes. EMBO Rep. 5, 686–691 (2004).

    Article  CAS  Google Scholar 

  2. Bell, S.P. & Dutta, A. DNA replication in eukaryotic cells. Annu. Rev. Biochem. 71, 333–374 (2002).

    Article  CAS  Google Scholar 

  3. Mechali, M. DNA replication origins: from sequence specificity to epigenetics. Nat. Rev. Genet. 2, 640–645 (2001).

    Article  CAS  Google Scholar 

  4. Burhans, W.C. et al. Identification of an origin of bidirectional DNA replication in mammalian chromosomes. Cell 62, 955–965 (1990).

    Article  CAS  Google Scholar 

  5. Abdurashidova, G. et al. Start sites of bidirectional DNA synthesis at the human lamin B2 origin. Science 287, 2023–2026 (2000).

    Article  CAS  Google Scholar 

  6. Vaughn, J.P., Dijkwel, P.A. & Hamlin, J.L. Replication initiates in a broad zone in the amplified CHO dihydrofolate reductase domain. Cell 61, 1075–1087 (1990).

    Article  CAS  Google Scholar 

  7. Dijkwel, P.A., Wang, S. & Hamlin, J.L. Initiation sites are distributed at frequent intervals in the Chinese hamster dihydrofolate reductase origin of replication but are used with very different efficiencies. Mol. Cell. Biol. 22, 3053–3065 (2002).

    Article  CAS  Google Scholar 

  8. Gencheva, M., Anachkova, B. & Russev, G. Mapping the sites of initiation of DNA replication in rat and human rRNA genes. J. Biol. Chem. 271, 2608–2614 (1996).

    Article  CAS  Google Scholar 

  9. Little, R.D., Platt, T.H. & Schildkraut, C.L. Initiation and termination of DNA replication in human rRNA genes. Mol. Cell. Biol. 13, 6600–6613 (1993).

    Article  CAS  Google Scholar 

  10. Dijkwel, P.A. & Hamlin, J.L. Sequence and context effects on origin function in mammalian cells. J. Cell. Biochem. 62, 210–222 (1996).

    Article  CAS  Google Scholar 

  11. Burhans, W.C. & Huberman, J.A. DNA replication origins in animal cells: a question of context? Science 263, 639–640 (1994).

    Article  CAS  Google Scholar 

  12. DePamphilis, M.L. Replication origins in metazoan chromosomes: fact or fiction? Bioessays 21, 5–16 (1999).

    Article  CAS  Google Scholar 

  13. Aladjem, M.I., Falaschi, A. & Kowalski, D. Eukaryotic DNA replication origins. in DNA Replication and Human Disease (ed. DePamphilis, M.L.) 31–61 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 2006).

    Google Scholar 

  14. Stefanovic, D. et al. In vitro protein-DNA interactions at the human lamin B2 replication origin. J. Biol. Chem. 278, 42737–42743 (2003).

    Article  CAS  Google Scholar 

  15. Delgado, S., Gomez, M., Bird, A. & Antequera, F. Initiation of DNA replication at CpG islands in mammalian chromosomes. EMBO J. 17, 2426–2435 (1998).

    Article  CAS  Google Scholar 

  16. Ladenburger, E.M., Keller, C. & Knippers, R. Identification of a binding region for human origin recognition complex proteins 1 and 2 that coincides with an origin of DNA replication. Mol. Cell. Biol. 22, 1036–1048 (2002).

    Article  CAS  Google Scholar 

  17. Beall, E.L. et al. Role for a Drosophila Myb-containing protein complex in site-specific DNA replication. Nature 420, 833–837 (2002).

    Article  CAS  Google Scholar 

  18. Bosco, G., Du, W. & Orr-Weaver, T.L. DNA replication control through interaction of E2F-RB and the origin recognition complex. Nat. Cell Biol. 3, 289–295 (2001).

    Article  CAS  Google Scholar 

  19. Abdurashidova, G. et al. Localization of proteins bound to a replication origin of human DNA along the cell cycle. EMBO J. 22, 4294–4303 (2003).

    Article  CAS  Google Scholar 

  20. Bousset, K. & Diffley, J.F. The Cdc7 protein kinase is required for origin firing during S phase. Genes Dev. 12, 480–490 (1998).

    Article  CAS  Google Scholar 

  21. Jiang, W., McDonald, D., Hope, T.J. & Hunter, T. Mammalian Cdc7-Dbf4 protein kinase complex is essential for initiation of DNA replication. EMBO J. 18, 5703–5713 (1999).

    Article  CAS  Google Scholar 

  22. Donaldson, A.D., Fangman, W.L. & Brewer, B.J. Cdc7 is required throughout the yeast S phase to activate replication origins. Genes Dev. 12, 491–501 (1998).

    Article  CAS  Google Scholar 

  23. Kumagai, H. et al. A novel growth- and cell cycle-regulated protein, ASK, activates human Cdc7-related kinase and is essential for G1/S transition in mammalian cells. Mol. Cell. Biol. 19, 5083–5095 (1999).

    Article  CAS  Google Scholar 

  24. Wu, X. & Lee, H. Human Dbf4/ASK promoter is activated through the Sp1 and MluI cell-cycle box (MCB) transcription elements. Oncogene 21, 7786–7796 (2002).

    Article  CAS  Google Scholar 

  25. Yamada, M. et al. A 63-base pair DNA segment containing an Sp1 site but not a canonical E2F site can confer growth-dependent and E2F-mediated transcriptional stimulation of the human ASK gene encoding the regulatory subunit for human Cdc7-related kinase. J. Biol. Chem. 277, 27668–27681 (2002).

    Article  CAS  Google Scholar 

  26. Guo, B. & Lee, H. Cloning and characterization of Chinese hamster homologue of yeast DBF4 (ChDBF4). Gene 264, 249–256 (2001).

    Article  CAS  Google Scholar 

  27. Giacca, M., Pelizon, C. & Falaschi, A. Mapping replication origins by quantifying relative abundance of nascent DNA strands using competitive polymerase chain reaction. Methods 13, 301–312 (1997).

    Article  CAS  Google Scholar 

  28. Kamath, S. & Leffak, M. Multiple sites of replication initiation in the human beta-globin gene locus. Nucleic Acids Res. 29, 809–817 (2001).

    Article  CAS  Google Scholar 

  29. Bielinsky, A.K. et al. Origin recognition complex binding to a metazoan replication origin. Curr. Biol. 11, 1427–1431 (2001).

    Article  CAS  Google Scholar 

  30. Gerbi, S.A. & Bielinsky, A.K. Replication initiation point mapping. Methods 13, 271–280 (1997).

    Article  CAS  Google Scholar 

  31. Burhans, W.C. et al. Emetine allows identification of origins of mammalian DNA replication by imbalanced DNA synthesis, not through conservative nucleosome segregation. EMBO J. 10, 4351–4360 (1991).

    Article  CAS  Google Scholar 

  32. Decker, R.S., Yamaguchi, M., Possenti, R. & DePamphilis, M.L. Initiation of simian virus 40 DNA replication in vitro: aphidicolin causes accumulation of early-replicating intermediates and allows determination of the initial direction of DNA synthesis. Mol. Cell. Biol. 6, 3815–3825 (1986).

    Article  CAS  Google Scholar 

  33. Bell, S.P. The origin recognition complex: from simple origins to complex functions. Genes Dev. 16, 659–672 (2002).

    Article  CAS  Google Scholar 

  34. Nguyen-Huynh, A.T. & Schaffer, P.A. Cellular transcription factors enhance herpes simplex virus type 1 oriS-dependent DNA replication. J. Virol. 72, 3635–3645 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Li, R. Stimulation of DNA replication in Saccharomyces cerevisiae by a glutamine- and proline-rich transcriptional activation domain. J. Biol. Chem. 274, 30310–30314 (1999).

    Article  CAS  Google Scholar 

  36. Yan, P. et al. HBV C promoter Sp1 binding sequence functionally substitutes for the yeast ARS1 ABF1 binding site. DNA Cell Biol. 21, 737–742 (2002).

    Article  CAS  Google Scholar 

  37. Kohara, Y., Tohdoh, N., Jiang, X.W. & Okazaki, T. The distribution and properties of RNA primed initiation sites of DNA synthesis at the replication origin of Escherichia coli chromosome. Nucleic Acids Res. 13, 6847–6866 (1985).

    Article  CAS  Google Scholar 

  38. Seufert, W. & Messer, W. Start sites for bidirectional in vitro DNA replication inside the replication origin, oriC, of Escherichia coli . EMBO J. 6, 2469–2472 (1987).

    Article  CAS  Google Scholar 

  39. Brown, T.A. et al. Replication of mitochondrial DNA occurs by strand displacement with alternative light-strand origins, not via a strand-coupled mechanism. Genes Dev. 19, 2466–2476 (2005).

    Article  CAS  Google Scholar 

  40. Yan, J. et al. The forkhead transcription factor FoxI1 remains bound to condensed mitotic chromosomes and stably remodels chromatin structure. Mol. Cell. Biol. 26, 155–168 (2006).

    Article  CAS  Google Scholar 

  41. Kumar, S. & Leffak, M. Conserved chromatin structure in c-myc 5′flanking DNA after viral transduction. J. Mol. Biol. 222, 45–57 (1991).

    Article  CAS  Google Scholar 

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Acknowledgements

We are grateful to S.-Y. Kim for her initial development of the ChIP assay protocol. This work was supported by grants from the Canadian Institutes of Health Research (MOP79473) to H.L. J.R. was supported in part by a graduate scholarship of the University of Ottawa.

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J.R. planned, developed protocols, carried out experiments, analyzed data and drafted the manuscript; H.L. conceived and guided the overall research project, analyzed and interpreted data, and wrote the final version of the manuscript.

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Correspondence to Hoyun Lee.

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Supplementary Figures 1–2, Supplementary Tables 1–3 and Supplementary Methods (PDF 584 kb)

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Romero, J., Lee, H. Asymmetric bidirectional replication at the human DBF4 origin. Nat Struct Mol Biol 15, 722–729 (2008). https://doi.org/10.1038/nsmb.1439

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