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One-way PCR-based mapping of a replication initiation point (RIP)

Abstract

The lamin B2 locus is the only mammalian origin whose replication initiation points (RIPs) have been mapped. Although this paper was published 8 years ago, no further mammalian RIP-mapping studies have been reported, largely due to technical difficulties of ligation-mediated (LM)-PCR used by the authors. Here, we report the development of a simple, one-way PCR-based protocol that allows one to accurately determine RIPs at mammalian origins. The procedure can be completed within 48 h from the time of cell lysis in the agarose gel. Nascent DNA is then isolated from the same gel after DNA is separated by alkaline gel electrophoresis. Subsequently, RIPs are determined by one-way PCR-based primer extension using labeled primers. Using this protocol, we have successfully mapped RIPs in the human DBF4 locus. As one-way PCR is routinely used by many scientists, this protocol will provide a powerful new tool for studying DNA replication in many organisms including mammalian cells.

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Figure 1: RIP mapping of the human DBF4 ori by one-way PCR-based primer extension.

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References

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  3. Bielinsky, A.K. & Gerbi, S.A. Chromosomal ARS1 has a single leading strand start site. Mol. Cell 3, 477–486 (1999).

    Article  CAS  PubMed  Google Scholar 

  4. Bielinsky, A.K. & Gerbi, S.A. Discrete start sites for DNA synthesis in the yeast ARS1 origin. Science 279, 95–98 (1998).

    Article  CAS  PubMed  Google Scholar 

  5. Gomez, M. & Antequera, F. Organization of DNA replication origins in the fission yeast genome. EMBO J. 18, 5683–5690 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  7. Matsunaga, F., Norais, C., Forterre, P. & Myllykallio, H. Identification of short 'eukaryotic' Okazaki fragments synthesized from a prokaryotic replication origin. EMBO Rep. 4, 154–158 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Robinson, N.P. et al. Identification of two origins of replication in the single chromosome of the archaeon Sulfolobus solfataricus . Cell 116, 25–38 (2004).

    Article  CAS  PubMed  Google Scholar 

  9. Sun, C., Zhou, M., Li, Y. & Xiang, H. Molecular characterization of the minimal replicon and the unidirectional theta replication of pSCM201 in extremely halophilic archaea. J. Bacteriol. 188, 8136–8144 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Romero, J. & Lee, H. Asymmetric bidirectional replication at the human DBF4 origin. Nat. Struct. Mol. Biol. 15, 722–729 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. 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  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Gerhardt, J. et al. Identification of new human origins of DNA replication by an origin-trapping assay. Mol. Cell Biol. 26, 7731–7746 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Liu, G., Bissler, J.J., Sinden, R.R. & Leffak, M. Unstable spinocerebellar ataxia type 10 (ATTCT*(AGAAT)) repeats are associated with aberrant replication at the ATX10 locus and replication origin-dependent expansion at an ectopic site in human cells. Mol. Cell Biol. 27, 7828–7838 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Henke, W. et al. Betaine improves the PCR amplification of GC-rich DNA sequences. Nucleic Acids Res. 25, 3957–3958 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Correspondence to Hoyun Lee.

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Romero, J., Lee, H. One-way PCR-based mapping of a replication initiation point (RIP). Nat Protoc 3, 1729–1735 (2008). https://doi.org/10.1038/nprot.2008.173

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