Abstract
The identification and isolation of origins of replication from mammalian genomes has been a demanding task owing to the great complexity of these genomes. However, two methods have been refined in recent years each of which allows significant enrichment of recently activated origins of replication from asynchronous cell cultures. In one of these, nascent strands are melted from the long template DNA, and the small, origin-centered strands are isolated on sucrose gradients. The second method involves the selective entrapment of bubble-containing fragments in gelling agarose and their subsequent recovery and isolation by molecular cloning. Libraries prepared by this method from Chinese hamster and human cells have been shown to be extremely pure, and provide a renewable resource of origins that can be used as probes on microarrays or sequenced by high-throughput techniques to localize them within the genomic source. The bubble-trapping method is described here for asynchronous mammalian cells that grow with reasonable doubling times and from which nuclear matrices can be reliably prepared. The method for nuclear matrix preparation and enrichment of replication intermediates is described in an accompanying chapter entitled “Purification of restriction fragments containing replication intermediates from mammalian cells for 2-D gel analysis” (Chapter 16).
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References
Kornberg A, Baker TA (1992) DNA Replication, 1st edn. W.H. Freeman, New York, NY
Chan CS, Tye BK (1980) Autonomously replicating sequences in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 77:6329–6333
Stinchcomb DT, Thomas M, Kelly J, Selker E, Davis RW (1980) Eukaryotic DNA segments capable of autonomous replication in yeast. Proc Natl Acad Sci U S A 77:4559–4563
Machida YJ, Hamlin JL, Dutta A (2005) Right place, right time, and only once: replication initiation in metazoans. Cell 123:13–24
Krysan PJ, Haase SB, Calos MP (1989) Isolation of human sequences that replicate autonomously in human cells. Mol Cell Biol 9:1026–1033
Haase SB, Calos MP (1991) Replication control of autonomously replicating human sequences. Nucleic Acids Res 19:5053–5058
Zannis-Hadjopoulos M, Persico M, Martin RG (1981) The remarkable instability of replication loops provides a general method for the isolation of origins of DNA replication. Cell 27:155–163
Heintz NH, Hamlin JL (1982) An amplified chromosomal sequence that includes the gene for dihydrofolate reductase initiates replication within specific restriction fragments. Proc Natl Acad Sci U S A 79:4083–4087
Kelly RE, DeRose ML, Draper BW, Wahl GM (1995) Identification of an origin of bidirectional DNA replication in the ubiquitously expressed mammalian CAD gene. Mol Cell Biol 15:4136–4148
Tribioli C, Biamonti G, Giacca M, Colonna M, Riva S, Falaschi A (1987) Characterization of human DNA sequences synthesized at the onset of S-phase. Nucleic Acids Res 15:10211–10232
James CD, Leffak M (1986) Polarity of DNA replication through the avian alpha-globin locus. Mol Cell Biol 6:976–984
Nawotka KA, Huberman JA (1988) Two-dimensional gel electrophoretic method for mapping DNA replicons. Mol Cell Biol 8:1408–1413
Muller M, Lucchini R, Sogo JM (2000) Replication of yeast rDNA initiates downstream of transcriptionally active genes. Mol Cell 5:767–777
Handeli S, Klar A, Meuth M, Cedar H (1989) Mapping replication units in animal cells. Cell 57:909–920
Burhans WC, Vassilev LT, Caddle MS, Heintz NH, DePamphilis ML (1990) Identification of an origin of bidirectional DNA replication in mammalian chromosomes. Cell 62:955–965
Vassilev LT, Johnson EM (1989) Mapping initiation sites of DNA replication in vivo using polymerase chain reaction amplification of nascent strand segments. Nucleic Acids Res 17:7693–7705
Pelizon C, Diviacco S, Falaschi A, Giacca M (1996) High-resolution mapping of the origin of DNA replication in the hamster dihydrofolate reductase gene domain by competitive PCR. Mol Cell Biol 16:5358–5364
Brewer BJ, Fangman WL (1987) The localization of replication origins on ARS plasmids in S. cerevisiae. Cell 51:463–471
Aladjem MI, Falaschi A, Kowalski D (2006) Eukaryotic DNA replication origins. In: DePamphilis M (ed) DNA replication and human disease. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 31–62
Mesner LD, Crawford EL, Hamlin JL (2006) Isolating apparently pure libraries of replication origins from complex genomes. Mol Cell 21:719–726
Todorovic V, Giadrossi S, Pelizon C, Mendoza-Maldonado R, Masai H, Giacca M (2005) Human origins of DNA replication selected from a library of nascent DNA. Mol Cell 19:567–575
Dijkwel PA, Vaughn JP, Hamlin JL (1991) Mapping of replication initiation sites in mammalian genomes by two-dimensional gel analysis: stabilization and enrichment of replication intermediates by isolation on the nuclear matrix. Mol Cell Biol 11:3850–3859
Little RD, Platt TH, Schildkraut CL (1993) Initiation and termination of DNA replication in human rRNA genes. Mol Cell Biol 13:6600–6613
Hanahan D, Jessee J, Bloom FR (1991) Plasmid transformation of Escherichia coli and other bacteria. Methods Enzymol 204:63–113
Labarca C, Paigen K (1980) A simple, rapid, and sensitive DNA assay procedure. Anal Biochem 102:344–352
Huberman JA, Riggs AD (1968) On the mechanism of DNA replication in mammalian chromosomes. J Mol Biol 32:327–341
Feinberg AP, Vogelstein B (1983) High specific activity labeling of DNA restriction fragments. Anal Biochem 132:6–13
Abdurashidova G, Deganuto M, Klima R, Riva S, Biamonti G, Giacca M, Falaschi A (2000) Start sites of bidirectional DNA synthesis at the human lamin B2 origin. Science 287:2023–2026
Dean WW, Dancis BM, Thomas CA Jr (1973) The trapping of circular DNA in agarose gels. Anal Biochem 56:417–427
Vaughn JP, Dijkwel PA, Hamlin JL (1990) Replication initiates in a broad zone in the amplified CHO dihydrofolate reductase domain. Cell 61:1075–1087
Acknowledgments
We thank Raul Torres for valuable contributions during the development of the procedure, and the other members of our laboratory for helpful discussions. This work was supported by a grant from the NIH to J.L.H. (RO1 HG002937).
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Mesner, L.D., Hamlin, J.L. (2015). Isolation of Restriction Fragments Containing Origins of Replication from Complex Genomes. In: Vengrova, S., Dalgaard, J. (eds) DNA Replication. Methods in Molecular Biology, vol 1300. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2596-4_17
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DOI: https://doi.org/10.1007/978-1-4939-2596-4_17
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