DNA replication initiates at multiple sites on plasmid DNA in Xenopus egg extracts

H. M. Mahbubani, T. Paull, J. K. Elder, J. J. Blow

    Research output: Contribution to journalArticle

    98 Citations (Scopus)

    Abstract

    Cell-free extracts of Xenopus eggs will replicate plasmid DNA molecules under normal cell cycle control. We have used the neutral/neutral 2-D gel technique to map the sites at which DNA replication initiates in this system. Three different plasmids were studied: one containing the Xenopus rDNA repeat, one containing single copy Xenopus genomic DNA, and another containing the yeast 2 microns replication origin. 2-D gel profiles show that many potential sites of initiation are present on each plasmid, and are randomly situated at the level of resolution of this technique (500-1000 bp). Despite the abundance of sites capable of supporting the initiation of replication, pulse-chase experiments suggest that only a single randomly situated initiation event occurs on each DNA molecule. Once initiation has taken place, conventional replication forks appear to move away from this site at a rate of about 10nt/second, similar to the rate observed in vivo.
    Original languageEnglish
    Pages (from-to)1457-1462
    Number of pages6
    JournalNucleic Acids Research
    Volume20
    Issue number7
    DOIs
    Publication statusPublished - 11 Apr 1992

    Fingerprint

    Xenopus
    DNA Replication
    Ovum
    Plasmids
    DNA
    Gels
    Replication Origin
    Ribosomal DNA
    Cell Cycle Checkpoints
    Cell Extracts
    Eggs
    Yeasts

    Cite this

    Mahbubani, H. M. ; Paull, T. ; Elder, J. K. ; Blow, J. J. / DNA replication initiates at multiple sites on plasmid DNA in Xenopus egg extracts. In: Nucleic Acids Research. 1992 ; Vol. 20, No. 7. pp. 1457-1462.
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    abstract = "Cell-free extracts of Xenopus eggs will replicate plasmid DNA molecules under normal cell cycle control. We have used the neutral/neutral 2-D gel technique to map the sites at which DNA replication initiates in this system. Three different plasmids were studied: one containing the Xenopus rDNA repeat, one containing single copy Xenopus genomic DNA, and another containing the yeast 2 microns replication origin. 2-D gel profiles show that many potential sites of initiation are present on each plasmid, and are randomly situated at the level of resolution of this technique (500-1000 bp). Despite the abundance of sites capable of supporting the initiation of replication, pulse-chase experiments suggest that only a single randomly situated initiation event occurs on each DNA molecule. Once initiation has taken place, conventional replication forks appear to move away from this site at a rate of about 10nt/second, similar to the rate observed in vivo.",
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    DNA replication initiates at multiple sites on plasmid DNA in Xenopus egg extracts. / Mahbubani, H. M.; Paull, T.; Elder, J. K.; Blow, J. J.

    In: Nucleic Acids Research, Vol. 20, No. 7, 11.04.1992, p. 1457-1462.

    Research output: Contribution to journalArticle

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    T1 - DNA replication initiates at multiple sites on plasmid DNA in Xenopus egg extracts

    AU - Mahbubani, H. M.

    AU - Paull, T.

    AU - Elder, J. K.

    AU - Blow, J. J.

    PY - 1992/4/11

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    AB - Cell-free extracts of Xenopus eggs will replicate plasmid DNA molecules under normal cell cycle control. We have used the neutral/neutral 2-D gel technique to map the sites at which DNA replication initiates in this system. Three different plasmids were studied: one containing the Xenopus rDNA repeat, one containing single copy Xenopus genomic DNA, and another containing the yeast 2 microns replication origin. 2-D gel profiles show that many potential sites of initiation are present on each plasmid, and are randomly situated at the level of resolution of this technique (500-1000 bp). Despite the abundance of sites capable of supporting the initiation of replication, pulse-chase experiments suggest that only a single randomly situated initiation event occurs on each DNA molecule. Once initiation has taken place, conventional replication forks appear to move away from this site at a rate of about 10nt/second, similar to the rate observed in vivo.

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