Mammalian nuclei become licensed for DNA replication during late telophase

Daniela S Dimitrova, Tatyana A Prokhorova, J. Julian Blow, Ivan T Todorov, David M. Gilbert

    Research output: Contribution to journalArticle

    81 Citations (Scopus)

    Abstract

    Mcm 2-7 are essential replication proteins that bind to chromatin in mammalian nuclei during late telophase. Here, we have investigated the relationship between Mcm binding, licensing of chromatin for replication, and specification of the dihydrofolate reductase (DHFR) replication origin. Approximately 20% of total Mcm3 protein was bound to chromatin in Chinese hamster ovary (CHO) cells during telophase, while an additional 25% bound gradually and cumulatively throughout G1-phase. To investigate the functional significance of this binding, nuclei prepared from CHO cells synchronized at various times after metaphase were introduced into Xenopus egg extracts, which were either immunodepleted of Mcm proteins or supplemented with geminin, an inhibitor of the Mcm-loading protein Cdt1. Within 1 hour after metaphase, coincident with completion of nuclear envelope formation, CHO nuclei were fully competent to replicate in both of these licensing-defective extracts. However, sites of initiation of replication in each of these extracts were found to be dispersed throughout the DHFR locus within nuclei isolated between 1 to 5 hours after metaphase, but became focused to the DHFR origin within nuclei isolated after 5 hours post-metaphase. Importantly, introduction of permeabilized post-ODP, but not pre-ODP, CHO nuclei into licensing-deficient Xenopus egg extracts resulted in the preservation of a significant degree of DHFR origin specificity, implying that the previously documented lack of specific origin selection in permeabilized nuclei is at least partially due to the licensing of new initiation sites by proteins in the Xenopus egg extracts. We conclude that the functional association of Mcm proteins with chromatin (i.e. replication licensing) in CHO cells takes place during telophase, several hours prior to the specification of replication origins at the DHFR locus.
    Original languageEnglish
    Pages (from-to)51-59
    Number of pages9
    JournalJournal of Cell Science
    Volume115
    Issue number1
    Publication statusPublished - 2002

    Fingerprint

    Telophase
    Tetrahydrofolate Dehydrogenase
    Licensure
    Cricetulus
    DNA Replication
    Ovary
    Metaphase
    Chromatin
    Ovum
    Replication Origin
    Xenopus
    Proteins
    Geminin
    Xenopus Proteins
    Nuclear Envelope
    G1 Phase

    Cite this

    Dimitrova, D. S., Prokhorova, T. A., Blow, J. J., Todorov, I. T., & Gilbert, D. M. (2002). Mammalian nuclei become licensed for DNA replication during late telophase. Journal of Cell Science, 115(1), 51-59.
    Dimitrova, Daniela S ; Prokhorova, Tatyana A ; Blow, J. Julian ; Todorov, Ivan T ; Gilbert, David M. / Mammalian nuclei become licensed for DNA replication during late telophase. In: Journal of Cell Science. 2002 ; Vol. 115, No. 1. pp. 51-59.
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    abstract = "Mcm 2-7 are essential replication proteins that bind to chromatin in mammalian nuclei during late telophase. Here, we have investigated the relationship between Mcm binding, licensing of chromatin for replication, and specification of the dihydrofolate reductase (DHFR) replication origin. Approximately 20{\%} of total Mcm3 protein was bound to chromatin in Chinese hamster ovary (CHO) cells during telophase, while an additional 25{\%} bound gradually and cumulatively throughout G1-phase. To investigate the functional significance of this binding, nuclei prepared from CHO cells synchronized at various times after metaphase were introduced into Xenopus egg extracts, which were either immunodepleted of Mcm proteins or supplemented with geminin, an inhibitor of the Mcm-loading protein Cdt1. Within 1 hour after metaphase, coincident with completion of nuclear envelope formation, CHO nuclei were fully competent to replicate in both of these licensing-defective extracts. However, sites of initiation of replication in each of these extracts were found to be dispersed throughout the DHFR locus within nuclei isolated between 1 to 5 hours after metaphase, but became focused to the DHFR origin within nuclei isolated after 5 hours post-metaphase. Importantly, introduction of permeabilized post-ODP, but not pre-ODP, CHO nuclei into licensing-deficient Xenopus egg extracts resulted in the preservation of a significant degree of DHFR origin specificity, implying that the previously documented lack of specific origin selection in permeabilized nuclei is at least partially due to the licensing of new initiation sites by proteins in the Xenopus egg extracts. We conclude that the functional association of Mcm proteins with chromatin (i.e. replication licensing) in CHO cells takes place during telophase, several hours prior to the specification of replication origins at the DHFR locus.",
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    Dimitrova, DS, Prokhorova, TA, Blow, JJ, Todorov, IT & Gilbert, DM 2002, 'Mammalian nuclei become licensed for DNA replication during late telophase', Journal of Cell Science, vol. 115, no. 1, pp. 51-59.

    Mammalian nuclei become licensed for DNA replication during late telophase. / Dimitrova, Daniela S; Prokhorova, Tatyana A; Blow, J. Julian; Todorov, Ivan T; Gilbert, David M.

    In: Journal of Cell Science, Vol. 115, No. 1, 2002, p. 51-59.

    Research output: Contribution to journalArticle

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    T1 - Mammalian nuclei become licensed for DNA replication during late telophase

    AU - Dimitrova, Daniela S

    AU - Prokhorova, Tatyana A

    AU - Blow, J. Julian

    AU - Todorov, Ivan T

    AU - Gilbert, David M.

    PY - 2002

    Y1 - 2002

    N2 - Mcm 2-7 are essential replication proteins that bind to chromatin in mammalian nuclei during late telophase. Here, we have investigated the relationship between Mcm binding, licensing of chromatin for replication, and specification of the dihydrofolate reductase (DHFR) replication origin. Approximately 20% of total Mcm3 protein was bound to chromatin in Chinese hamster ovary (CHO) cells during telophase, while an additional 25% bound gradually and cumulatively throughout G1-phase. To investigate the functional significance of this binding, nuclei prepared from CHO cells synchronized at various times after metaphase were introduced into Xenopus egg extracts, which were either immunodepleted of Mcm proteins or supplemented with geminin, an inhibitor of the Mcm-loading protein Cdt1. Within 1 hour after metaphase, coincident with completion of nuclear envelope formation, CHO nuclei were fully competent to replicate in both of these licensing-defective extracts. However, sites of initiation of replication in each of these extracts were found to be dispersed throughout the DHFR locus within nuclei isolated between 1 to 5 hours after metaphase, but became focused to the DHFR origin within nuclei isolated after 5 hours post-metaphase. Importantly, introduction of permeabilized post-ODP, but not pre-ODP, CHO nuclei into licensing-deficient Xenopus egg extracts resulted in the preservation of a significant degree of DHFR origin specificity, implying that the previously documented lack of specific origin selection in permeabilized nuclei is at least partially due to the licensing of new initiation sites by proteins in the Xenopus egg extracts. We conclude that the functional association of Mcm proteins with chromatin (i.e. replication licensing) in CHO cells takes place during telophase, several hours prior to the specification of replication origins at the DHFR locus.

    AB - Mcm 2-7 are essential replication proteins that bind to chromatin in mammalian nuclei during late telophase. Here, we have investigated the relationship between Mcm binding, licensing of chromatin for replication, and specification of the dihydrofolate reductase (DHFR) replication origin. Approximately 20% of total Mcm3 protein was bound to chromatin in Chinese hamster ovary (CHO) cells during telophase, while an additional 25% bound gradually and cumulatively throughout G1-phase. To investigate the functional significance of this binding, nuclei prepared from CHO cells synchronized at various times after metaphase were introduced into Xenopus egg extracts, which were either immunodepleted of Mcm proteins or supplemented with geminin, an inhibitor of the Mcm-loading protein Cdt1. Within 1 hour after metaphase, coincident with completion of nuclear envelope formation, CHO nuclei were fully competent to replicate in both of these licensing-defective extracts. However, sites of initiation of replication in each of these extracts were found to be dispersed throughout the DHFR locus within nuclei isolated between 1 to 5 hours after metaphase, but became focused to the DHFR origin within nuclei isolated after 5 hours post-metaphase. Importantly, introduction of permeabilized post-ODP, but not pre-ODP, CHO nuclei into licensing-deficient Xenopus egg extracts resulted in the preservation of a significant degree of DHFR origin specificity, implying that the previously documented lack of specific origin selection in permeabilized nuclei is at least partially due to the licensing of new initiation sites by proteins in the Xenopus egg extracts. We conclude that the functional association of Mcm proteins with chromatin (i.e. replication licensing) in CHO cells takes place during telophase, several hours prior to the specification of replication origins at the DHFR locus.

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    VL - 115

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    JO - Journal of Cell Science

    JF - Journal of Cell Science

    SN - 0021-9533

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    ER -

    Dimitrova DS, Prokhorova TA, Blow JJ, Todorov IT, Gilbert DM. Mammalian nuclei become licensed for DNA replication during late telophase. Journal of Cell Science. 2002;115(1):51-59.