Skip to main content
Article thumbnail
Location of Repository

Replication initiation complex formation in the absence of nuclear function in Xenopus

By Liliana Krasinska and Daniel Fisher


In this article, we study how intercalation-induced changes in chromatin and DNA topology affect chromosomal DNA replication using Xenopus egg extracts. Unexpectedly, intercalation by ethidium or doxorubicin prevents formation of a functional nucleus: although nucleosome formation occurs, DNA decondensation is arrested, membranous vesicles accumulate around DNA but do not fuse to form a nuclear membrane, active transport is abolished and lamins are found on chromatin, but do not assemble into a lamina. DNA replication is inhibited at the stage of initiation complex activation, as shown by molecular combing of DNA and by the absence of checkpoint activation. Replication of single-stranded DNA is not prevented. Surprisingly, in spite of the absence of nuclear function, DNA-replication proteins of pre-replication and initiation complexes are loaded onto chromatin. This is a general phenomenon as initiation complexes could also be seen without ethidium in membrane-depleted extracts which do not form nuclei. These results suggest that DNA or chromatin topology is required for generation of a functional nucleus, and activation, but not formation, of initiation complexes

Topics: Genome Integrity, Repair and Replication
Publisher: Oxford University Press
OAI identifier:
Provided by: PubMed Central
Download PDF:
Sorry, we are unable to provide the full text but you may find it at the following location(s):
  • http://www.pubmedcentral.nih.g... (external link)
  • Suggested articles


    1. (1990). A cdc2-like protein is involved in the initiation of DNA replication in Xenopus egg extracts.
    2. (2006). A Cdt1-geminin complex licenses chromatin for DNA replication and prevents rereplication during S phase in Xenopus.
    3. (2001). A role for nuclear lamins in nuclear envelope assembly.
    4. (2003). Anthracycline-dependent heat-induced transition from positive to negative supercoiled DNA.
    5. (2004). ATR and ATM regulate the timing of DNA replication origin firing.
    6. (1996). Both cyclin A and cyclin E have S-phase promoting (SPF) activity in Xenopus egg extracts.
    7. (2003). CDK- and Cdc45-dependent priming of the MCM complex on chromatin during S-phase in Xenopus egg extracts: possible Nucleic Acids
    8. (2007). CDK-dependent phosphorylation of
    9. (2000). Central role for cdc45 in establishing an initiation complex of DNA replication in Xenopus egg extracts.
    10. (2001). Chromatin docking and exchange activity enhancement of RCC1 by histones
    11. (1996). Chromotin binding, nuclear localization and phosphorylation of Xenopus cdc21 are cell-cycle dependent and associated with the control of initiation of DNA replication.
    12. (2004). Control of replication origin density and firing time in Xenopus egg extracts: role of a caffeine-sensitive, ATR-dependent checkpoint.
    13. (1997). Daunomycin disrupts nuclear assembly and the coordinate initiation of DNA replication in Xenopus egg extracts.
    14. (2000). Disruption of nuclear lamin organization blocks the elongation phase of DNA replication.
    15. (2004). DNA intercalators differentially affect chromatin structure and DNA replication in Xenopus egg extract.
    16. (2003). DNA replication of mitotic chromatin in Xenopus egg extracts.
    17. (1997). Dynamic molecular combing: stretching the whole human genome for high-resolution studies.
    18. (2004). Echinomycin inhibits chromosomal DNA replication and embryonic development in vertebrates.
    19. (1979). Effect of drugs on deoxyribonucleic acid synthesis in isolated mammalian cell nuclei. Comparison with partially purified deoxyribonucleic acid polymerases.
    20. (1978). Effect of ethidium bromide on the digestion of chromatin DNA with micrococcal nuclease.
    21. (1983). Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components.
    22. (2005). Functional uncoupling of MCM helicase and DNA polymerase activities activates the ATR-dependent checkpoint.
    23. (1995). ICRF-193, an inhibitor of topoisomerase II, demonstrates that DNA replication in sperm nuclei reconstituted in Xenopus egg extracts does not require chromatin decondensation.
    24. (2002). Identification of the nuclear localization signal in Xenopus cyclin e and analysis of its role in replication and mitosis.
    25. (1986). Initiation of DNA replication in nuclei and purified DNA by a cell-free extract of Xenopus eggs.
    26. (2004). Initiation of DNA replication in Xenopus egg extracts.
    27. (2000). Initiation of eukaryotic DNA replication: origin unwinding and sequential chromatin association of Cdc45, RPA, and DNA polymerase alpha.
    28. (2008). levels determine the efficiency of replication origin firing in Xenopus.
    29. (2004). Mcm2 is a direct substrate of ATM and ATR during DNA damage and DNA replication checkpoint responses.
    30. (2008). Multiple mechanisms contribute to S. pombe ORC-DNA interactions.
    31. (2008). Nuclear lamins: major factors in the structural organization and function of the nucleus and chromatin.
    32. (1987). Nuclear reconstitution in vitro: stages of assembly around protein-free DNA.
    33. (1992). Nucleoplasmin remodels sperm chromatin in Xenopus egg extracts.
    34. (2007). Phosphorylation of Sld2 and Sld3 by cyclin-dependent kinases promotes DNA replication in budding yeast.
    35. (2008). Plx1 is required for chromosomal DNA replication under stressful conditions.
    36. (2001). Ran GTPase: a master regulator of nuclear structure and function during the eukaryotic cell division cycle?
    37. (1998). Regulated chromosomal DNA replication in the absence of a nucleus.
    38. (2000). Response of Xenopus Cds1 in cell-free extracts to DNA templates with double-stranded ends.
    39. (1989). Selective displacement of nuclear proteins by antitumor drugs having affinity for nucleic acids.
    40. (2004). Sleeping policemen for DNA replication?
    41. (1976). The binding of echinomycin to deoxyribonucleic acid.
    42. (1974). The degree of unwinding of the DNA helix by ethidium. I. Titration of twisted PM2 DNA molecules in alkaline cesium chloride density gradients.
    43. (1978). The inhibition of mitochondrial DNA polymerase gamma from animal cells by intercalating drugs.
    44. (1998). Xenopus Cdc45-dependent loading of DNA polymerase alpha onto chromatin under the control of S-phase Cdk.

    To submit an update or takedown request for this paper, please submit an Update/Correction/Removal Request.