Replication initiation complex formation in the absence of nuclear function in Xenopus.
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.
Krasinska
Liliana
L
CNRS, UMR 5535-Institut de Génétique Moléculaire de Montpellier (IGMM), 34293 Montpellier cedex 5, France.
Fisher
Daniel
D
D000818:Animals; D002467:Cell Nucleus / Q000235:genetics*; D002843:Chromatin / Q000378:metabolism; D004261:DNA Replication / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000378:metabolism; D004996:Ethidium / Q000494:pharmacology; D007364:Intercalating Agents / Q000494:pharmacology; D009685:Nuclear Envelope / Q000187:drug effects; D034881:Nuclear Lamina / Q000187:drug effects; D014981:Xenopus
D002843:Chromatin; D004268:DNA-Binding Proteins; D007364:Intercalating Agents; D004996:Ethidium