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.
Replication initiation complex formation in the absence of nuclear function in Xenopus
Krasinska, L.; Fisher, D.
Nucleic Acids Res
2009-04 / vol 37 / pages 2238-48
gkp081 [pii] 10.1093/nar/gkp081
IGMM team(s) involved in this publication
Nuclear Control of Cell Proliferation
Animals; Chromatin/metabolism; Xenopus; Cell Nucleus/*genetics; DNA-Binding Proteins/metabolism; *DNA Replication/drug effects; Ethidium/pharmacology; Intercalating Agents/pharmacology; Nuclear Envelope/drug effects; Nuclear Lamina/drug effects