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Nucleic Acids Res April 1, 2009; 37 (7): 2238-48.

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

Krasinska L , Fisher D .

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.

PubMed ID: 19237397
PMC ID: PMC2673427
Article link: Nucleic Acids Res

Species referenced: Xenopus
Genes referenced: cdc6 cdk1 cdk2 fubp1 gem gmnn h2ac21 h2axl igf2bp3 lmnb3 mcm7 pcna rcc1 rpa1

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References [+] :
Arias, Initiation of DNA replication in Xenopus egg extracts. 2004, Pubmed, Xenbase