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

Abstract

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.