Specification of a DNA replication origin by a transcription complex

Abstract

In early Xenopus development, transcription is repressed and DNA replication initiates at non-specific sites. Here, we show that a site-specific DNA replication origin can be induced in this context by the assembly of a transcription domain. Deletion of the promoter element abolishes site-specific initiation, and its relocalization to an ectopic site induces a new origin of replication. This process does not require active transcription, and specification of the origin occurs mainly through a decrease in non-specific initiation at sites distant from the promoter. Finally, chromatin immunoprecipitation experiments suggest that site-specific acetylation of histones favours the selection of the active DNA replication origin. We propose that the specification of active DNA replication origins occurs by secondary epigenetic events and that the programming of chromatin for transcription during development contributes to this selection in higher eukaryotes.

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Figure 1: pGAL–myc is assembled in a DNAse I hypersensitive chromatin domain, transcribed and replicated in a regulated fashion after injection into Xenopus eggs.
Figure 2: Specification of an origin of DNA replication by TBP and GAL4–VP16.
Figure 3: Specification of an origin requires promoter sequences.
Figure 4: Site-specific initiation of DNA replication at the promoter element in an ectopic position.
Figure 5: GAL4–VP16 or a natural transcription activator are sufficient to confer site-specific initiation of DNA replication.
Figure 6: Methylation is not required for specification of DNA replication origins.
Figure 7: Distribution of GAL4–VP16, acetylated histone H3 and ORC2 on the plasmid chromatin.

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Acknowledgements

We thank D. Fisher for critical reading of this manuscript. This study has been supported by grants from the CNRS, Human Frontier Science Program, Association pour la Recherche sur le Cancer, the Fondation pour la Recherche Médicale (FRM) and the Ligue Nationale Contre le Cancer.

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Correspondence to Marcel Méchali.

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Fig. S1, Fig. S2, Fig. S3,,Fig. S4, Fig. S5 and supplementary methods (PDF 1172 kb)

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Danis, E., Brodolin, K., Menut, S. et al. Specification of a DNA replication origin by a transcription complex. Nat Cell Biol 6, 721–730 (2004). https://doi.org/10.1038/ncb1149

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