In Xenopus Egg Extracts, DNA Replication Initiates Preferentially at or near Asymmetric AT Sequences

Abstract

Previous observations led to the conclusion that in Xenopus eggs and during early development, DNA replication initiates at regular intervals but with no apparent sequence specificity. Conversely, here, we present evidence for site-specific DNA replication origins in Xenopus egg extracts. Using λ DNA, we show that DNA replication origins are activated in clusters in regions that contain closely spaced adenine or thymine asymmetric tracks used as preferential initiation sites. In agreement with these data, AT-rich asymmetric sequences added as competitors preferentially recruit origin recognition complexes and inhibit sperm chromatin replication by increasing interorigin spacing. We also show that the assembly of a transcription complex favors origin activity at the corresponding site without necessarily eliminating the other origins. Thus, although Xenopus eggs have the ability to replicate any kind of DNA, AT-rich domains or transcription factors favor the selection of DNA replication origins without increasing the overall efficiency of DNA synthesis. These results suggest that asymmetric AT-rich regions might be default elements that favor the selection of a DNA replication origin in a transcriptionally silent complex, whereas other epigenetic elements linked to the organization of domains for transcription may have further evolved over this basal layer of regulation.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://mcb.asm.org/ .

ACKNOWLEDGMENTS

We thank P. Pasero and E. Ralph for useful discussion and critical reading of the manuscript. We acknowledge the Montpellier DNA Combing Facility for providing the silanized surfaces. We also acknowledge N. Montel and S. Bocquet for technical help.

S.S. is supported by the Fondation pour la Recherche Medicale. This work is supported by the CNRS, the Association pour la Recherche sur le Cancer, the Ligue contre le Cancer, and the National Agency for Research (ANR).