Origin-specific initiation of mammalian nuclear DNA replication in a Xenopus cell-free system.
The introduction of Chinese hamster ovary (CHO) cell nuclei into Xenopus egg extracts provides the only cell-free system that can efficiently initiate replication at a specific metazoan replication origin. With intact late-G1-phase nuclei as a substrate, the pattern of initiation sites for replication at the CHO dihydrofolate reductase (DHFR) locus is indistinguishable from that observed in cultured cells. By contrast, with early-G1-phase nuclei or with late-G1-phase nuclei that have damaged nuclear envelopes, these same extracts efficiently initiate replication at apparently random sites. Thus, at a distinct point during G1 phase [origin decision point (ODP)], nuclei experience a transition that is required for specific recognition of the DHFR origin by Xenopus egg cytosol. Described here are the basic requirements to achieve origin-specific initiation, which include: 1) a cell line that can be synchronized in G1 phase, 2) a method to prepare intact nuclei, 3) a technique to map origins with a few million cells, and 4) a small colony of Xenopus laevis. Immunodepletion of specific gene products allows one to test hypotheses about the requirements for origin recognition. Here we show that depletion of the Xenopus origin recognition complex subunit XORC2 from Xenopus egg extracts has no influence on the efficiency of replication or the pattern of initiation sites with either pre-ODP or post-ODP nuclei.
Wu
J R
JR
Department of Biochemistry and Molecular Biology, State University of New York Health Science Center, Syracuse 13210, USA.
Yu
G
G
Gilbert
D M
DM
D000818:Animals; D002457:Cell Extracts / Q000235:genetics; D002474:Cell-Free System; D002478:Cells, Cultured; D004261:DNA Replication; D005821:Genetic Techniques; D010063:Ovum / Q000378:metabolism*; D018741:Replication Origin; D014982:Xenopus laevis / Q000235:genetics*
D002457:Cell Extracts