Origin-Specific Initiation of Mammalian Nuclear DNA Replication in aXenopusCell-Free System

Abstract

The introduction of Chinese hamster ovary (CHO) cell nuclei intoXenopusegg 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 byXenopusegg 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 ofXenopus laevis.Immunodepletion of specific gene products allows one to test hypotheses about the requirements for origin recognition. Here we show that depletion of theXenopusorigin recognition complex subunit XORC2 fromXenopusegg extracts has no influence on the efficiency of replication or the pattern of initiation sites with either pre-ODP or post-ODP nuclei.