Analysis of mammalian origin specification in ORC-depleted Xenopus egg extracts.

BACKGROUND Xenopus egg extracts initiate replication specifically at the Chinese Hamster Ovary (CHO) cell dihydrofolate reductase (DHFR) origin with CHO G1-phase nuclei as a substrate, providing that these nuclei have intact nuclear envelopes and are isolated from cells that have passed through a distinct transition (origin decision point; ODP) early in G1-phase. With intact pre-ODP nuclei, or with post-ODP nuclei that have permeabilized nuclear envelopes, replication initiates efficiently but, at apparently random sites. We have investigated whether the Xenopus embryonic origin recognition complex (XORC) influences origin specification in this system. RESULTS Xenopus egg extracts were immunodepleted of XORC, eliminating their ability to assemble pre-initiation complexes. These extracts were deficient in the replication of CHO metaphase chromosomes but supported efficient DNA replication within both pre- and post-ODP hamster G1-phase nuclei, even after permeabilization and extraction of soluble nuclear proteins. XORC-depleted extracts initiated replication specifically at the DHFR origin with intact post-ODP nuclei but still initiated at apparently random sites with intact pre-ODP nuclei or permeabilized post-ODP nuclei. CONCLUSIONS Xenopus embryonic ORC is clearly not required for random origin site selection in Xenopus egg extracts. We conclude that a modification of Chinese Hamster chromatin takes place shortly after metaphase that complements a lack of XORC activity. This modification most likely represents an interaction of mammalian ORC with chromatin that is required for replication but, that is not sufficient for origin specification.