Previous studies have suggested that cell cycle-dependent changes in the affinity of the origin recognition complex (ORC) for chromatin are involved in regulating initiation of DNA replication. To test this hypothesis, chromatin lacking functional ORCs was isolated from metaphase hamster cells and incubated in Xenopus egg extracts to initiate DNA replication. Intriguingly, Xenopus ORC rapidly bound to hamster somatic chromatin in a Cdc6-dependent manner and was then released, concomitant with initiation of DNA replication. Once pre-replication complexes (pre-RCs) were assembled either in vitro or in vivo, further binding of XIORC was inhibited. Neither binding nor release of XIORC was affected by inhibitors of either cyclin-dependent protein kinase activity or DNA synthesis. In contrast, inhibition of pre-RC assembly, either by addition of Xenopus geminin or by depletion of XIMcm proteins, augmented ORC binding by inhibiting ORC release. These results demonstrate a programmed release of XIORC from somatic cell chromatin as it enters S phase, consistent with the proposed role for ORC in preventing re-initiation of DNA replication during S phase.
|頁（從 - 到）||1437-1446|
|出版狀態||Published - 15 3月 2002|