Changes in association of the Xenopus origin recognition complex with chromatin on licensing of replication origins

During late mitosis and early G(1), a series of proteins are assembled onto replication origins that results in them becoming 'licensed' for replicati on in the subsequent S phase. In Xenopus this first involves the assembly o nto chromatin of the Xenopus origin recognition complex XORC, and then XCdc 6, and finally the RLF-M component of the replication licensing system. In this paper we examine changes in the way that XORC associates with chromati n in the Xenopus cell-free system as origins become licensed. Restricting t he quantity of XORC on chromatin reduced the extent of replication as expec ted if a single molecule of XORC is sufficient to specify a single replicat ion origin. During metaphase, XOrc1 associated only weakly with chromatin, In early interphase, XOrc1 formed a strong complex with chromatin, as evide nced by its resistance to elution by 200 mM salt, and this state persisted when XCdc6 was assembled onto the chromatin, As a consequence of origins be coming licensed the association of XOrc1 and XCdc6 with chromatin was desta bilised, and XOrc1 became susceptible to removal from chromatin by exposure to either high salt or high Cdk levels. At this stage the essential functi on for XORC and XCdc6 in DNA replication had already been fulfilled. Since high Cdk levels are required for the initiation of DNA replication, this 'l icensing-dependent origin inactivation' may contribute to mechanisms that p revent re-licensing of replication origins once S phase has started.