Xenopus Cdc7 function is dependent on licensing but not on XORC, XCdc6, or CDK activity and is required for XCdc45 loading

Abstract

The assembly and disassembly of protein complexes at replication origins play a crucial role in the regulation of chromosomal DNA replication. The sequential binding of the origin recognition complex (ORC), Cdc6, and the minichromosome maintenance (MCM/P1) proteins produces a licensed replication origin. Before the initiation of replication can occur, each licensed origin must be acted upon by S phase-inducing CDKs and the Cdc7 protein kinase. In the present report we describe the role of Xenopus Cdc7 (XCdc7) in DNA replication using cell-free extracts of Xenopus eggs. We show that XCdc7 binds to chromatin during G(1) and S phase. XCdc7 associates with chromatin only once origins have been licensed, but this association does not require the continued presence of XORC or XCdc6 once they have fulfilled their essential role in licensing. Moreover, XCdc7 is required for the subsequent CDK-dependent loading of XCdc45 but is not required for the destabilization of origins that occurs once licensing is complete. Finally, we show that CDK activity is not necessary for XCdc7 to associate with chromatin, induce MCM/P1 phosphorylation, or perform its essential replicative function. From these results we suggest a simple model for the assembly of functional initiation complexes in the Xenopus system.