The RLF-B Component of the Replication Licensing System is Distinct from Cdc6 and Functions After Cdc6 has Bound to Chromatin

Replication Licensing Factor (RLF) was originally defined as an essential initiation factor whose regulation prevents re-replication of DNA in a single cell cycle [ 1 , reviewed in 2 ]. It is required for the initiation of DNA replication, binds to chromatin early in the cell cycle, is removed from chromatin as DNA replicates and is unable to re-bind replicated chromatin until the following mitosis. Chromatography of replication licensing factor (RLF) from Xenopus extracts has shown that it consist of two components termed RLF-B and RLF-M [ 3 ]. RLF-M consists of complexes of all 6 Xenopus MCM/P1 proteins (XMcm2,3,4,5,6 and 7), which bind to chromatin in late mitosis and are removed as replication occurs [ 3 - 7 ]. The identity of RLF-B is currently unknown. At least two factors must be present on chromatin before licensing can occur: the Xenopus Origin Recognition Complex (XORC) [ 8 , 9 ] and Xenopus Cdc6 (XCdc6) [ 10 ]. XORC saturates Xenopus sperm chromatin at approximately 1 copy per replication origin and is required for licensing by RLF-B and RLF-M to occur [ 9 - 11 ]. XCdc6 binds to chromatin only if XORC is bound first, and the binding of MCM/P1 proteins to chromatin is dependent on XCdc6 [ 10 ]. Although XORC has been shown to be a distinct activity from RLF-B [ 9 ], the relationship between XCdc6 and RLF-B is currently unclear. Here we show that active XCdc6 is loaded onto chromatin in RLF-defective extracts, and show that both RLF-M and RLF-B are still required for the licensing of XCdc6-containing chromatin. Further, RLF-B can be separated from XCdc6 by immunoprecipitation and standard chromatography. These experiments demonstrate that RLF-B is both functionally and physically distinct from XCdc6, and that XCdc6 is loaded onto chromatin before RLF-B function is executed.