The RLF-B component of the replication licensing system is distinct from Cdc6 and functions after Cdc6 binds to chromatin

Replication licensing factor (RLF) is an essential initiation factor that c an prevent re-replication of DNA in a single cell cycle [1,2]. It is requir ed for the initiation of DNA replication, binds to chromatin early in the c ell cycle, is removed from chromatin as DNA replicates and is unable to re- bind replicated chromatin until the following mitosis, Chromatography of RL F from Xenopus extracts has shown that it consists of two components termed RLF-B and RLF-M [3]. The RLF-M component consists of complexes of all six Xenopus minichromosome maintenance (MCM/P1) proteins (XMcm2-7), which bind to chromatin in late mitosis and are removed as replication occurs [3-7]. T he identity of RLF-B is currently unknown. At least two factors must be pre sent on chromatin before licensing can occur: the Xenopus origin recognitio n complex (XORC) [8,9] and Xenopus Cdc6 (XCdc6) [10]. XORC saturates Xenopu s sperm chromatin at approximately one copy per replication origin whereas XCdc6 binds to chromatin only if XORC is bound first [9-11]. Although XORC has been shown to be a distinct activity from RLF-B [9], the relationship b etween XCdc6 and RLF-B is currently unclear. Here, we show that active XCdc 6 is loaded onto chromatin in extracts with defective RLF, and that both RL F-M and RLF-B are still required for the licensing of XCdc6-containing chro matin. Furthermore, RLF-B can be separated from XCdc6 by immunoprecipitatio n and standard chromatography. These experiments demonstrate that RLF-B is both functionally and physically distinct from XCdc6, and that XCdc6 is loa ded onto chromatin before RLF-B function is executed.