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

• Published in: Current biology Vol. 9; no. 4; pp. 211 - 215
• Main Authors: Tada, Shusuke, Chong, James P.J., Mahbubani, Hiro M., Blow, J.Julian
• Format: Journal Article
• Language: English
• Published: England Elsevier Inc 25.02.1999
• Subjects: Animals; Cell Cycle; Cell Cycle Proteins - isolation & purification; Cell Cycle Proteins - metabolism; Cell Nucleus - physiology; Chromatin - metabolism; Chromosomal Proteins, Non-Histone - metabolism; DNA Replication; Female; Male; Ovum - physiology; Saccharomyces cerevisiae Proteins; Spermatozoa - physiology; Transcription Factors - chemistry; Transcription Factors - isolation & purification; Transcription Factors - metabolism; Xenopus; Xenopus Proteins; Zinc Fingers
• ISSN: 0960-9822; 1879-0445
• DOI: 10.1016/S0960-9822(99)80092-X

Replication licensing factor (RLF) is an essential initiation factor that can prevent re-replication of DNA in a single cell cycle [1,2]. 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 RLF 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]. 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 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 between XCdc6 and RLF-B is currently unclear. Here, we show that active XCdc6 is loaded onto chromatin in extracts with defective RLF, and that both RLF-M and RLF-B are still required for the licensing of XCdc6-containing chromatin. Furthermore, 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.