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

S Tada, J P Chong, H M Mahbubani, J J Blow

Research output: Contribution to journalArticlepeer-review

Abstract

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] [4] [5] [6] [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] [10] [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.
Original languageEnglish
Pages (from-to)211-4
Number of pages4
JournalCurrent Biology
Volume9
Issue number4
Publication statusPublished - 1999

Keywords

  • Animals
  • Cell Cycle
  • Cell Cycle Proteins
  • Cell Nucleus
  • Chromatin
  • Chromosomal Proteins, Non-Histone
  • DNA Replication
  • Female
  • Male
  • Ovum
  • Saccharomyces cerevisiae Proteins
  • Spermatozoa
  • Transcription Factors
  • Xenopus
  • Xenopus Proteins
  • Zinc Fingers

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