Biphasic chromatin binding of histone chaperone FACT during eukaryotic chromatin DNA replication

Lena R. Kundu, Masayuki Seki, Nanae Watanabe, Hiromu Murofushi, Asako Furukohri, Shou Waga, Alan J. Score, J. Julian Blow, Masami Horikoshi, Takemi Enomoto, Shusuke Tada

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    7 Citations (Scopus)

    Abstract

    The facilitates chromatin transcription (FACT) complex affects nuclear DNA transactions in a chromatin context. Though the involvement of FACT in eukaryotic DNA replication has been revealed, a clear understanding of its biochemical behavior during DNA replication still remains elusive. Here, we analyzed the chromatin-binding dynamics of FACT using Xenopus egg extract cell-free system. We found that FACT has at least two distinct chromatin-binding phases: (1) a rapid chromatin-binding phase at the onset of DNA replication that did not involve origin licensing and (2) a second phase of chromatin binding that initiated after origin licensing. Intriguingly, early-binding FACT dissociated from chromatin when DNA replication was blocked by the addition of Cdc6 in the licensed state before origin firing. Cdc6-induced removal of FACT was blocked by the inhibition of origin licensing with geminin, but not by suppressing the activity of DNA polymerases, CDK, or Cdc7. Furthermore, chromatin transfer experiments revealed that impairing the later binding of FACT severely compromises DNA replication activity. Taken together, we propose that even though FACT has rapid chromatin-binding activity, the binding pattern of FACT on chromatin changes after origin licensing, which may contribute to the establishment of its functional link to the DNA replication machinery. (C) 2011 Elsevier B.V. All rights reserved.

    Original languageEnglish
    Pages (from-to)1129-1136
    Number of pages8
    JournalBiochimica et Biophysica Acta. Molecular Cell Research
    Volume1813
    Issue number6
    DOIs
    Publication statusPublished - Jun 2011

    Keywords

    • FACT
    • Spt16
    • SSRP1
    • DUF
    • DNA replication
    • Origin licensing
    • ORIGIN RECOGNITION COMPLEX
    • CELL-FREE-EXTRACTS
    • POLYMERASE ALPHA
    • S-PHASE
    • FUNCTIONAL COOPERATION
    • LICENSING SYSTEM
    • MCM-HELICASE
    • IN-VITRO
    • XENOPUS
    • INITIATION

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