title: Biphasic chromatin binding of histone chaperone FACT during eukaryotic chromatin DNA replication.
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.
authors.affiliation: Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan.
authors.forename: Lena R
authors.initials: LR
authors.lastname: Kundu
authors.forename: Masayuki
authors.initials: M
authors.lastname: Seki
authors.forename: Nanae
authors.initials: N
authors.lastname: Watanabe
authors.forename: Hiromu
authors.initials: H
authors.lastname: Murofushi
authors.forename: Asako
authors.initials: A
authors.lastname: Furukohri
authors.forename: Shou
authors.initials: S
authors.lastname: Waga
authors.forename: Alan J
authors.initials: AJ
authors.lastname: Score
authors.forename: J Julian
authors.initials: JJ
authors.lastname: Blow
authors.forename: Masami
authors.initials: M
authors.lastname: Horikoshi
authors.forename: Takemi
authors.initials: T
authors.lastname: Enomoto
authors.forename: Shusuke
authors.initials: S
authors.lastname: Tada
mesh_terms: D000818:Animals; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D002843:Chromatin / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005057:Eukaryotic Cells / Q000378:metabolism; D005260:Female; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006609:High Mobility Group Proteins / Q000235:genetics / Q000378:metabolism*; D056488:Histone Chaperones / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D008297:Male; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism; D011485:Protein Binding; D013094:Spermatozoa / Q000378:metabolism; D013997:Time Factors; D035602:Transcriptional Elongation Factors / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: C103706:CDC6 protein, human; D018797:Cell Cycle Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; D006609:High Mobility Group Proteins; D056488:Histone Chaperones; D009687:Nuclear Proteins; C073537:SSRP1 protein, human; D035602:Transcriptional Elongation Factors; D005982:Glutathione Transferase