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.

The role of FACT in making and breaking nucleosomes

“…The Spt16 subunit of FACT in yeast can be ubiquitylated by an E3 ligase based on the cullin Rtt101, and this modification appears to target a population of FACT molecules for a function in DNA replication [74]. Ubiquitylation of Spt16 is associated with enhanced localization of both FACT and the MCM helicase with replication origins, supporting previous results showing that FACT has an important role at replication origins [23], and that FACT and MCM collaborate to bind to origins [80]. Ubiquitylation appears to have a role in origin firing, as some FACT defects and loss of Rtt101 each caused diminished origin function [74].…”

“…While mutating POB3 also causes defects in transcription, Pob3 was first identified as a Po l1- b inding factor physically associated with the catalytic subunit of DNA polymerase α in yeast [5], implicating FACT in DNA replication. Subsequent work has strengthened ties to transcription factors and to replication machinery [3, 13, 20–23], and has in addition suggested a role in centromere function [14]. These activities can all be ascribed to FACT’s core ability to alter the properties of chromatin by binding histones and nucleosomes, but the range of processes involved suggests that the acronym “FACT” should be reinterpreted to mean “ fa cilitates c hromatin t ransactions.”…”

A Role for H2B Ubiquitylation in DNA Replication

“…The MCM helicase contributes to fork progression by unwinding DNA and displacing nucleosomes ahead of the fork by its association with FACT, a histone chaperone and nucleosome re-organization factor (Abe et al, 2011; Han et al, 2010; Kundu et al, 2011; Tan et al, 2006; Tan et al, 2010). H2Bub1 and Spt16, the histone-binding subunit of yeast FACT, co-operate to restore nucleosome occupancy during transcription elongation.…”

“…Alternatively, H2Bub1 could affect the association of MCM with FACT. FACT stimulates the helicase activity of MCM and together the two factors promote replication on chromatin templates (Abe et al, 2011; Han et al, 2010; Kundu et al, 2011; Tan et al, 2006; Tan et al, 2010). Because FACT is not stably associated with origin-adjacent chromatin in the absence of H2Bub1, the lower levels of FACT could in turn affect the activity and stability of MCM, impeding fork progression and leading to replisome destabilization.…”

Emerging players in the initiation of eukaryotic DNA replication

“…This suggests that FACT-MCM interaction functions not only in the initiation phase but also the elongation stage of DNA replication. Similarly in Xenopus egg extracts FACT displays two phases of chromatin binding: one before origin licensing and the other binding during replication [157]. …”