The fork protection complex recruits FACT to reorganize nucleosomes during replication

Abstract

Chromosome replication depends on efficient removal of nucleosomes by accessory factors to ensure rapid access to genomic information. Here, we show this process requires recruitment of the nucleosome reorganization activity of the histone chaperone FACT. Using single-molecule FRET, we demonstrate that reorganization of nucleosomal DNA by FACT requires coordinated engagement by the middle and C-terminal domains of Spt16 and Pob3 but does not require the N-terminus of Spt16. Using structure-guided pulldowns, we demonstrate instead that the N-terminal region is critical for recruitment by the fork protection complex subunit Tof1. Using in vitro chromatin replication assays, we confirm the importance of these interactions for robust replication. Our findings support a mechanism in which nucleosomes are removed through the coordinated engagement of multiple FACT domains positioned at the replication fork by the fork protection complex.

Mechanism of curaxin-dependent nucleosome unfolding by FACT

“…Partial DNA uncoiling that must occur during transcription and replication exposes FACT-binding sites on the octamer and provides a target for FACT binding. Indeed, FACT is associated with transcribed genes and the replication fork ( Belotserkovskaya et al, 2003 ; Hsieh et al, 2013 ; Kulaeva et al, 2013 ; Safaric et al, 2022 ); since destabilized nucleosomes have exposed binding sites for FACT, nucleosome unfolding likely occurs during these processes ( Figure 8A ). It has been proposed that nucleosome unfolding could facilitate nucleosome survival during transcription ( van Holde et al, 1992 ); although there is no direct evidence for unfolding of these nucleosomes, FACT facilitates nucleosome survival during transcription in vitro ( Hsieh et al, 2013 ).…”

“…FACT complex and ATP-dependent chromatin remodeling complexes participate in the nuclear processes involving the nucleosome eviction ( Ray-Gallet & Almouzni, 2022 ; Safaric et al, 2022 ). Therefore, curaxins cause the destabilization of nucleosomal structure and possibly could stimulate nucleosome unfolding or sliding by other remodelers as well as by FACT.…”

Embracing Heterogeneity: Challenging the Paradigm of Replisomes as Deterministic Machines

“…Furthermore, there are known interactions between the replisome and histones/chaperones that contribute to chromatin replication, , but their exact mechanism of working remains unknown. To investigate how histone chaperone FACT reorganizes nucleosomes and its role in chromatin replication, smFRET was used to visualize dynamic changes in nucleosomal DNA during engagement by FACT . Large-scale structural changes and reorganization of yeast nucleosomes were observed in the presence of FACT.…”

Mechanism of Curaxin-dependent Nucleosome Unfolding by FACT

“…Partial DNA uncoiling that must occur during transcription and replication exposes FACT-binding sites on the octamer and provides a target for FACT binding. Indeed, FACT is associated with transcribed genes and the replication fork (25)(26)(27)(28); since destabilized nucleosomes have exposed binding sites for FACT, nucleosome unfolding likely occurs during these processes (Fig. S10A).…”