Single-molecule imaging reveals control of parental histone recycling by free histones during DNA replication

Abstract

During replication, nucleosomes are disrupted ahead of the replication fork, followed by their reassembly on daughter strands from the pool of recycled parental and new histones. However, because no previous studies have managed to capture the moment that replication forks encounter nucleosomes, the mechanism of recycling has remained unclear. Here, through real-time single-molecule visualization of replication fork progression in Xenopus egg extracts, we determine explicitly the outcome of fork collisions with nucleosomes. Most of the parental histones are evicted from the DNA, with histone recycling, nucleosome sliding, and replication fork stalling also occurring but at lower frequencies. Critically, we find that local histone recycling becomes dominant upon depletion of endogenous histones from extracts, revealing that free histone concentration is a key modulator of parental histone dynamics at the replication fork. The mechanistic details revealed by these studies have major implications for our understanding of epigenetic inheritance.

Studying chromosome biology with single-molecule resolution in Xenopus laevis egg extracts

“…This justifies development of new single-molecule approaches in extracts to give a detailed insight into pathways like non-NHEJ DNA repair mechanisms and transcription [50]. Further study of the intersection between chromosome replication, repair and organisation in egg extracts will be interesting [26,33].…”

“…[22]. (B) Real-time imaging of replication forks in extracts with Fen1-mKikGR, which binds nascent DNA [25], and adaptation of this system to visualise the fate of labelled nucleosomes reconstituted on λ DNA [26]. The right hand panel shows a kymogram, a stack of images from a single position of interest, with a growing replication bubble marked by Fen1-mKikGR.…”

“…Real-time imaging of DNA replication with Fen1-mKikGR was adapted to probe dynamics of individual nucleosomes after replication fork collision [26]. Histones and corresponding post-translational modifications must be transferred to daughter DNA strands during replication, but the molecular mechanism of histone inheritance is poorly understood.…”

“…Xenopus egg extracts are a powerful in vitro system as large volumes of cell-free extract synchronised in the cell cycle can reconstitute complex reactions involving chromosomes. Extending assays in extracts to provide single-molecule resolution gives an ability to follow discrete interactions in a single experiment [33], measure frequencies of heterogenous events [26,30] and measure dynamic structural rearrangements of proteins and DNA [6,15,40]. The use of TIRF microscopy to monitor reactions on surface tethered DNAs has proven particularly valuable.…”

Replication-Coupled Chromatin Remodeling: An Overview of Disassembly and Assembly of Chromatin during Replication

“…Indeed, the authors observed the local redeposition of the parental nucleosome through DNA replication in repressed chromatin domains, while active chromatin domains exhibited the positional dispersion of parental nucleosomes in a replication-dependent manner. Interestingly, in addition to the structure of chromatin in the recycling of parental histones, in vitro analyses of real-time single-molecule in Xenopus laevis egg extracts revealed that the fate of the parental nucleosome did not follow a unique mechanism [ 84 ]. In these experimental conditions, the nucleosome in front of the replication fork exhibited different behaviors: sliding along the DNA fiber, stalling replication fork, histone transfer or histone eviction.…”