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

D T Gruszka,S Xie,H Kimura,H Yardimci

doi:10.1126/sciadv.abc0330

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.

Highlights

Eukaryotic genomes are organized into chromatin, which influences many cellular processes, ranging from DNA replication and repair to gene transcription
To determine the outcome of replication fork encounters with parental nucleosomes, we focused on low–nucleosome density templates containing either H3-K36CCy5 or H4-E63CA647, because of their high fluorophore labeling efficiency and lower exchange rates, compared to H2A-H2B, during licensing in high-speed supernatant (HSS)
On the basis of these data, we conclude that the efficiency of localized histone recycling at the replication fork depends on the concentration of soluble histones. Chromatin domains and their constituent histones with specific posttranslational modifications (PTMs) define the transcriptional program of the cell and must be faithfully replicated through cell division

Summary

Introduction

Eukaryotic genomes are organized into chromatin, which influences many cellular processes, ranging from DNA replication and repair to gene transcription. Nucleosome dynamics control DNA accessibility and are regulated by complex interplay of numerous factors, such as chromatin remodelers, histone chaperones, modifying enzymes, and polymerases [2]. Chromatin is partitioned into domains, which either promote (euchromatin) or block (heterochromatin) transcription and determine the cellular identity. Nucleosomes in transcriptionally active and silenced chromatin domains carry specific histone posttranslational modifications (PTMs) and/or distinct histone sequence variants [3, 4]. Maintenance of cellular identity through mitotic cell division relies on faithful transfer of information encoded in both DNA sequence (genetic inheritance) and nucleosome landscape (epigenetic inheritance). Semiconservative DNA replication ensures genetic inheritance, but it presents a major challenge to chromatin, which undergoes substantial structural reorganization, starting from disassembly of parental nucleosomes and ending in restoration of nucleosome landscape on daughter strands [5, 6]

Objectives

Methods

Results

Conclusion