# Molecular mechanisms of replication-coupled chromatin assembly

> **NIH NIH R01** · SLOAN-KETTERING INST CAN RESEARCH · 2021 · $510,075

## Abstract

Summary
 Faithful duplication of eukaryotic chromosomes during cell division requires the accurate replication
of both chromosomal DNA and its associated chromatin states. However, DNA replication results in the
disassembly of nucleosomes ahead of replication forks and thus poses a significant challenge to the
integrity of chromatin. Chromatin restoration following DNA replication is initiated by pathways that recycle
parental histones or that assemble nucleosomes de novo from newly synthesized histones. As parental
histones carry epigenetic modifications, the recycling of parental histones into the daughter genomes is of
particular significance for the transmission of epigenetic information across generations. The redundancy of
cellular chromatin assembly pathways and the lack of functional biochemical assay systems has limited the
mechanistic study of replication-coupled chromatin assembly. We have recently reconstituted the eukaryotic
DNA replication reaction using purified budding yeast proteins and yeast origin-containing plasmid
templates. The system recapitulates key features of cellular DNA replication, including regulated origin firing
and canonical leading and lagging strand synthesis. Parental nucleosomes are also efficiently recycled on
the newly replicated DNA by core replisomes in this system. However, extended nucleosome arrays are not
reestablished, presumably due to the absence of the de novo nucleosome assembly pathway. In
unpublished results, we have purified the budding yeast orthologs of histone chaperones implicated in
replication-coupled chromatin assembly in vivo and demonstrate their ability to coordinately assemble
nucleosomes from bound histone H3-H4 and H2A-H2B dimers. Combined, the reconstituted DNA
replication and nucleosome assembly reactions put us in position to reconstitute replication-coupled
chromatin assembly. Aim 1 focuses on mechanisms of parental histone recycling at the replication fork. We
will use our recently reconstituted chromatin replication system to test the template commitment during
parental histone transfer, identify histone acceptors within the replisome, and test the role of replisome
components in the process. The goal of Aim 2 is to interrogate the mechanism of parental nucleosome
segregation during DNA replication in vitro. We will employ a strand-specific sequencing approach to
determine the distribution pattern of parental nucleosomes to the leading and lagging arms of the fork and
how this distribution is controlled by replisome proteins. We will also assess parental nucleosome positions
before and after replication to identify the rules governing nucleosome positioning. In Aim3 we will focus on
the reconstitution and characterization of the de novo replication-coupled nucleosome assembly pathway
using biochemical, structural, and next generation sequencing approaches.

## Key facts

- **NIH application ID:** 10242799
- **Project number:** 5R01GM127428-03
- **Recipient organization:** SLOAN-KETTERING INST CAN RESEARCH
- **Principal Investigator:** Dirk Remus
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $510,075
- **Award type:** 5
- **Project period:** 2019-09-01 → 2023-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10242799

## Citation

> US National Institutes of Health, RePORTER application 10242799, Molecular mechanisms of replication-coupled chromatin assembly (5R01GM127428-03). Retrieved via AI Analytics 2026-05-21 from https://api.ai-analytics.org/grant/nih/10242799. Licensed CC0.

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