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The Diffley Lab
Chromosome Replication
Epigenetic Inheritance
How is chromatin architecture inherited and maintained during DNA replication?
Eukaryotic DNA is packaged into nucleosomes, characterised by highly stable histone-DNA interactions. To replicate DNA, the eukaryotic replisome must first disrupt nucleosomes ahead of the replication fork to facilitate DNA unwinding and fork progression. The evicted parental histones must then be reassembled on roughly the same genomic position on daughter DNA strands, so that epigenetic information, stored as covalent histone modifications, is accurately retained after replication. The restoration of chromatin architecture, together with the accurate replication of DNA, ensures the stable propagation of both genetic and epigenetic information across cell divisions.
Figure. Chromatin replication summary
Mechanisms of replisome progression through chromatin
To study how replisomes progress through chromatin, we have reconstituted complete chromatin replication using purified budding yeast proteins. This in vitro system allowed us to identify factors that are important for maintaining replication fork speed through nucleosomes, including histone chaperones and nucleosome remodellers. Since these factors are involved in a wide range of chromatin-based processes, their specific roles in replication are difficult to discern through genetics and in vivo studies. The reconstituted replication system allows us to investigate mechanistic details of how these factors coordinate with replisome components to process histones at forks.
Understanding replication-coupled parental histone recycling and downstream chromatin maturation
Genome-wide sequencing studies indicate that parental histones are immediately re-deposited behind the replication fork, supporting the view that parental histone recycling is coupled with replication. To understand the exact mechanism of this recycling, we are now developing nanopore sequencing approaches to understand how parental nucleosomes are specifically inherited to the leading and lagging strands during replication.
Since DNA content is doubled during replication, histones from the parental nucleosomes can make up no more than 50% of the nucleosomes on the daughter molecules. The remainder come from newly synthesised histones assembled by a replication-coupled de novo assembly pathway. We are also investigating how parental histone recycling and de novo histone deposition are coordinated to establish fully chromatinised daughter molecules.
Key Research Questions
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How are nucleosomes disrupted ahead of the fork?
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How are nucleosomes transferred behind the fork?
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How is this transfer of parental nucleosomes coordinated with deposition of newly synthesised histones?
Relevant Papers
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Kurat et al. Chromatin Controls DNA Replication Origin Selection, Lagging-Strand Synthesis, and Replication Fork Rates. Mol Cell, 2017. 65(1): p. 117-130.
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