Investigating the mechanisms of epigenetic inheritance during DNA replication

This is a summer student position supervised by Sophie Williams in John Diffley's lab.

Introduction to the science

Our lab aims to understand the mechanisms of how our DNA is precisely duplicated in each cell cycle. In eukaryotes, all our DNA is packaged into chromatin which makes up the epigenetic landscape. During DNA replication, the chromatin must be disassembled to allow access to the DNA sequence. We aim to understand how this chromatin architecture gets disrupted and subsequently restored during the process of DNA replication to ensure the stable propagation of both genetic and epigenetic information across cell divisions. Our lab studies this by biochemically reconstituting the steps of DNA replication and chromatin assembly using purified components in a test tube (in vitro) [1].

About the project

The fundamental repeating unit of chromatin is the nucleosome. This is made up of an octamer of proteins called histones, which the DNA is wrapped around. During each cell cycle, these nucleosomes must be disassembled to enable access to the DNA for it to be replicated. These displaced parental histones are re-deposited on the newly synthesised DNA behind the replication fork. Since the DNA content is doubled during the process of replication, only up to 50% of the histones on the nascent DNA can come from the parental nucleosomes. The remaining nucleosomes are assembled from newly synthesised histones in a replication-coupled de novo assembly pathway. We are investigating how these two pathways (re-deposition of parental histones and de novo deposition of new histones) are coordinated with one another to ensure accurate epigenetic inheritance. One of the key proteins involved in this process is a histone chaperone called CAF-1 [2]. CAF-1 is targeted to replication forks through an interaction with a key component of the replication machinery called PCNA. Once recruited to the nascent DNA, CAF-1 dimerises to enable deposition of newly synthesised histones onto the DNA. In this project, we will aim to better understand how CAF-1 functions in the de novo histone deposition pathway. To do this, we will generate mutations that: (i) perturb the interaction of CAF-1 with PCNA, and (ii) inhbit CAF-1 dimerisation. We will then assess how these mutations affect the function of CAF-1 and the assembly of chromatin during DNA replication. This will involve:

• Cloning constructs and generating yeast strains for the expression of CAF-1 mutants
• Expressing and purifying the generated mutants from yeast cells
• Testing purified wild-type and mutant proteins in biochemical assays including protein-protein interaction assays

Candidate background