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Cryo-EM Imaging of Histone Recycling at the Replication Fork

Project description

Understanding eukaryotic replication machinery with the help of cryo-electron microscopy

Eukaryotic DNA is densely packed in nucleosome arrays to form chromatin, protecting the genetic material and regulating access to DNA. The replication machinery dismantles nucleosomes at the replication fork and reassembles them on nascent DNA strands, coordinating the redeposition of parental and newly synthesised histones. The post-translational histone modifications provide an epigenetic code that modulates the activation and silencing of specific chromosomal regions. The EU-funded Cryo-H-Rec project employs cryo-electron microscopy, solution NMR spectroscopy and cross-linking mass spectrometry to study DNA duplication and parental histone recycling. These data will uncover the role of replisome components in nucleosome reshuffling at the replication fork and shed light on the molecular mechanism of chromosome replication and epigenetic inheritance.

Objective

DNA replication is essential for cell proliferation. In eukaryotic cells, DNA is densely packaged in nucleosome arrays that form chromatin. Such organisation protects the genetic material and controls access to DNA, thus providing an important mechanism for regulating gene expression. The eukaryotic replication machinery has evolved to unravel nucleosomes in order to access and duplicate DNA, while also maintaining chromatin density on newly duplicated DNA. To achieve this, the replisome dismantles nucleosomes ahead of the replication fork and reassembles them on nascent DNA strands, by coordinating redeposition of parental and newly synthesized histones. Histone proteins are subject to an array of post-translational modifications (PTMs), providing an epigenetic code that modulates activation and silencing of specific chromosomal regions. Redeposition of parental histones with their PTMs on both nascent DNA strands is, thus, pivotal in transmission of the epigenetic marks to daughter cells. I intend to perform in vitro reconstitution of the replisome on a chromatinised template and use cryo-electron microscopy to image DNA duplication and parental histone recycling at the replication fork. I seek to describe different structural intermediates in the process of nucleosome disassembly, DNA duplication and histone incorporation into new nucleosomes. To capture and characterise intermediate states of the replication machinery during this concerted process, I will employ a multidisciplinary approach and resort to structural techniques complementary to cryo-EM like solution NMR spectroscopy and crosslinking-mass spectrometry (XL-MS). My results will help dissect the role of different replisome components in nucleosome reshuffling at the replication fork, and elucidate the molecular mechanism that underpins chromosome replication and epigenetic inheritance.

Coordinator

THE FRANCIS CRICK INSTITUTE LIMITED
Net EU contribution
€ 212 933,76
Address
1 MIDLAND ROAD
NW1 1AT London
United Kingdom

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Region
London Inner London — West Camden and City of London
Activity type
Research Organisations
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Total cost
€ 212 933,76