Ben-Shahar, Tamar Tom Rolef; (2004) Nucleosome assembly and histone H3 methylation during DNA replication. Doctoral thesis (Ph.D), UCL (University College London)..

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Abstract

Nucleosome assembly is essential for viability and entails a controlled mechanism by which newly synthesised histones are specifically deposited onto replicating DNA by assembly factors such as Chromatin Assembly Factor 1 (CAF1). CAF1 binds directly to Proliferating Cell Nuclear Antigen (PCNA), a processivity factor for DNA replication and repair, and this interaction is necessary to target its 'cargo', histones H3/H4, to newly synthesised DNA. Chapter 3 in this work presents the characterisation of the interaction between CAF1 and PCNA. This interaction is surprisingly complex, involving two distinct consensus PCNA binding motifs in the p150 subunit of CAF1, which are functionally distinct. Similar motifs are found in many other proteins involved in cell cycle control, DNA replication, DNA modification and various forms of DNA repair. I found some specificity in binding to PCNA, underscoring the existence of a regulatory mechanism which ensures that CAF1 and other PCNA-binding proteins act in a well-orchestrated manner at the replication fork. In Chapters 4 and 5, I employed two different techniques to identify other factors involved in chromatin assembly in human cells and in yeast. Micro-array analysis in budding yeast revealed global changes in gene expression in chromatin assembly factors null mutants. Through this screen I identified Periodic Tryptophan Protein 1 (Pwp1) as a putative chromatin assembly factor. The faithful replication and transfer of hereditary material to daughter cells includes not only accurate duplication of the parental DNA and chromosome architecture, but also the propagation of an epigenetic 'signature'. DNA replication offers a window of opportunity for cells to duplicate or modify the epigenetic state. For instance, DNA methylation is propagated during replication by the binding of the maintenance DNA Methyltransferase DNMT1 to PCNA. In Chapter 6, I used the budding yeast to examine a possible role for PCNA in directing the specialised histone H3 lysine 4 methyltransferase Set1 to newly assembled chromatin.

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