SFI UREKA Site in Molecular Recognition
Translation Design into Application

Project 2:  Chromatin Flexibility Dependent on Intermolecular Histone Interfaces

Supervisor:  Dr Andrew Flaus, Department of Biochemistry, NUI Galway.

Project Summary

As the fundamental repeating unit of chromatin structure, the nucleosome acts as the substrate for most processes accessing the genomes of eukaryotes. The last two decades have seen rapid progress towards identifying the many factors which modify or manipulate chromatin structure to manage DNA accessibility. Full appreciation of the role of chromatin depends on understanding how nucleosomes behave and how factors bind in the nucleosomal context.

The nucleosome is composed of an octamer composed of histone proteins around which DNA is wrapped in a tight superhelix. This distorts the conformation of DNA, altering its recognition by DNA binding proteins. Since the DNA is in an energetically unfavorable conformation, it is potentially readily released from the nucleosome by other factors which bind. Non sequence-specific nucleosome binding proteins can therefore potentially manipulate nucleosome stability by specifically recognising nucleosomal DNA.

To study the effect of non-specific nucleosome binding proteins in detail, we will express and purify a number of representative examples. This will involve recombinant DNA cloning and protein expression in E. coli followed by chromatographic purification. We will also prepare nucleosomes using expressed histone proteins and DNA available in our laboratory. We will monitor nucleosomal binding on the non sequence-specific proteins using gel shift assays and DNA footprinting techniques. This will allow us to establish assays for monitoring the effect of the binding proteins on nucleosomes containing a large range of histone mutants being prepared in our laboratory.

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