The role of protein-protein interactions in regulation of DNA double-strand break repair in chromatin

"Nuclear DNA is compacted through the association with histone proteins to form chromatin. The compaction of DNA into chromatin affects all nuclear processes involving DNA. Previous experiments indicated that Non-Homologous End Joining (NHEJ) machinery repairs double strand breaks within the nucleus by interacting with and modifying linker histones to manipulate chromatin. Linker histones are a large family of similar proteins: 10 linker histone proteins have been found in humans, 7 of them in all somatic cells. The experiments performed led to the discovery that one specific linker histone variant is critical for the cellular repair of Double-Strand Breaks (DSB). Cells in which the production of this protein has been targeted by siRNA technology are hypersensitive to ionizing radiation which significantly decreases their capacity to repair DSB. The depleted cells all exhibit the hallmarks of a novel DNA repair-deficient phenotype, independent of previously described phenotypes. Although the specific mechanistic details of linker histone interactions with the cellular DNA repair machinery remain to be elucidated, we have already shown that this specific variant is very efficiently phosphorylated in vitro by the DNA-dependent protein kinase (DNA-Pk; the core signalling protein complex in NHEJ).
The aims and objectives of this project are to elucidate further the function of this linker histone in repair of DSB by focussing on:
The identification of molecular partners using already developed tagged over-expression systems and proteomic analysis.
Detailed mechanistic analysis of DNA break repair enzymology by in vitro reconstitution of DSB repair reaction from purified components.
Analysis of the functional impact of post-translational modifications.
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