The basic unit of eukaryotic chromatin is the nucleosome core, which contains 147 base pairs of DNA wrapped around an octamer of core histone proteins. Linker histones bind through their globular domain at the nucleosome dyad and to internucleosomal DNA through their C-terminal basic tail. The Saccharomyces cerevisiae linker histone homologue, Hho1p, contains two domains, GI and GII, that have sequence similarity to the globular domain of the canonical linker histone H1. The individual domains of Hho1p differ in their structural and functional properties, for example in 10 mM sodium phosphate GI is folded while GII exists as two species: folded and 'unfolded'. In Chapter 2 the structure of the second globular domain of Hho1p, GII, is further investigated. NMR studies indicate residual structure in the 'unfolded' form of GII, especially at the start of helices I and III. Chapter 3 considers the structural roles of Hho1p within chromatin. Semi-quantitative Western blotting is used to measure the abundance of Hho1p relative to nucleosomes in yeast. Analysis of reconstituted nucleosome arrays containing NGIL (Hho1p with the second globular domain removed) are indistinguishable from those containing full-length Hho1p, in gel-based assays and by analytical ultracentrifugation, suggesting the GII domain may not have a major role in chromatin compaction. Chapter 4 focuses on the interaction of Hho1p with chromatin proteins. Chemical cross-linking and gel filtration indicate that Hho1p does not interact significantly with the putative HMGB1 homologues Hmo1p and Nhp6ap in vitro. Hho1p and Htz1p, the yeast histone H2A.Z subtype, do not appear to interact directly in co-immunoprecipitation and chemical cross-linking assays, while chromatin immunoprecipitation studies show no evidence of colocalisation across the ADH2 and PHO5 genes. Hho1p and Sir2p cross-link in solution, but purification difficulties precluded further investigation. The effect of phosphorylation on the interaction of Hho1p and related truncation proteins with DNA and chromatin are investigated in Chapter 5. Phosphorylation reduces their affinity for linear DNA, but has different effects on the binding to four-way junction DNA for Hho1p and NGIL, compared with LGII (the linker region and GII domain of Hho1p). Phosphorylation has no obvious effect on the affinity of these proteins for chromatin in sucrose gradient centrifugation assays. NMR spectroscopy studies show that the linker region is mostly unstructured, with a short region showing some α-helical character. Phosphorylation of the linker domain changes its structural character.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:541827 |
| Date | January 2010 |
| Creators | Osmotherly, Lara May |
| Contributors | Thomas, Jean O. |
| Publisher | University of Cambridge |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.repository.cam.ac.uk/handle/1810/228702 |
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