Biophysical analysis of the Nucleosome Assembly Protein and its interaction with histones

Abstract

Eukaryotic DNA must undergo high levels of compaction with a complex topology to package the entire genome into the relatively small nucleus. Exquisitely controlled, local de-compaction of the DNA is also essential for fundamental cellular processes such as DNA replication and transcription. An understanding of the biological and structural nature of these processes is central to unravelling how cellular life is regulated. The primary level of compaction involves the double stranded DNA wrapping around a core of histone proteins, two each of the H2A/H2B and H3/H4 dimers, to form the nucleosome core particle. Multiple factors are involved in controlling the assembly and disassembly of the nucleosome core particle including proteins such as the histone chaperones. NAP1 is one such protein which is involved in many cellular functions including histone shuttling, cell cycle control, transcriptional regulation, histone variant exchange and facilitating nucleosome sliding.
Following the purification of the Xenopus laevis (x) NAP and histones, H2A/H2B and H3/H4, characterisation of xNAP and the associated xNAP·Histone complexes was undertaken. Data obtained from analytical ultracentrifugation (AUC) and size exclusion chromatography with multi-angle laser light scattering (SEC-MALLS) indicated that xNAP formed a stable dimer in solution. In physiologically relevant conditions the xNAP dimer further oligomerised into multiple high-order species. Utilising analytical size exclusion in combination with gel electrophoresis, the binding stoichiometry of one xNAP dimer to one histone dimer was identified. These stoichiometric units (xNAP₂·H2A/H2B and xNAP₂·H3/H4) were also found to oligomerise into multiple large mega-Dalton complexes, as identified by SEC-MALLS and AUC sedimentation velocity experiments. AUC sedimentation equilibrium and small angle scattering data revealed that the predominant species formed was an oblate decamer with a maximum dimension of approximately 250 Å, consistent with electron microscopy images.
All of the data obtained from the complementary techniques employed are supportive of the idea that the stoichiometric sub-units oligomerise into pentameric or hexameric complexes, which in turn self-associate into decamer or dodecamer assemblies respectively. It is likely that this NAP·Histone oligomerisation is dependent upon multiple factors such as the phosphorylation state of NAP, post translational modifications of the histones and the binding of several non-histone protein binding partners. The oligomerisation state of NAP may form the basis of a mechanism which alters the localisation and function of NAP within the cell.

Date of Award	Jan 2011
Original language	English
Awarding Institution	University of Portsmouth
Supervisor	John McGeehan (Supervisor) & Geoff Kneale (Supervisor)