Carty, Alice Elizabeth; (2021) Biophysics of archaeal chromatin studied at the single molecule level. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

DNA compaction is a universal requirement across the three domains of life. The proteins responsible for DNA compaction in archaea and eukaryotes are structurally distinct from those found in bacteria. Archaea and eukaryotes share a conserved protein fold for DNA compaction: the histone fold. This implies that the nucleosome, which is the fundamental building block of eukaryotic chromatin, evolved before the archaeal and eukaryotic domains of life separated. Despite their low sequence homology, recent structural studies indicate that the canonical nucleosome core can be formed by archaeal histones. However, the archaeal nucleosome might assemble into a continuous, solenoid-like, fibre called a ‘hypernucleosome’, which is unlike the discrete, regularly-spaced, nucleosomes formed in eukaryotes. I have used a combination of single molecule approaches (magnetic tweezers and total internal reflection fluorescence microscopy) to investigate the mechanics of DNA binding, wrapping and compaction by the abundant histone protein (‘A3’) from the hyperthermophilic archaeon, Methanocaldococcus jannaschii. I have found that assembly of the histones on the DNA is concentration dependent, and that the stability under moderate load of the resulting fibre is improved by increasing histone concentration. I have found that negative supercoiling also confers stability to the hypernucleosome structure, and that the chromatin fibre shows a sharp buckling transition between positive and negative supercoiling regimes. Furthermore, I have demonstrated that the hypernucleosome can be disrupted by force, within the physiological range, with the fibre reverting to the worm-like chain model for naked DNA under sufficient load. I also propose a model for the force-extension of archaeal chromatin based upon these observations. Significantly, I have proposed a binding mechanism that may enable processing enzymes to access the DNA by shunting or displacing nucleosomes, without the need for active chromatin remodelling proteins - which have not yet been identified or characterised in the archaeal domain.

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