Histone Stability and Nucleosomes in Hyperthermophilic Archaea
John N. REEVE*, Wen-Tyng LI, Suzette L. PEREIRA, Divya Soaresand, and Kathleen SANDMAN
Department of Microbiology, The Ohio State University, Columbus, OH43210, USA
Many hyperthermophilic Archaea grow at temperatures above the melting temperature of their DNA in vitro. Pursuit of this problem led to the discovery of archaeal histones that bind and wrap DNA into nucleosome-like structures in vitro (1), which substantially increase the temperature of heat-denaturation of the bound DNA. Results will be reported that document the isolation and characterization of archaeal nucleosomes assembled in vivo, in Methanothermus fervidus and Methanobacterium thermoautotrophicum, that contain archaeal histone tetramers which, in vitro, protect 〜60bp of DNA from micrococcal nuclease digestion. The regions of genomic DNA present in archaeal nucleosomes, isolated from the methanogens growing under different substrate-supply conditions, have been identified demonstrating that archaeal nucleosomes are not randomly located in vivo. DNA sequences have been isolated and characterized that direct archaeal nucleosome positioning in vitro.
The 3D structure established for (rHMfB)2, a dimer of the recombinant version of histone B from M. fervidus confirmed the presence of the histone fold (2). Based on this structure, predictions were made for residues that participate in DNA binding, and for residues that provide rHMfB with structural stability which have been tested by site-specific mutagenesis with gel-shift assays for DNA binding, and circular dichroism and differential scanning calorimetry assays for the stability of the resulting variants. The results obtained demonstrate that the locations of changes in rHMfB that result in a loss of DNA binding are entirely consistent with the structure of the eukaryal nucleosome (3), and that differences in the inherent stabilities of histones from Archaea that grow at different temperatures result primarily from differences in the hydrophobic packing of the histone dimer core. Changing just one residue, replacing an hydrophobic methioninyl residue with a lysyl residue reduced the melting temperature of (rHMfB)2 by〜15℃, and introducing the reciprocal change into (rHFoB)2, a dimer of the ancestrally homologous histone from the mesophilic species Methanobacterium formicicum, resulted in an〜15℃ increase in melting temperature.
1. Grayling, R.A., Sandman, K. and Reeve, J.N. (1996) Histones and chromatin structure in hyperthermophile Archaea. FEMS Microbiol. Rev., 18,203-213.
2. Starich, M.R., Sandman, K., Reeve, J.N. and Summers, M.F.(1996) NMR structure of HMfB from the hyperthermophile Methanothermus fervidus, confirms that this archaeal protein is a histone. J. Mol. Biol. 255,187-203.
3. Luger, K., Mader, A.W., Richmond, R.K., Sargent, D.F., and Richmond, T.J. 1997. Crystal structure of the nucleosome core particle at 2.8A resolution. Nature, 389, 251-260.
