Mutational analysis of archaeal histone-DNA interactions

Site-specific mutagenesis of the hmfB gene cloned from the archaeon Methano thermus fervidus, followed by expression in Escherichia coli, has been used to generate similar to 90 recombinant (r) variants of the archaeal histone HMfB. The abilities of these variants to form stable archaeal nucleosome-c ontaining complexes with linear pBR322 DNA, and with an 89 bp restriction f ragment of this DNA have been determined. Variants that failed to form such complexes, based on negative gel-shift assays, had substitutions at the N terminus or within the alpha 1, L1 and L2 regions of the rHMfB histone fold , at sites predicted to be homologous to eucaryal histone fold residues tha t contact the DNA in the eucaryal nucleosome. Variants that failed to give gel shifts were further assayed for their abilities to facilitate ligase-ca talyzed circularization of a linear 88 bp DNA molecule, and to reduce the e llipticity of a DNA solution at 275 nm (theta(275)). Consistent with cooper ative but independent sites of DNA binding, a combination of three residue substitutions, one each in alpha 1, L1 and L2, was required to generate a r HMfB variant with no detectable DNA binding based on gel shift, circulariza tion and theta(275) reduction assays. (C) 2000 Academic Press.