Thermostable and site-specific DNA binding of the gene product ORF56 from the Sulfolobus islandicus plasmid pRN1, a putative archael plasmid copy control protein

There is still a lack of information on the specific characteristics of DNA -binding proteins from hyperthermophiles, Here we report on the product of the gene orf56 from plasmid pRN1 of the acidophilic and thermophilic archae on Sulfolobus islandicus. orf56 has not been characterised yet but low sequ ence similarity to-several eubacterial plasmid-encoded genes suggests that this 6.5 kDa protein is a sequence-specific DNA-binding protein. The DNA-bi nding properties of ORF56, expressed in Escherichia coli, have been investi gated by EMSA experiments and by fluorescence anisotropy measurements. Reco mbinant ORF56 binds to double-stranded DNA, specifically to an inverted rep eat located within the promoter of orf56, Binding to this site could downre gulate transcription of the orf56 gene and also of the overlapping orf904 g ene, encoding the putative initiator protein of plasmid replication, By gel filtration and chemical crosslinking we have shown that ORF56 isa:dimeric protein, Stoichiometric fluorescence anisotropy :titrations further indicat e that ORF56 binds as a tetramer to the inverted repeat of its target bindi ng site, CD spectroscopy points to a significant increase in ordered second ary structure of ORF56 upon binding DNA, ORF56 binds without apparent coope rativity: to its target DNA with a dissociation constant in the nanomolar r ange. Quantitative analysis of binding isotherms performed at various salt concentrations and at different temperatures indicates that approximately s even ions are released upon complex formation and that complex formation is accompanied by a change in heat capacity of -6.2 kJ/mol. Furthermore,-:rec ombinant ORF56 proved to be highly thermostable and is able to bind DNA up to 85 degreesC.