Overproduction and improved strategies to purify the three native forms ofnuclease-free HU protein

The heterodimeric HU protein was isolated from Escherichia coli as one of t he most abundant DNA binding proteins associated with the bacterial nucleoi d. HU alpha beta is composed of two very homologous subunits, but HU can al so be present in E. coli under its two homodimeric forms, HU alpha(2) and H U beta(2). This protein is conserved either in its heterodimeric form or in one of its homodimeric forms in all bacteria, in plant chloroplasts and in some viruses. HU can participate, like the histones, in the maintenance of DNA supercoiling and in DNA condensation. This protein which does not reco gnize any specific sequence on double-stranded DNA, has been shown to bind specifically to cruciform DNA as does the eukaryotic HMG1 protein and to a series of structures which are found as intermediates of DNA repair, e.g., nick, gap, 3'overhang, etc. The strong binding of HU to these diverse DNA s tructures could explain, in part at least, its pleiotropic role in the bact erial cell. To understand all the facets of its interactions with nucleic a cids, it was necessary to develop a procedure which allowed the purificatio n of the three forms of HU under their native form and without the nuclease activity strongly associated with the protein. We describe here such a pro cedure as well as demonstrating that the three histidine-tagged HUs we have produced, have conserved the binding characteristics of native HUs. Intere stingly, by two complementation tests, we show that the histidine-tagged HU s are fully active in vivo. (C) 2000 Societe francaise de biochimie et biol ogie moleculaire / Editions scientifiques et medicales Elsevier SAS.