RESUMO
The heterodimeric HU protein was isolated from Escherichia coli as one of the most abundant DNA binding proteins associated with the bacterial nucleoid. HUalphabeta is composed of two very homologous subunits, but HU can also be present in E. coli under its two homodimeric forms, HUalpha(2) and HUbeta(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 recognize 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 structures could explain, in part at least, its pleiotropic role in the bacterial cell. To understand all the facets of its interactions with nucleic acids, it was necessary to develop a procedure which allowed the purification 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 procedure as well as demonstrating that the three histidine-tagged HUs we have produced, have conserved the binding characteristics of native HUs. Interestingly, by two complementation tests, we show that the histidine-tagged HUs are fully active in vivo.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/isolamento & purificação , Escherichia coli/metabolismo , Proteínas de Bactérias/genética , Cromatografia de Afinidade , Proteínas de Ligação a DNA/genética , Dimerização , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Teste de Complementação Genética , Peso Molecular , Subunidades Proteicas , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/isolamento & purificaçãoRESUMO
We have shown recently that the relative abundance of the three dimeric forms (alpha2, alphabeta and beta2) of the HU protein from Escherichia coli varies during growth and in response to environmental changes. Using gel retardation assays we have compared the DNA binding properties of the three dimers with different DNA substrates. The determination of their DNA binding parameters shows that the relative affinities of HUalphabeta and HUalpha2 are comparable. Both recognize, with a high degree of affinity under stringent conditions, cruciform structures or DNA molecules with a nick or a gap, whereas they bind to linear DNA only at low salt. DNA containing a gap of two nucleotides is in fact the substrate recognized with the highest degree of affinity by these two forms under all conditions. Conversely, HUbeta2 binds very poorly to duplex DNA and shows a much lower affinity for nicked or gapped DNAs. However, HUbeta2 binds to cruciform DNA structures almost as well as HUalphabeta and HUalpha2. This almost exclusive binding of HUbeta2 to a unique substrate is surprising in regards of the quasi identity, in the three forms, of the flexible arms considered as the DNA-binding domains of the three forms of HU. Cruciform DNA may stabilize HUbeta2 structure which could be structurally defective.
