Sequence recognition, cooperative interaction, and dimerization of the initiator protein DnaA of Streptomyces

Using a combined PCR-gel retardation assay, the preferred recognition seque nce of the Streptomyces initiator protein DnaA was determined. The protein showed a preference toward DNA containing two Escherichia coli-like DnaA bo xes in a head-to-head arrangement (consensus sequence TTATCCACA, whereas th e consensus sequence of the DnaA boxes found in the Streptomyces oriC regio n is TTGTCCACA). In quantitative band shift experiments, the kinetics of th e Streptomyces DnaA-DnaA box interaction was characterized. The DnaA protei n can form dimers while binding to a single DnaA box; dimer formation is me diated by the domain III of the protein, and the dissociation constant of t his process was between 35 and 115 nM. Streptomyces initiator protein DnaA interacts in a cooperative manner with DNA containing multiple binding site s. For the cooperativity effect, which seems to be independent of the dista nce separating the DnaA boxes, domain I (or I and II) is responsible. The c ooperativity constant is moderate and is in the range of 20-110.