J. Majka et al., Sequence recognition, cooperative interaction, and dimerization of the initiator protein DnaA of Streptomyces, J BIOL CHEM, 276(9), 2001, pp. 6243-6252
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.