Architecture of the Streptomyces lividans DnaA protein-replication origin complexes

The Streptomyces oriC region contains two clusters of 19 DnaA boxes separat ed by a spacer (134 bp). The Streptomyces DnaA protein consists, like all o ther DnaA proteins, of four domains: domain III and the carboxyterminal par t (domain IV) are responsible for binding of ATP and DNA, respectively. Bin ding of the DnaA protein to the entire oriC region analysed by electron mic roscopy showed that the DnaA. protein forms separate complexes at each of t he clusters of DnaA boxes, but not at the spacer separating them. In vivo m utational analysis revealed that the number of DnaA boxes and the presence of the spacer linking both groups of DnaA boxes seem to be important for a functional Streptomyces origin. We suggest that the arrangement of DnaA box es allows the DNA-bound DnaA protein to induce bending and looping of the o riC region. As it was shown by electrophoretic mobility shift assay and "on e hybrid system", two domains, I and III, facilitate interactions between D naA molecules. We postulate that domain I and domain III could be involved in cooperativity at distant and at closely spaced DnaA boxes, respectively. The long domain II extends the range over which N termini (domain I) of DN A-bound DnaA protein can form dimers. Thus, interactions between DnaA molec ules may bring two clusters of DnaA boxes separated by the spacer into func tional contact by loop formation. Removal of the spacer region or deletion of domains I and II resulted, respectively, in nucleoprotein complexes whic h are not fully developed, or huge nucleoprotein aggregates. (C) 2000 Acade mic Press.