Streptomycetes differ from other prokaryotic organisms in their mycelial life cycle (3) and in possessing a linear, GC-rich chromosome (8 Mb) (4, 17-19) which is nearly twice as large as the one from Escherichia coli. Central to the processes regulating prokaryotic DNA replication are the events that occur at the chromosomal replication origin, oriC (32). The initiator protein DnaA plays a key role in the initiation of DNA replication in bacteria (for reviews, see references 21 and 30). Prokaryotic chromosomal replication is best understood for E. coli, in which it has been demonstrated that the DnaA protein (52 kDa) binds ATP and interacts specifically with the replication origin. The DnaA protein has the highest affinity for the DnaA box motifs with the asymmetric consensus sequence 5Ј-TT(A /T)TNCACA (28). About 10 to 20 DnaA molecules interact with five DnaA boxes located within the E. coli oriC region, resulting in the formation of the initial complex (16,21). This interaction causes a local unwinding within AT-rich motifs, creating an open complex (1, 10). The unwound region then serves as an entry site for helicase (DnaB/DnaC) followed by primase (DnaG) (16).The chromosomal replication origin (oriC) of Streptomyces lividans was identified as an autonomously replicating fragment of approximately 0.7 kb (36, 38). It carries 17 DnaA boxes which are arranged in two clusters. Replication origins that contain clusters with multiple DnaA boxes have also been found in other organisms (34,35). Recently, dnaA genes from several bacteria have been cloned and sequenced. Comparisons of the corresponding deduced proteins (21, 25, 30) resulted in the identification of four domains: (i) a short Nterminal part, (ii) a second domain, which varies in length and amino acid composition, (iii) the highly conserved domain III with an ATP binding motif, and (iv) the C-terminal DNA binding domain IV. The DNA binding domain (94 amino acids) of E. coli contains two potential amphipathic ␣-helices (A and B) and a third ␣-helix (C). The helices A and B might form a helix-loop-helix (HLH) DNA binding motif (25). It is interesting that the DnaA protein of S. lividans (2, 38) is significantly larger than the corresponding proteins from E. coli (11), Bacillus subtilis (9), and several other bacteria (30).In light of the recent discoveries that the Streptomyces oriC region is centrally located in the linear chromosome (4, 23) and shows higher complexity than the oriC regions of unicellular bacteria (36), it is interesting to gain a better understanding of the initiation of Streptomyces DNA replication.In this study we describe the purification of the S. lividans DnaA protein and analyze its interaction with the DnaA box motifs located within the oriC region. Moreover, by mutational analysis we have also characterized two domains from S. lividans DnaA.Overproduction and purification of the Streptomyces lividans DnaA fusion protein. To characterize the interaction of the S. lividans DnaA protein in detail, it was produced as a Histagged prot...