Purification and characterization of the Streptomyces lividans initiator protein DnaA

Abstract

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...

Sequence Recognition, Cooperative Interaction, and Dimerization of the Initiator Protein DnaA of Streptomyces

“…Proteins and DNA-The His-tagged wild type DnaA proteins of S. lividans and its truncated mutants DnaA(III-IV) comprising the domain III and the DNA binding domain were purified on a Ni 2ϩ -nitrilotriacetic acid-agarose column (Qiagen) as described earlier (15). The DNA binding domain of the DnaA protein DnaA(BD) was expressed as a C-terminal glutathione S-transferase fusion and purified using glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech) followed by factor X a cleavage as described earlier (16).…”

“…A DNase I footprint of the oriC region with DnaA showed protection of all boxes. However, when analyzed individually by surface plasmon resonance (SPR), only some DnaA boxes were specifically recognized by the DnaA protein (14,15). To characterize in detail the recognition properties of the Streptomyces DnaA protein, we applied a binding site selection technique based on the combined EMSA and polymerase chain reactions ( Fig.…”

“…In contrast to the other bacteria, the Streptomyces DnaA protein is larger (70 -73 kDa), since it comprises an additional stretch (ϳ230 amino acids) of predominantly acidic or hydrophobic amino acids within domain II. The residues lower the isoelectric point of the entire Streptomyces DnaA protein (pI ϭ 5.7) (15,16). As it was shown for the E. coli and Bacillus subtilis DnaA proteins, domain III and the C-terminal part (domain IV) of the Streptomyces DnaA protein are responsible for binding of ATP and DNA, respectively (15,17).…”

“…The residues lower the isoelectric point of the entire Streptomyces DnaA protein (pI ϭ 5.7) (15,16). As it was shown for the E. coli and Bacillus subtilis DnaA proteins, domain III and the C-terminal part (domain IV) of the Streptomyces DnaA protein are responsible for binding of ATP and DNA, respectively (15,17). The consensus sequence of the Streptomyces DnaA box in oriC is 5Ј-TTGTCCACA-3Ј, which differs at the third position (AЈG) from the E. coli DnaA box (13).…”

Characterization of the mycobacterial chromosome segregation protein ParB and identification of its target in Mycobacterium smegmatis

“…All the PCR-derived clones were analysed by DNA sequencing to check their fidelity. The fusion proteins, glutathione S-transferase (GST)-ParA or 6His-ParB were purified on a glutathione Sepharose 4B or on a Ni 2+ -NTA-agarose column (Qiagen), respectively, as described previously (Majka et al, 1999;Zawilak et al, 2004). For removal of the GST part, the bound fusion proteins were treated with the PreScission protease (Amersham-Pharmacia Biotech) and the ParA proteins were released from the beads.…”