Identification of the chromosomal replication origin from Thermus thermophilus and its interaction with the replication initiator DnaA.
The chromosomal replication origin oriC and the gene encoding the replication initiator protein DnaA from Thermus thermophilus have been identified and cloned into an Escherichia coli vector system. The replication origin is composed of 13 characteristically arranged DnaA boxes, binding sites for the DnaA protein, and an AT-rich stretch, followed by the dnaN gene. The dnaA gene is located upstream of the origin and expresses a typical DnaA protein that follows the division into four domains, as with other members of the DnaA protein family. Here, we report the purification of Thermus-DnaA (Tth-DnaA) and characterize the interaction of the purified protein with the replication origin, with regard to the binding kinetics and stoichiometry of this interaction. Using gel retardation assays, surface plasmon resonance (SPR) and electron microscopy, we show that, unlike the E. coli DnaA, Tth-DnaA does not recognize a single DnaA box, instead a cluster of three tandemly repeated DnaA boxes is the minimal requirement for specific binding. The highest binding affinities are observed with full-length oriC or six clustered, tandemly repeated DnaA boxes. Furthermore, high-affinity DNA-binding of Tth-DnaA is dependent on the presence of ATP. The Thermus DnaA/oriC interaction will be compared with oriC complex formation generated by other DnaA proteins.
Schaper
S
S
Max-Planck-Institut für molekulare Genetik, Ihnestrasse 73, Berlin, D-14195, Germany.
Nardmann
J
J
Lüder
G
G
Lurz
R
R
Speck
C
C
Messer
W
W
D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000648:ultrastructure; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D001483:Base Sequence; D001665:Binding Sites; D002876:Chromosomes, Bacterial / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D005798:Genes, Bacterial / Q000235:genetics; D006868:Hydrolysis; D007700:Kinetics; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018741:Replication Origin / Q000235:genetics*; D020349:Surface Plasmon Resonance; D020080:Tandem Repeat Sequences / Q000235:genetics; D013816:Thermodynamics; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases