CHARACTERIZATION OF ESCHERICHIA-COLI DNAACOS PROTEIN IN REPLICATION SYSTEMS RECONSTITUTED WITH HIGHLY PURIFIED PROTEINS

Excessive initiation of chromosomal replication occurs in the dnaAcos mutant at 30 degrees C. Whereas purified wild-type DnaA protein binds ATP and ADP tightly, DnaAcos protein is defective for such nucleotide binding. As initiation is a multistep reaction and DnaA protein functi ons at each step, activities of DnaAcos protein need to be examined pr ecisely. DnaAcos protein specifically bound a DNA fragment containing the chromosomal replication origin with an affinity similar to that se en with the wild-type protein. In a system reconstituted with purified proteins at 30 degrees C, the mutant protein initiated replication of single-stranded DNA that contains a DnaA-binding hairpin structure. T hus, DnaAcos protein basically sustains affinity to a DnaA-binding seq uence and functions in the loading of DnaB helicase onto single-strand ed DNA. Thermal stabilities of wild-type DnaA and DnaAcos activities w ere comparable. Unlike wild-type DnaA protein, DnaAcos protein was ina ctive for minichromosomal replication in systems reconstituted with pu rified proteins in which the ATP-bound form of DnaA protein is require d for initiation. Taken together, the data indicate that the prominent defect in DnaAcos protein appears to be the inability to bind nucleot ide.