Characterization of Escherichia coli DnaAcos protein in replication systems reconstituted with highly purified proteins

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Abstract
Excessive initiation of chromosomal replication occurs in the dnaAcos mutant at 30 degrees C. Whereas purified wild-type DnA protein binds ATP and ADP tightly, DnaAcos protein is defective for such nucleotide binding. As initiation is a multistep reaction and DnaA protein functions at each step, activities of DnaAcos protein need to be examined precisely. DnaAcos protein specifically bound a DNA fragment containing the chromosomal replication origin with an affinity similar to that seen 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 DnA-binding hairpin structure. Thus, DnaAcos protein basically sustains affinity to a DnaA-binding sequence and functions in the loading of DnaB helicase onto single-stranded DNA. Thermal stabilities of wild-type DnA and DnaAcos activities were comparable. Unlike wild-type DnaA protein, DnaAcos protein was inactive for minichromosomal replication in systems reconstituted with purified proteins in which the ATP-bound form of DnaA protein is required for initiation. Taken together, the data indicate that the prominent defect in DnaAcos protein appears to be the inability to bind nucleotide.

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