Molecular mechanism of DNA replication‐coupled inactivation of the initiator protein in Escherichia coli: interaction of DnaA with the sliding clamp‐loaded DNA and the sliding clamp‐Hda complex

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Abstract
In Escherichia coli, the ATP‐DnaA protein initiates chromosomal replication. After the DNA polymerase III holoenzyme is loaded on to DNA, DnaA‐bound ATP is hydrolysed in a manner depending on Hda protein and the DNA‐loaded form of the DNA polymerase III sliding clamp subunit, which yields ADP‐DnaA, an inactivated form for initiation. This regulatory DnaA‐inactivation represses extra initiation events. In this study, in vitro replication intermediates and structured DNA mimicking replicational intermediates were first used to identify structural prerequisites in the process of DnaA‐ATP hydrolysis. Unlike duplex DNA loaded with sliding clamps, primer RNA‐DNA heteroduplexes loaded with clamps were not associated with DnaA‐ATP hydrolysis, and duplex DNA provided in trans did not rescue this defect. At least 40‐bp duplex DNA is competent for the DnaA‐ATP hydrolysis when a single clamp was loaded. The DnaA‐ATP hydrolysis was inhibited when ATP‐DnaA was tightly bound to a DnaA box‐bearing oligonucleotide. These results imply that the DnaA‐ATP hydrolysis involves the direct interaction of ATP‐DnaA with duplex DNA flanking the sliding clamp. Furthermore, Hda protein formed a stable complex with the sliding clamp. Based on these, we suggest a mechanical basis in the DnaA‐inactivation that ATP‐DnaA interacts with the Hda‐clamp complex with the aid of DNA binding.