Abstract
Escherichia coli DnaA protein, a member of the AAA+ superfamily, initiates replication from the chromosomal origin oriC in an ATP-dependent manner. Nucleoprotein complex formed on oriC with the ATP-DnaA multimer but not the ADP-DnaA multimer is competent to unwind the oriC duplex. The oriC region contains ATP-DnaA-specific binding sites termed I2 and I3, which stimulate ATP-DnaA-dependent oriC unwinding. In this study, we show that the DnaA R285A mutant is inactive for oriC replication in vivo and in vitro and that the mutation is associated with specific defects in oriC unwinding. In contrast, activities of DnaA R285A are sustained in binding to the typical DnaA boxes and to ATP and ADP, formation of multimeric complexes on oriC, and loading of the DnaB helicase onto single-stranded DNA. Footprint analysis of the DnaA-oriC complex reveals that the ATP form of DnaA R285A does not interact with ATP-DnaA-specific binding sites such as the I sites. A subgroup of DnaA molecules in the oriC complex must contain the Arg-285 residue for initiation. Sequence and structural analyses suggest that the DnaA Arg-285 residue is an arginine finger, an AAA+ family-specific motif that recognizes ATP bound to an adjacent subunit in a multimeric complex. In the context of these and previous results, the DnaA Arg-285 residue is proposed to play a unique role in the ATP-dependent conformational activation of an initial complex by recognizing ATP bound to DnaA and by modulating the structure of the DnaA multimer to allow interaction with ATP-DnaA-specific binding sites in the complex.
Highlights
DnaA protein plays an essential role in the initiation of Escherichia coli chromosomal replication [1,2,3,4]
The pol III holoenzyme consists of the  clamp subunit, which is directly loaded onto the primed site, and the pol III* subassembly, which binds to the  clamp
We identified a DnaA amino acid residue that plays a specific role in an ATP-specific conformational change in the initiation complex
Summary
DnaA protein plays an essential role in the initiation of Escherichia coli chromosomal replication [1,2,3,4]. When the ATP-bound form of DnaA is included in the initiation complex, a region within oriC containing AT-rich 13-mers is unwound, resulting in open complex formation. Speck and Messer [14] reported that ATP-DnaA, but not ADP-DnaA, binds with low affinity to specific sites located in a region that includes the AT-rich 13-mers. They termed these sites ATP-DnaA boxes, with the consensus sequence AG(A)(T)(C)(T). Leonard and co-workers [15] recently reported that specific sites, termed I2 and I3, bind ATP-DnaA, but not ADP-DnaA, with low affinity These sites are located between the DnaA M and R4 boxes and stimulate the formation of an open complex. Analyses of AAAϩ protein sequences and of the crystal structure of domains III–IV of the hyperthermophilic bacterium Aquifex aeolicus DnaA homolog suggest that arginine 281 or 285 of E. coli DnaA corresponds to the arginine finger [23, 24, 28]
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