Formation of an ATP-DnaA-specific Initiation Complex Requires DnaA Arginine 285, a Conserved Motif in the AAA+ Protein Family

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-DnaAspecific 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 ATPDnaA-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-DnaAspecific binding sites in the complex.DnaA protein plays an essential role in the initiation of Escherichia coli chromosomal replication (1-4). The protein forms a homomultimer on the replication origin oriC to form an initiation complex. When the ATP-bound form of DnaA is included in the initiation complex, a region within oriC containing AT-rich 13-mers is specifically unwound, resulting in open complex formation. ADP-DnaA forms a multimeric complex on oriC that does not promote open complex formation. On the exposed single-stranded (ss) 1 DNA of the 13-mer region, DnaB helicase is loaded via ordered interactions with the DnaC helicase loader and DnaA. DnaG primase forms a complex with DnaB that promotes DNA duplex unwinding and RNA primer synthesis, which allow loading of the DNA polymerase (pol) III holoenzyme. 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. Pol III* includes the clamp-loader ␥ complex and the pol III core complex, which contains the catalytic center of the polymerase.

Near-Atomic Structural Model for Bacterial DNA Replication Initiation Complex and its Functional Insights

“…The orientation of the remaining low-affinity site τ2 in the left half has yet to be examined because of sequence degeneracy (14,15). DnaA binding to the τ1 box, which is located at the left side of R5M, is observed with a low affinity only for a linear form of oriC but not for a replication-active supercoiled form (14,15); we thus excluded this box in this study. IHF is one of the bacterial architectural proteins and sharply bends dsDNA (20).…”

“…The ATP-bound AAA+ domain can form stable helical homooligomers by assembling in a head-to-tail manner (Fig. 1B, Bottom cartoon) (14,24). The arginine-finger in an interface (Arg-finger interface) of one AAA+ domain coordinates to the ATP molecule bound on another interface (ATPbound interface) of an adjacent AAA+ domain, which explains the nucleotide-dependent head-to-tail oligomer formation on oriC (Fig.…”

“…The six right boxes (R2, C3, C2, I3, C1, and R4) share the same orientation of the motifs, whereas the four motifs in the left half, i.e., R1, R5M, and I1-2, have a motif orientation opposite to that of the right half. The orientation of the remaining low-affinity site τ2 in the left half has yet to be examined because of sequence degeneracy (14,15). DnaA binding to the τ1 box, which is located at the left side of R5M, is observed with a low affinity only for a linear form of oriC but not for a replication-active supercoiled form (14, 15); we thus excluded this box in this study.…”

“…The 11 DnaA boxes (R1-2, R4, R5M, I1-3, C1-3, and τ2) have differing affinities to DnaA and motif orientations (indicated by triangles in Fig. 1A): The two terminal boxes R1 and R4 have especially high affinities (dissociation constants 1-6 nM for R1 and ∼1 nM for R4), whereas others have modest (R2) to low affinities (I1-3, C1-3, and R5M and τ2; dissociation constants for R5M are >200 nM) (12)(13)(14)(15)(16)(17)(18)(19). The 11 DnaA boxes have been divided into two groups on the left-and right-half oriC.…”

“…1A) (1)(2)(3)(4)(5)(11)(12)(13)(14)(15). The DnaA boxes contain 9-mer nucleotides (consensus sequence TTATNCACA, where N can be any base) (16).…”