Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2002 Sep 16;21(18):4763-73.
doi: 10.1093/emboj/cdf496.

The Structure of Bacterial DnaA: Implications for General Mechanisms Underlying DNA Replication Initiation

Affiliations
Free PMC article

The Structure of Bacterial DnaA: Implications for General Mechanisms Underlying DNA Replication Initiation

Jan P Erzberger et al. EMBO J. .
Free PMC article

Abstract

The initiation of DNA replication is a key event in the cell cycle of all organisms. In bacteria, replication initiation occurs at specific origin sequences that are recognized and processed by an oligomeric complex of the initiator protein DnaA. We have determined the structure of the conserved core of the Aquifex aeolicus DnaA protein to 2.7 A resolution. The protein comprises an AAA+ nucleotide-binding fold linked through a long, helical connector to an all-helical DNA-binding domain. The structure serves as a template for understanding the physical consequences of a variety of DnaA mutations, and conserved motifs in the protein suggest how two critical aspects of origin processing, DNA binding and homo-oligomerization, are mediated. The spatial arrangement of these motifs in DnaA is similar to that of the eukaryotic-like archaeal replication initiation factor Cdc6/Orc1, demonstrating that mechanistic elements of origin processing may be conserved across bacterial, archaeal and eukaryotic domains of life.

Figures

None
Fig. 1. DnaA domain organization and secondary structure. (A) Comparison of the domain organization of E.coli and A.aeolicus DnaA proteins. Domains are numbered according to earlier boundary predictions (Schaper and Messer, 1997). The boundary between domains III and IV has been adjusted using the DnaA structure. Black arrows define the extent of the conserved DnaA core region described in this study that spans the C-terminal 324 residues of A.aeolicus DnaA. There is 35% identity and 65% similarity between A.aeolicus DnaA and the E.coli ortholog across this region. Full-length A.aeolicus DnaA lacks domain II and has a poorly conserved domain I (15% identity compared with E.coli). (B) Sequence alignment and secondary structure elements of the conserved core of A.aeolicus and E.coli DnaA. Aquifex aeolicus residue numbers and their E.coli equivalents (in parentheses) are indicated. Secondary structure elements are in excellent agreement with previously published predictions (Messer et al., 1999). The coloring of the boxes above the residues reflects the degree of conservation at each position among all 67 currently available DnaA sequences, as determined by Clustal X Q scores, with dark blue representing high conservation and lighter blue shadings indicating progressively less conservation. Residues with a Q score <0.5 remain uncolored (see Materials and methods). In addition, invariant residues are highlighted in magenta and residues with strong chemical conservation in pink. Black bars indicate conserved sequence motifs: A, Walker A; B, Walker B; I, Sensor I; II, Sensor II; B-loop, DnaA basic loop; A-sig, DnaA signature sequence.
None
Fig. 2. Structure of DnaA. (A) Stereo RIBBONS diagram of the DnaA model. Domains are colored as follows: IIIa, green; IIIb, red; IV, gold. The bound nucleotide is shown as a ball-and-stick model. Key nucleotide-binding motifs and the HTH motif of domain IV are labeled. (B) Stereo RIBBONS diagram of the nucleotide binding cleft of DnaA. Residues located within 4 Å of the bound nucleotide and the coordinated Mg2+ ion are labeled and shown as ball-and-stick models.
None
Fig. 3. (A) RIBBONS diagram highlighting the position of E.coli DnaA mutations mapped to the A.aeolicus DnaA model (blue spheres). Identification and phenotype of mutations can be found in Table II. The shaded oval (gray) marks the location of the α12 hinge region reported to play a role in cardiolipin-mediated effects. (B) RIBBONS diagrams of DnaA domain IV and trp repressor DNA-binding domain/DNA complex (Otwinowski et al., 1988) highlighting the closely related HTH motif in gold. Highly conserved residues in the basic loop and the DnaA signature sequences are indicated by spheres. Residues determined by mutagenesis to be critical for DNA binding in E.coli that map to the HTH and the basic loop motifs are highlighted in red. (C) RIBBONS model of the DnaA–DNA complex based on the trp repressor/DNA complex. The positions of the signature sequence motif and the basic loop are indicated.
None
Fig. 4. (A) RIBBONS diagram of DnaA and p97 (residues 191–458) showing the high degree of structural conservation across the AAA+ domain of the two proteins (the r.m.s.d. between the two proteins is 3.2 Å over 192 Cα positions). (B) RIBBONS diagram of the AAA+ region of a p97 dimer excised from the hexameric structure (inset) (Zhang et al., 2000) depicting the typical oligomeric arrangement of AAA+ protomers. The ADP bound at the interface is shown in black. The helix containing the Box VII motif is shown in cyan and the key arginine residue present at the dimerization interface is depicted as a magenta ball-and-stick model. (C) Inset: surface depiction (Nicholls et al., 1991) of the DnaA dimer modeled on p97, showing the high degree of complementarity between the monomers. The structural alignment of the AAA+ regions was generated using least-squares fitting of the DnaA AAA+ domain on each of two neighboring p97 AAA+ domains from the p97 hexamer. The exploded view reveals the clustering of conserved residues at the dimerization interface to form the bipartite nucleotide-interaction site. The degree of conservation among all known DnaA sequences is indicated by the degree of blue shading (Figure 1B); invariant (magenta) and chemically conserved residues (pink) are also highlighted. (D) RIBBONS diagram of the DnaA AAA+ domain (residues D77S130–G290N348) model dimer shown in blue and gold. Critical elements present at the predicted dimer interface are highlighted as in Figure 4B.
None
Fig. 5. Model for DnaA assembly on oriC. (Top) oriC is recognized by DnaA (red, green and yellow) and architectural factors such as IHF and HU (purple). (Middle) Concomitant with oriC binding, the AAA+ domains oligomerize, stabilizing the nucleoprotein complex through intermonomer contacts around the ATP-binding site. Additional stability may be provided by domain I self-oligomerization (light blue). (Bottom) Self-assembly of DnaA molecules eventually leads to formation of the complete nucleoprotein complex. Note that the DnaA oligomer could conceivably accommodate either a closed ring (left) or a helical filament (right) arrangement of monomers. DUE opening may occur spontaneously through local strain induced by assembly of the nucleoprotein complex in the presence of ATP.
None
Fig. 6. Structural comparison of DnaA and Cdc6/Orc1. (A) RIBBONS diagrams of DnaA and its closest structural homolog, Cdc6/Orc1. Domains are labeled as in Figure 2A, except that the HTH motifs are highlighted in yellow. (B) RIBBONS diagram superimposing the AAA+ regions of DnaA (light gray) and Cdc6/Orc1 (pale yellow). Elements critical for the spatial conservation of the bipartite ATP-interaction cleft are highlighted in magenta (DnaA) and blue (Cdc6/Orc1). The two proteins exhibit an r.m.s.d. of 2.3 Å across 139 of the 213 Cα residues that are present in core secondary structure elements. (C) Comparisons of DnaA and Cdc6/Orc1 OLEs. The RIBBONS diagram shows the related HTH topology (in yellow) of the C-terminal modules next to secondary structure topology diagrams that indicate how the HTH motif is anchored within each C-terminal fold. Secondary structure comparisons were performed using the sequential structure alignment program SSAP (Orengo and Taylor, 1996).

Similar articles

See all similar articles

Cited by 93 articles

See all "Cited by" articles

Publication types

Associated data

LinkOut - more resources

Feedback