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. 2003 Apr 15;31(8):2077-86.
doi: 10.1093/nar/gkg309.

Structural Basis of Replication Origin Recognition by the DnaA Protein

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Structural Basis of Replication Origin Recognition by the DnaA Protein

Norie Fujikawa et al. Nucleic Acids Res. .
Free PMC article

Abstract

Escherichia coli DnaA binds to 9 bp sequences (DnaA boxes) in the replication origin, oriC, to form a complex initiating chromosomal DNA replication. In the present study, we determined the crystal structure of its DNA-binding domain (domain IV) complexed with a DnaA box at 2.1 A resolution. DnaA domain IV contains a helix-turn-helix motif for DNA binding. One helix and a loop of the helix- turn-helix motif are inserted into the major groove and 5 bp (3' two-thirds of the DnaA box sequence) are recognized through base-specific hydrogen bonds and van der Waals contacts with the C5-methyl groups of thymines. In the minor groove, Arg399, located in the loop adjacent to the motif, recognizes three more base pairs (5' one-third of the DnaA box sequence) by base-specific hydrogen bonds. DNA bending by approximately 28 degrees was also observed in the complex. These base-specific interactions explain how DnaA exhibits higher affinity for the strong DnaA boxes (R1, R2 and R4) than the weak DnaA boxes (R3 and M) in the replication origin.

Figures

Figure 1
Figure 1
(Opposite) Crystal structure of DnaA domain IV complexed with DnaA box DNA. (A) Alignment of the DnaA box sequences, R1, R2, R3, R4 and M, in E.coli oriC. The bottom row shows the DnaA box sequence found in Streptomyces lividans. Numbers indicate base positions from the 5′ end of the top strand. Capital letters indicate the consensus sequence of the DnaA boxes and small letters indicate the two flanking bases of the DnaA boxes. Red letters indicate bases that are not conserved among those in the five DnaA box sequences. The box shows position 7, which is not recognized by DnaA domain IV in the crystal structure. (B) The DNA containing the R1 sequence of the DnaA box used in the structural analysis of the DnaA domain IV–DNA complex. The box indicates the DnaA box consensus sequence. Numbers without asterisks indicate base positions from the 5′ end of the top strand, and numbers with asterisks indicate base positions from the 3′ end of the bottom strand. (C) The secondary structure of the E.coli DnaA domain IV is presented in the top row and that of the A.aeolicus DnaA domain IV is presented in the bottom row. The amino acid sequence of the A.aeolicus DnaA domain IV is aligned to the E.coli DnaA domain IV. Conserved amino acid residues are indicated by red letters. (D) Overall structure of the DnaA domain IV–DNA complex. Three views from different angles are shown. The colors of the protein and the DNA strands correspond to those in (B) and (C). (E) The superimposed model of the E.coli DnaA domain IV and the A.aeolicus DnaA domain III/IV structure (17). The E.coli domain IV in the complex with DNA is colored orange. The A.aeolicus DnaA domains IIIa, IIIb and IV are colored blue, pink and red, respectively.
Figure 2
Figure 2
Schematic diagram summarizing the DNA contacts by DnaA domain IV. The essential base pairs in the DnaA box consensus sequence are colored yellow. Water molecules are denoted as light blue spheres. Open circles represent phosphate groups. Base-specific hydrogen bonds are indicated with red lines and van der Waals contacts with the C5-methyl groups of thymines are indicated by blue lines. Hydrogen bonds and salt bridges with the backbone phosphate groups are indicated with black lines. The colors and the numbers of the DNA strands correspond to those in Figure 1.
Figure 3
Figure 3
Stereo views of the interactions in the major groove. The DNA strands shown in green and yellow correspond to those in Figure 1. (A) Base-specific interactions. Dotted lines in red represent hydrogen bonds and those in blue represent van der Waals interactions. Waters are represented by light blue spheres. Side chains forming hydrogen bonds are colored purple and those forming van der Waals bonds are colored red. (B) Interactions with the backbone phosphate groups. Dotted lines in black represent hydrogen bonds and those in blue represent water-mediated hydrogen bonds. Waters are represented by light blue spheres. Side chains forming hydrogen bonds are colored purple.
Figure 4
Figure 4
Stereo views of the interactions in the minor groove. The DNA strands shown in green and yellow correspond to those in Figure 1. (A) Base-specific interactions. Dotted lines in red represent hydrogen bonds. Waters are represented by light blue spheres. The Arg399 side chain forming hydrogen bonds is colored purple. (B) Interactions with the backbone phosphate groups. Dotted lines in black represent hydrogen bonds and those in blue represent water-mediated hydrogen bonds. Waters are represented by light blue spheres. Side chains forming hydrogen bonds are colored purple.
Figure 5
Figure 5
Structural comparisons of the DnaA domain IV–DNA complex (A) with the complexes of the Thermus aquaticus σA protein (B) (34), the human CENP-B domain 1 (C) (35) and the E.coli trp repressor (TrpR) (D) (33). The local DNA axis was calculated by the program CURVES (43) and is shown as a red line.
Figure 6
Figure 6
(Next page) The base-specific interactions with His434 and Arg399. (A) Stereo view of the (2|Fo| – |Fc|) electron density map for His434 and C10:G10*. The hydrogen bond between the His434 Nε and G10* O6 atoms is indicated by a dotted red line and those between the C:G base pair are indicated by dotted yellow lines. (B) Stereo view of a model for the interaction between the His434 Nε and thymine O4 atoms in the R3 sequence. The A:T base pair (colored blue) of position 10 in the R3 sequence is superimposed on the C10:G10* base pair (colored red) of the R1 sequence. (C) Stereo view of the (2|Fo| – |Fc|) electron density map for Arg399 and T4:A4*. The hydrogen bonds between the Arg399 Nη and T4 O2 or A4* N3 atoms are indicated by a dotted red line and those between the T:A base pair are indicated by dotted yellow lines. (D) Stereo view of a model for the interaction between the Arg399 Nη and cytosine O2 atoms in the M sequence. The C:G base pair (colored blue) of position 4 in the M sequence is superimposed on the T4:A4* base pair (colored red) of the R1 sequence. The van der Waals radii of the oxygen (1.4 Å) of the water molecule and the NH2 group (1.65 Å) are indicated in light blue and green circles, respectively.

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