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. 2005 Jul 15;389(Pt 2):471-81.
doi: 10.1042/BJ20050143.

Architecture of Bacterial Replication Initiation Complexes: Orisomes From Four Unrelated Bacteria

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Free PMC article

Architecture of Bacterial Replication Initiation Complexes: Orisomes From Four Unrelated Bacteria

Anna Zawilak-Pawlik et al. Biochem J. .
Free PMC article

Abstract

Bacterial chromosome replication is mediated by single initiator protein, DnaA, that interacts specifically with multiple DnaA boxes located within the origin (oriC). We compared the architecture of the DnaA-origin complexes of evolutionarily distantly related eubacteria: two Gram-negative organisms, Escherichia coli and Helicobacter pylori, and two Gram-positive organisms, Mycobacterium tuberculosis and Streptomyces coelicolor. Their origins vary in size (from approx. 200 to 1000 bp) and number of DnaA boxes (from 5 to 19). The results indicate that: (i) different DnaA proteins exhibit various affinities toward single DnaA boxes, (ii) spatial arrangement of two DnaA boxes is crucial for the H. pylori and S. coelicolor DnaA proteins, but not for E. coli and M. tuberculosis proteins, and (iii) the oriC regions are optimally adjusted to their cognate DnaA proteins. The primary functions of multiple DnaA boxes are to determine the positioning and order of assembly of the DnaA molecules. Gradual transition from the sequence-specific binding of the DnaA protein to binding through co-operative protein-protein interactions seems to be a common conserved strategy to generate oligomeric initiator complexes bound to multiple sites within the chromosomal, plasmid and virial origins.

Figures

Figure 1
Figure 1. Comparison of the oriC regions of the analysed bacteria
Genome size and GC content are given in parenthesis. For each analysed oriC region, its size and the consensus sequence for the DnaA box is presented. The insert presents features of the analysed DnaA proteins.
Figure 2
Figure 2. Interactions of DnaA proteins with a single DnaA box
(A) Gel retardation assay. The assay was performed using a 32P-labelled double-stranded oligonucleotide containing the single E. coli DnaA box (EcDnaAbox) (20 fmol). The DNA fragment was incubated with increasing amounts of H. pylori (tracks 2–8) or E. coli (tracks 9–14) DnaA. Tracks: 1, 0; 2 and 9, 1; 3, 5; 4 and 10, 10; 5 and 11, 25; 6 and 12, 50; 7 and 13, 100; 8 and 14, 250 nM DnaA. (B) SPR analysis. The top part shows the BIAcore sensorgram of the H. pylori or the E. coli DnaA protein binding to the E. coli DnaA box (EcDnaAbox). The biotinylated double-stranded oligonucleotide was immobilized on a streptavidin-coated chip of the BIAcore apparatus and then incubated with increasing amounts of DnaA protein. The concentrations of the DnaA protein (from bottom to top) were, for H. pylori, 1, 5, 10, 25, 50, 100 and 500 nM, and, for E. coli, 1.2–300 nM in 2-fold increments. The bottom part shows the affinity of the DnaA proteins toward different types of single DnaA box. Kd (app) values were determined by gel retardation assay and/or by SPR as described in detail in [28,34]. The M. tuberculosis and S. coelicolor DnaA boxes have the same consensus sequence (TTGTCCACA).
Figure 3
Figure 3. Interaction of DnaA proteins with two DnaA boxes
Gel retardation assay was performed using 32P-labelled double-stranded oligonucleotides containing two DnaA boxes (10 fmol): Hpboxes-wt, Hpboxes-rv, Hpboxes-wt+10bp, Scboxes-wt, Scboxes-rv and Scboxes-wt+10bp (Table 1). DNA fragments were incubated with increasing amounts of the H. pylori or S. coelicolor DnaA protein. The DNA–protein complexes were separated on a 6% polyacrylamide gel. The concentrations of the DnaA protein were, for H. pylori, 1.95–250 nM and, for S. coelicolor, 0.06–250 nM in 2-fold increments.
Figure 4
Figure 4. Interaction of DnaA proteins with oriC regions
Gel-retardation assay was pre-formed using 32P-labelled oriC fragments (2–5 fmol) that were incubated with increasing amounts of the H. pylori, E. coli, M. tuberculosis or S. coelicolor DnaA protein. The DNA–protein complexes were separated on a 4% polyacrylamide gel. The structure of the oriC regions (for details, see Figure 1) are shown above the gels. Tracks: 1, 0; 2, 1; 3, 2.5; 4, 5; 5, 10; 6, 25; 7, 50; 8, 100 nM DnaA. Kd (app) was determined using the method described by Carey [28] (see the Materials and methods section).
Figure 5
Figure 5. Competition gel retardation assay
All four 32P-labelled oriC fragments (∼2 fmol of each oriC fragment) were incubated together with increasing amounts of the H. pylori, E. coli, M. tuberculosis or S. coelicolor DnaA protein. Tracks: 1, 0; 2, 1; 3, 2.5; 4, 5; 5, 10; 6, 25; 7, 50; 8, 100 nM DnaA. The H. pylori oriC DNA fragment used for this analysis was longer (360 bp) than the oriC region of H. pylori (the fragment does not contain any DnaA box outside the oriC region, therefore the affinity of the HpDnaA protein towards the oriC region was not changed [20]).
Figure 6
Figure 6. Bending of the S. coelicolor oriC region by DnaA proteins – probing of DnaA–oriC complexes with SfiI
Plasmid pGEM®-T Easy-SfiIScoriCSfiI containing the S. coelicolor oriC region flanked by SfiI restriction sites was incubated with different amounts of the E. coli, the M. tuberculosis or the S. coelicolor DnaA protein and then subjected to SfiI enzyme cleavage. The SfiI enzyme digestion activity in the absence of DnaA protein was set at 100.
Figure 7
Figure 7. Comparison of the amino acid sequences of the DNA-binding domains of DnaA from H. pylori (Hp), E. coli (Ec), M. tuberculosis (Mt) and S. coelicolor (Sc)
(A) Protein alignment of DNA-binding domains. Grey bars above the sequence symbolize α-helices (numbered α1–α6, according to the crystal structure [16]). The positions of the HTH motif, the signature sequence motif and the basic loop are indicated [16]. Residues involved directly in DNA binding are underlined [16]. For Hp, Mt and Sc, residues identical with or similar to the E. coli residues involved directly in DNA binding are indicated respectively by bold or italic bold letters; non-closely related residues are encircled. (B) Schematic diagram summarizing the DNA (R1 DnaA box) contacts by the E. coli DNA-binding domain (according to Fujikawa et al. [16]). The essential base pairs in the R1 DnaA box are bold [16]. Similar and divergent (encircled) residues of other analysed organisms are indicated. (C) Comparison of affinities of the analysed proteins toward the E. coli R1 DnaA box. Dissociation constants (Kd) for the DnaA protein interaction with the E. coli R1 DnaA box were measured in gel retardation and SPR assays (for details see [34]). The left-hand part of the Table shows the number of amino acid substitutions in the fragment of the binding domain that is involved directly in DnaA box binding; residues involved directly in base recognition (base specific) and responsible for interaction with DNA backbone phosphate groups (phosph. group) are specified separately (see also B). C, conservative substitution; D, divergent substitution; nb, not bound.

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