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. 2010 Sep 3;285(36):28229-39.
doi: 10.1074/jbc.M110.147975. Epub 2010 Jul 1.

Origin Remodeling and Opening in Bacteria Rely on Distinct Assembly States of the DnaA Initiator

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Free PMC article

Origin Remodeling and Opening in Bacteria Rely on Distinct Assembly States of the DnaA Initiator

Karl E Duderstadt et al. J Biol Chem. .
Free PMC article

Abstract

The initiation of DNA replication requires the melting of chromosomal origins to provide a template for replisomal polymerases. In bacteria, the DnaA initiator plays a key role in this process, forming a large nucleoprotein complex that opens DNA through a complex and poorly understood mechanism. Using structure-guided mutagenesis, biochemical, and genetic approaches, we establish an unexpected link between the duplex DNA-binding domain of DnaA and the ability of the protein to both self-assemble and engage single-stranded DNA in an ATP-dependent manner. Intersubunit cross-talk between this domain and the DnaA ATPase region regulates this link and is required for both origin unwinding in vitro and initiator function in vivo. These findings indicate that DnaA utilizes at least two different oligomeric conformations for engaging single- and double-stranded DNA, and that these states play distinct roles in controlling the progression of initiation.

Figures

FIGURE 1.
FIGURE 1.
DnaA organization. A, domain configuration of E. coli and A. aeolicus DnaA. Key motifs are indicated. B, surface representation of 12 protomers of the AaDnaA assembly (Protein Data Bank code 2HCB) with the DBDs (310–399) colored either dark or light gray and the AAA+ domains (77–310) colored either dark blue or pale blue. Exposed interfaces on either end of the helical oligomer are color-coded by region.
FIGURE 2.
FIGURE 2.
Assembly and ssDNA binding of AaDnaA. A, glutaraldehyde-cross-linked samples of AaDnaA (13.2 pmol/reaction) bound to different nucleotides. B, comparison of cross-linked species from A. The fraction of monomer through tetramer obtained for each nucleotide is expressed as a ratio compared with ATP. The sum of all species from monomer to tetramer was normalized to 1.0 for each nucleotide tested before calculating the ratio. C, binding of fluorescein-tagged, single-stranded oligonucleotides (10 nm), monitored as a function of AaDnaA concentration by fluorescence polarization. ADP·BeF3 was present in all cases. Oligonucleotide sequences are listed in supplemental Table S3. D, fluorescence polarization measurements for the binding of F-dT25 (10 nm) by different AaDnaA concentrations in the presence of different nucleotides. For C and D, data are normalized so that the bottom and top plateaus are at zero and one, respectively.
FIGURE 3.
FIGURE 3.
Assembly and ssDNA binding of AaDnaA truncations. A, glutaraldehyde-cross-linking data for full-length and truncated forms of AaDnaA (13.2 pmol/reaction). Cross-linking time and nucleotide state are indicated. B, comparison of cross-linked species from A. The fraction of monomer through tetramer obtained for each construct is expressed as a ratio compared with wild-type (WT), ADP·BeF3-bound DnaA. The sum of all species from monomer to tetramer was normalized to 1.0 for each construct tested before calculating the ratio. C, fluorescence polarization measurements for the binding of F-dT25 (10 nm) by different concentrations of various AaDnaA constructs in the presence of ADP·BeF3.
FIGURE 4.
FIGURE 4.
AaDnaA interface mutants. A, interaction surface. Color-coded AaDnaAAAA+/DBD interfaces, as seen in Fig. 1B, are highlighted with mutated residues labeled. The fraction of the total buried surface area is displayed for each interface region. B, assembly propensity. Comparison of cross-linked AaDnaAAAA+/DBD species is plotted as the ratio of each mutant (ADP·BeF3-bound) to wild-type DnaA (ADP·BeF3-bound) for all species up to tetramer (for representative gels, see supplemental Fig. S4). The sum of all four oligomer species was normalized to 1.0 for each lane before calculating the ratio, and each bar represents the average of three independent experiments. Samples are arranged from the least to the greatest amount of observed cross-linking and are color-coded by interface region. ADP- and ADP·BeF3-bound AaDnaAAAA+/DBD controls are included at each end for reference. C, ssDNA binding. Fluorescence polarization measurements are shown for the binding of F-dT25 (10 nm) by different concentrations of various AaDnaAAAA+/DBD mutants in the presence of ADP·BeF3. Data were fit to determine the Kd,app of ssDNA binding for each mutant bound to ADP·BeF3, and are shown compared with values obtained for wild-type AaDnaAAAA+/DBD bound to either ADP or ADP·BeF3 (supplemental Table S2). Samples are arranged from least to greatest affinity and color-coded by interface. For B and C, orange asterisks indicate the four mutants that have the greatest effects on both assembly and ssDNA affinity.
FIGURE 5.
FIGURE 5.
Effect of interface mutants in vivo and in vitro. A, mutations in EcDnaA assembly interfaces cause growth defects in vivo. Cultures of the temperature-sensitive E. coli dnaA5 strain transformed with a plasmid carrying the indicated EcDnaA mutants were serially diluted (1:10) from saturated cultures grown at the permissive temperature, spotted onto plates, and grown overnight at the indicated temperatures. WT, wild-type; △, empty vector. Subscripts for each mutant correspond to the AaDnaA residue number. B, mutations in the EcDnaA DBD/AAA+ interface disrupt oriC melting in vitro. The maximal melting activity of each mutant, as assessed by P1 nuclease, is shown compared with that of wild-type DnaA. Each value represents the average of two independent experiments.
FIGURE 6.
FIGURE 6.
DnaA assembly is incompatible with binding dsDNA. A, left, AMPPCP-bound AaDnaA filament structure (Protein Data Bank code 2HCB) (red and purple) docked with dsDNA from the EcDnaAIV DNA-bound structure (Protein Data Bank code 1J1V) (yellow). A, right, surface representation with dsDNA omitted. The DBD/AAA+ interface and ssDNA-binding site are highlighted in green and blue, respectively. B, left, model for the binding of dsDNA, based on a rigid body rotation of the DBD about the linker helix similar to that seen for ADP-DnaA (supplemental Fig. S1B). B, right, surface representation with dsDNA omitted. Coloring is identical to A).
FIGURE 7.
FIGURE 7.
Double-stranded DNA effects on ssDNA binding and DnaA assembly. A, assembly of EcDnaA alone, in the presence of an ssDUE fragment and in the presence of both an ssDUE fragment and a dsDNA R1 box substrate. Each sample (8 pmol of protein) was cross-linked with glutaraldehyde, and the resulting species were separated by denaturing PAGE. B, comparison of cross-linked species from A. The fraction of monomer through tetramer obtained for each nucleotide is expressed as a ratio compared with the sample containing ATP and the ssDUE oligonucleotide. The sum of all species from monomer to tetramer was normalized to 1.0 for each nucleotide tested before calculating the ratio. Each bar represents the average of three independent experiments. C, disassembly of EcDnaA·ssDUE complexes (800 and 40 nm, respectively), monitored as a function of dsDNA concentration by fluorescence polarization. The E. coli ssDUE fragment is fluorescein-labeled (supplemental Table S3).
FIGURE 8.
FIGURE 8.
Two-state DnaA assembly model during initiation. Top, oriC engagement. DnaA monomers (extended state) associate with the high affinity dsDNA boxes in the origin. Middle, assembly. In the presence of ATP, DnaA self-assembles (initial complex). As the DUE melts, DnaA molecules transition from an extended to a compact state. Bottom, oriC opening. DnaA molecules (compact state) stabilize a melted ssDUE state (open complex). Only the AAA+ domains and DBDs are depicted; helicase-binding domains are omitted for clarity.

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