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. 2011 Oct 2;478(7368):209-13.
doi: 10.1038/nature10455.

DNA Stretching by Bacterial Initiators Promotes Replication Origin Opening

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DNA Stretching by Bacterial Initiators Promotes Replication Origin Opening

Karl E Duderstadt et al. Nature. .
Free PMC article

Abstract

Many replication initiators form higher-order oligomers that process host replication origins to promote replisome formation. In addition to dedicated duplex-DNA-binding domains, cellular initiators possess AAA+ (ATPases associated with various cellular activities) elements that drive functions ranging from protein assembly to origin recognition. In bacteria, the AAA+ domain of the initiator DnaA has been proposed to assist in single-stranded DNA formation during origin melting. Here we show crystallographically and in solution that the ATP-dependent assembly of Aquifex aeolicus DnaA into a spiral oligomer creates a continuous surface that allows successive AAA+ domains to bind and extend single-stranded DNA segments. The mechanism of binding is unexpectedly similar to that of RecA, a homologous recombination factor, but it differs in that DnaA promotes a nucleic acid conformation that prevents pairing of a complementary strand. These findings, combined with strand-displacement assays, indicate that DnaA opens replication origins by a direct ATP-dependent stretching mechanism. Comparative studies reveal notable commonalities between the approach used by DnaA to engage DNA substrates and other, nucleic-acid-dependent, AAA+ systems.

Figures

Figure 1
Figure 1. The ATPase pore of assembled DnaA binds ssDNA
a, Side view of the asymmetric unit, with DnaA subunits differentially colored. Single-stranded DNA is displayed as red sticks. AMPPCP and Mg2+, bound to chain A, are shown as spheres colored by element and in magenta, respectively; AMPPCP•Mg2+ bound to chains B-D are occluded in this view. b, Side and top views of oligomerized DnaA, reconstructed through crystal packing, showing twelve DnaA subunits and three strands of ssDNA. Coloring as per panel A. c, Side view of the DnaA tetramer with helices α3/α4 and α5/α6 highlighted in orange and yellow, respectively (“ISM” – initiator specific motif). Single-stranded DNA is shown as a transparent stick-and-surface representation colored by element; phosphates are further highlighted as red spheres. d, Protein-DNA contacts. Protein chains B (left) and C (right) are displayed with the same coloring as in c. Single-stranded DNA is colored by element.
Figure 2
Figure 2. DnaA engages ssDNA in a manner similar to RecA
a, View of a DnaA-AMPPCP-ssDNA pentamer (consisting of one full tetramer, as well as chain A (DnaAA') and its associated triplet from the adjacent asymmetric unit). AMPPCP•Mg2+ is shown as spheres colored by atom, ssDNA as red sticks. b, View of a RecA-ADP-AlF4-ssDNA pentamer (PDB ID 3CMW). ADP•AlF4•Mg2+ is shown as spheres colored by atom, ssDNA as red sticks. c, Comparison of ssDNA bound to DnaA (orange), RecA (green) and a strand of B-DNA (yellow). d, Close-up view of triplet bound to DnaA (chain C) with magenta dashed lines indicating key contacts. e, Close-up view of triplet bound to RecA (protomer 2) with magenta dashed lines indicating key contacts. f, Side (left) and top (right) views of the triplets displayed in d and e aligned with each other.
Figure 3
Figure 3. DnaA extends ssDNA in solution
a, Cartoon of ssDNA extension assay. b, Emission scan (donor excitation) of FR-dT21 in the presence of 10 μM DnaA with either ADP•BeF3 (top) or ADP (bottom). c, Emission scan (donor excitation) of FR-dT21 in the presence of 10 μM RecA with either ATPγS (top) or ADP (bottom). Reported transfer efficiencies and distances were calculated using donor emission as described in the Methods.
Figure 4
Figure 4. DnaA directly melts duplex DNA
a, Schematic of strand displacement assay. The green circle represents the Cy3 fluorescent end-label used to follow the status of one DNA strand. Complementary strands of duplex substrates are colored grey and black. b, Strand displacement assay conducted with 15 and 20mer duplex substrates (C3–15mer and C3–20mer) in the presence and absence of different nucleotides. DnaA concentrations used are indicated above each lane. c, (left) Cartoon model showing how complementary base triplets (yellow) would pair (in a B-DNA manner) with ssDNA bound to DnaA (red). The orientation of successive DnaA-bound triplets is such that it prevents the formation of a continuous base-paired strand favoring duplex separation. (right) Same DNA view, but as seen in RecA, where triplets are oriented to allow pairing of an extended complementary strand to promote duplex formation and strand exchange (PDB ID 3CMX).
Figure 5
Figure 5. Common DNA recognition strategies of AAA+ proteins
Structures of DNA-bound assemblies (top) and individual domains (bottom) for AAA+ proteins involved in replication. All recognize DNA using the same face of the AAA+ fold (violet) (bottom). a, Bacterial clamp-loader (γδδ′) complex (AAA+ domains – differentially colored) bound to primer-template DNA (PDB ID 3GLF). b, Archaeal initiators Orc1–1 (gray) and Orc1–3 (AAA+ domain - green) bound to origin DNA (PDB ID 2QBY). c, Bacterial initiator DnaA (AAA+ domains – gray/blue) bound to ssDNA. d, Viral initiator/helicase E1 (AAA+ domains – orange/gray) bound to ssDNA (PDB ID 2GXA). For all panels, DNA is shown as either red spheres (top), or as a red/grey cartoon (bottom). Nucleotide co-factors bound to AAA+ domains (bottom) are represented as spheres colored by atom.

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