Two oppositely oriented arrays of low-affinity recognition sites in oriC guide progressive binding of DnaA during Escherichia coli pre-RC assembly

Rozgaja, Tania A.; Grimwade, Julia E.; Iqbal, Mohammad Perwaiz; Czerwonka, Christopher; Vora, Mansi Pankaj; Leonard, Alan C.

doi:10.1111/j.1365-2958.2011.07827.x

Cited by 74 publications

(195 citation statements)

References 57 publications

(115 reference statements)

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“…We found that maintaining N pd = 5 was more difficult than the τ2l case. This result suggests that, for the left-half subcomplex, the complex is more stable with the same orientations of all five bindings, which is in agreement with the report by Rozgaja et al (15).…”

Section: Resultssupporting

confidence: 83%

“…We demonstrated that the low-affinity site τ2 shares its binding orientation with other sites in the left-half subcomplex, which is in agreement with a recent DMS footprint assay (15). It is suggested in ref.…”

Section: Discussionsupporting

confidence: 78%

“…S11), which is consistent with the idea that specific domain III-domain III interaction was allowed in this mutant. These results are also consistent with the previous report indicating that efficient binding between DnaA molecules requires 2-to 5-bp spacing between the DnaA boxes (15). The space between the R2 and C3 sites of the Δ10 mutant was 10 bp, whereas that of the Δ18 mutant is 2 bp.…”

Section: Conformational Change Between Domains III and Iv Upon Complexsupporting

confidence: 82%

“…It is suggested in ref. 15 that DnaA loading to τ2 is assisted by cooperative interaction of DnaA molecules. Interactions through domain III, which forms homooligomers, would restrain binding sites and the orientation of domain IV of τ2-binding DnaA.…”

Section: Discussionmentioning

confidence: 99%

“…Also, there remains a question concerning the importance of specific DnaADnaA interactions to depositing DnaA with specific directionality on low-affinity binding sites. In the right-half oriC, the R2 box is 9 and 20 bp apart from the left and right adjacent DnaA boxes, whereas most of the boxes are only 2 bp apart from each other, which is the most favored distance between two DnaA boxes in DnaA-DnaA interactions (15). These provoke a question of whether the R2-bound DnaA is involved in helical oligomer formation.…”

mentioning

confidence: 89%

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Near-Atomic Structural Model for Bacterial DNA Replication Initiation Complex and its Functional Insights

Shimizu

Noguchi

Sakiyama

et al. 2017

Biophysical Journal

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Upon DNA replication initiation in Escherichia coli, the initiator protein DnaA forms higher-order complexes with the chromosomal origin oriC and a DNA-bending protein IHF. Although tertiary structures of DnaA and IHF have previously been elucidated, dynamic structures of oriC-DnaA-IHF complexes remain unknown. Here, combining computer simulations with biochemical assays, we obtained models at almost-atomic resolution for the central part of the oriC-DnaA-IHF complex. This complex can be divided into three subcomplexes; the left and right subcomplexes include pentameric DnaA bound in a head-to-tail manner and the middle subcomplex contains only a single DnaA. In the left and right subcomplexes, DnaA ATPases associated with various cellular activities (AAA+) domain III formed helices with specific structural differences in interdomain orientations, provoking a bend in the bound DNA. In the left subcomplex a continuous DnaA chain exists, including insertion of IHF into the DNA looping, consistent with the DNA unwinding function of the complex. The intervening spaces in those subcomplexes are crucial for DNA unwinding and loading of DnaB helicases. Taken together, this model provides a reasonable near-atomic level structural solution of the initiation complex, including the dynamic conformations and spatial arrangements of DnaA subcomplexes.DnaA | molecular simulation | coarse-grained model | oriC C hromosomal DNA replication is initiated by unwinding the dsDNA of the replication origin, which requires formation of higher-order protein-DNA complexes, typically referred to as the initiation complexes (1). DnaA is a major replication initiation protein conserved in the initiation complex of most eubacterial species. In a model organism, Escherichia coli, DnaA forms homooligomers on the replication origin oriC, which promotes dsDNA unwinding. The resulting ssDNA is captured by DnaB helicase, followed by formation of the replisomes (2-5). Molecular mechanisms of how DnaA facilitates dsDNA unwinding are still unclear, although some models have been proposed (1, 6-10). A high-resolution structure model of the initiation complex, discovered using computational modeling based on experimental data, would provide significant insight into the molecular mechanism.The E. coli minimal oriC region contains the AT-rich DNA unwinding element (DUE), at least 11 DnaA-binding motifs (termed DnaA boxes) and a single binding site for the integration host factor (IHF) (Fig. 1A) (1-5, 11-15). The DnaA boxes contain 9-mer nucleotides (consensus sequence TTATNCACA, where N can be any base) (16). The 11 DnaA boxes (R1-2, R4, R5M, I1-3, C1-3, and τ2) have differing affinities to DnaA and motif orientations (indicated by triangles in Fig. 1A): The two terminal boxes R1 and R4 have especially high affinities (dissociation constants 1-6 nM for R1 and ∼1 nM for R4), whereas others have modest (R2) to low affinities (I1-3, C1-3, and R5M and τ2; dissociation constants for R5M are >200 nM) (12-19). The 11 DnaA boxes have been divided into two groups...

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Section: Resultssupporting

confidence: 83%

Section: Discussionsupporting

confidence: 78%

Section: Conformational Change Between Domains III and Iv Upon Complexsupporting

confidence: 82%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 89%

See 3 more Smart Citations

Near-Atomic Structural Model for Bacterial DNA Replication Initiation Complex and its Functional Insights

Shimizu

Noguchi

Sakiyama

et al. 2017

Biophysical Journal

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DNA Replication Origins

Rodríguez‐Martínez

Méchali

2014

Encyclopedia of Life Sciences

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The onset of genomic DNA synthesis requires precise interactions of specialized initiator proteins with DNA at sites where the replication machinery can be loaded. These sites, defined as replication origins, are found at a few unique locations in all of the prokaryotic chromosomes examined so far. However, replication origins are dispersed among tens of thousands of loci in metazoan chromosomes, thereby raising questions regarding the role of specific nucleotide sequences and chromatin environment in origin selection and the mechanisms used by initiators to recognize replication origins. Close examination of bacterial and archaeal replication origins reveals an array of DNA sequence motifs that position individual initiator protein molecules and promote initiator oligomerization on origin DNA. Conversely, the need for specific recognition sequences in eukaryotic replication origins is relaxed. In fact, the primary rule for origin selection appears to be flexibility, a feature that is modulated either by structural elements or by epigenetic mechanisms at least partly linked to the organization of the genome for gene expression.

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How bacteria initiate DNA replication comes into focus

Rashid,

Berger

2024

BioEssays

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The ability to initiate DNA replication is a critical step in the proliferation of all organisms. In bacteria, this process is mediated by an ATP‐dependent replication initiator protein, DnaA, which recognizes and melts replication origin (oriC) elements. Despite decades of biochemical and structural work, a mechanistic understanding of how DnaA recognizes and unwinds oriC has remained enigmatic. A recent study by Pelliciari et al. provides important new structural insights into how DnaA from Bacillus subtilis recognizes and processes its cognate oriC, showing how DnaA uses sequence features encoded in the origin to engage melted DNA. Comparison of the DnaA‐oriC structure with archaeal/eukaryl replication origin complexes based on Orc‐family proteins reveals a high degree of similarity in origin engagement by initiators from di domains of life, despite fundamental differences in origin melting mechanisms. These findings provide valuable insights into bacterial replication initiation and highlight the intriguing evolutionary history of this fundamental biological process.

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Two oppositely oriented arrays of low-affinity recognition sites in oriC guide progressive binding of DnaA during Escherichia coli pre-RC assembly

Cited by 74 publications

References 57 publications

Near-Atomic Structural Model for Bacterial DNA Replication Initiation Complex and its Functional Insights

Near-Atomic Structural Model for Bacterial DNA Replication Initiation Complex and its Functional Insights

DNA Replication Origins

How bacteria initiate DNA replication comes into focus

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