Highly organized DnaA– oriC complexes recruit the single-stranded DNA for replication initiation

Ozaki, Shogo; Katayama, Tsutomu

doi:10.1093/nar/gkr832

Cited by 98 publications

(278 citation statements)

References 68 publications

(131 reference statements)

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“…Next, we performed biochemical experiments to analyze the DUE unwinding activity of oriC mutants bearing altered τ2 box sequences. This box resides in the left-half oriC, which sustains the entire activity in DUE unwinding (9,19). To identify which definition, i.e., (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…As mentioned above, DnaA forms at least two subcomplexes corresponding to the left and right halves of oriC (9,10,15). The left-half oriC subcomplex sustains the entire activity in DUE unwinding; i.e., deletion of the region spanning R2 to R4 does not impair the unwinding activity (9).…”

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confidence: 93%

“…The resulting ssDNA is captured by DnaB helicase, followed by formation of the replisomes (2)(3)(4)(5). Molecular mechanisms of how DnaA facilitates dsDNA unwinding are still unclear, although some models have been proposed (1,(6)(7)(8)(9)(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.…”

mentioning

confidence: 99%

“…The left-half oriC subcomplex sustains the entire activity in DUE unwinding; i.e., deletion of the region spanning R2 to R4 does not impair the unwinding activity (9). Each subcomplex binds a DnaB helicase for loading onto the single-stranded region (9,10). DnaA oligomers are formed on the regions spanning R1-I2 and R4-C3 on oriC in a manner depending on the Arg finger-ATP interaction and other specific interactions (9,10,14,15,27).…”

mentioning

confidence: 99%

“…Each subcomplex binds a DnaB helicase for loading onto the single-stranded region (9,10). DnaA oligomers are formed on the regions spanning R1-I2 and R4-C3 on oriC in a manner depending on the Arg finger-ATP interaction and other specific interactions (9,10,14,15,27). DnaA binding to the two terminal, high-affinity DnaA boxes has been proposed to elicit sequential binding of ATP-DnaA molecules in low-affinity binding sites (15,18,27).…”

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confidence: 99%

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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: Resultsmentioning

confidence: 99%

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confidence: 93%