The Arg Fingers of Key DnaA Protomers Are Oriented Inward within the Replication Origin oriC and Stimulate DnaA Subcomplexes in the Initiation Complex

Noguchi, Yasunori; Sakiyama, Yukari; Kawakami, Hironori; Katayama, Tsutomu

doi:10.1074/jbc.m115.662601

Cited by 31 publications

(100 citation statements)

References 65 publications

(163 reference statements)

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“…For bidirectional replication from oriC, a pair of DnaB helicases is loaded on the unwound DNA of the oriC region in opposite directions (42). Each subcomplex might exert specific dynamics to coordinately load a pair of DnaB helicases (9,27). The vicinity of the R2 box has exceptionally long intervening spaces, which do not have DnaA binding sequences; the space to the I2 box is 9 bp, whereas that to the C3 box is 20 bp (Fig.…”

Section: Conformational Change Between Domains III and Iv Upon Complexmentioning

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%

“…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). The Argfinger interface, but not the ATP-bound interface, of the terminal DnaA protomers orients inward within oriC and interacts with the ATP-bound interface of the adjacent DnaA protomer (27).…”

mentioning

confidence: 99%

“…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). The Argfinger interface, but not the ATP-bound interface, of the terminal DnaA protomers orients inward within oriC and interacts with the ATP-bound interface of the adjacent DnaA protomer (27). However, the entire oriC-DnaA complex structure model is still unknown.…”

mentioning

confidence: 99%

“…Given that highresolution structures are known for many parts of the systems and that numerous biochemical functional assays have already been accumulated (8,16,20,21,27), we set up molecular simulations to integrate these data for generating structures of the initiation complex. Because the entire system is too large to be modeled by straightforward atomistic molecular dynamics (MD) simulations, we took a multiscale approach.…”

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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: Conformational Change Between Domains III and Iv Upon Complexmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

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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Initiation of DNA Replication at the Chromosomal Origin of E. coli, oriC

Katayama

2017

Advances in Experimental Medicine and Biology

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The Escherichia coli chromosomal origin consists of a duplex-unwinding region and a region bearing a DNA-bending protein, IHF-binding site, and clusters of binding sites for the initiator protein DnaA. ATP-DnaA molecules form highly organized oligomers in a process stimulated by DiaA, a DnaA-binding protein. The resultant ATP-DnaA complexes promote local unwinding of oriC with the aid of IHF, for which specific interaction of DnaA with the single-stranded DNA is crucial. DnaA complexes also interact with DnaB helicases bound to DnaC loaders, promoting loading of DnaB onto the unwound DNA strands for bidirectional replication. Initiation of replication is strictly regulated during the cell cycle by multiple regulatory systems for oriC and DnaA. The activity of oriC is regulated by its methylation state, whereas that of DnaA depends on the form of the bound nucleotide. ATP-DnaA can be yielded from initiation-inactive ADP-DnaA in a timely manner depending on specific chromosomal DNA elements termed DARS (DnaA-reactivating sequences). After initiation, DnaA-bound ATP is hydrolyzed by two systems, yielding ADP-DnaA. In this review, these and other mechanisms of initiation and its regulation in E. coli are described.

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