Ordered and Sequential Binding of DnaA Protein to  , the Chromosomal Origin of

Margulies, Carla; Kaguni, Jon M.

doi:10.1074/jbc.271.29.17035

Cited by 92 publications

(112 citation statements)

References 34 publications

Supporting

Mentioning

102

Contrasting

Order By: Relevance

“…As electron microscopy observation suggests that 20 -30 DnaA molecules are included in an initiation complex (10), we incubated variable amounts of DnaA, ranging up to about 50 input DnaA molecules per oriC, before carrying out gel electrophoresis. As previously reported (13,36), we observed that ATP-bound wild-type DnaA forms multimeric DnaA complexes with oriC (Fig. 7A).…”

Section: Resultssupporting

confidence: 69%

“…7A). ADP-DnaA multimers formed on oriC may be less stable than ATP-DnaA multimers (18,36). When 3.2-26 DnaA molecules per oriC were used, slight differences were also seen for ATP-DnaA and ADP-DnaA in the mobility of oriC complexes, but these were less reproducible.…”

Section: Resultsmentioning

confidence: 91%

“…Experiments using a minimal oriC-containing fragment were performed according to methods previously described, with minor modifications (13,36). The oriC DNA (469 bp) was isolated as an AvaI fragment from pBSoriC.…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Formation of an ATP-DnaA-specific Initiation Complex Requires DnaA Arginine 285, a Conserved Motif in the AAA+ Protein Family

Kawakami¹,

Keyamura

Katayama

2005

Journal of Biological Chemistry

137

283

View full text Add to dashboard Cite

Escherichia coli DnaA protein, a member of the AAA؉ superfamily, initiates replication from the chromosomal origin oriC in an ATP-dependent manner. Nucleoprotein complex formed on oriC with the ATP-DnaA multimer but not the ADP-DnaA multimer is competent to unwind the oriC duplex. The oriC region contains ATP-DnaAspecific binding sites termed I2 and I3, which stimulate ATP-DnaA-dependent oriC unwinding. In this study, we show that the DnaA R285A mutant is inactive for oriC replication in vivo and in vitro and that the mutation is associated with specific defects in oriC unwinding. In contrast, activities of DnaA R285A are sustained in binding to the typical DnaA boxes and to ATP and ADP, formation of multimeric complexes on oriC, and loading of the DnaB helicase onto single-stranded DNA. Footprint analysis of the DnaA-oriC complex reveals that the ATP form of DnaA R285A does not interact with ATPDnaA-specific binding sites such as the I sites. A subgroup of DnaA molecules in the oriC complex must contain the Arg-285 residue for initiation. Sequence and structural analyses suggest that the DnaA Arg-285 residue is an arginine finger, an AAA؉ family-specific motif that recognizes ATP bound to an adjacent subunit in a multimeric complex. In the context of these and previous results, the DnaA Arg-285 residue is proposed to play a unique role in the ATP-dependent conformational activation of an initial complex by recognizing ATP bound to DnaA and by modulating the structure of the DnaA multimer to allow interaction with ATP-DnaAspecific binding sites in the complex.DnaA protein plays an essential role in the initiation of Escherichia coli chromosomal replication (1-4). The protein forms a homomultimer on the replication origin oriC to form an initiation complex. When the ATP-bound form of DnaA is included in the initiation complex, a region within oriC containing AT-rich 13-mers is specifically unwound, resulting in open complex formation. ADP-DnaA forms a multimeric complex on oriC that does not promote open complex formation. On the exposed single-stranded (ss) 1 DNA of the 13-mer region, DnaB helicase is loaded via ordered interactions with the DnaC helicase loader and DnaA. DnaG primase forms a complex with DnaB that promotes DNA duplex unwinding and RNA primer synthesis, which allow loading of the DNA polymerase (pol) III holoenzyme. The pol III holoenzyme consists of the ␤ clamp subunit, which is directly loaded onto the primed site, and the pol III* subassembly, which binds to the ␤ clamp. Pol III* includes the clamp-loader ␥ complex and the pol III core complex, which contains the catalytic center of the polymerase.

show abstract

Section: Resultssupporting

confidence: 69%

Section: Resultsmentioning

confidence: 91%

See 1 more Smart Citation

Formation of an ATP-DnaA-specific Initiation Complex Requires DnaA Arginine 285, a Conserved Motif in the AAA+ Protein Family

Kawakami¹,

Keyamura

Katayama

2005

Journal of Biological Chemistry

137

283

View full text Add to dashboard Cite

show abstract

“…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)(13)(14)(15)(16)(17)(18)(19). The 11 DnaA boxes have been divided into two groups on the left-and right-half oriC.…”

mentioning

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%

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

Shimizu

Noguchi

Sakiyama

et al. 2017

Biophysical Journal

View full text Add to dashboard Cite

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...

show abstract

Box

Schneider¹

2004

Dictionary of Bioinformatics and Computational Biology

View full text Add to dashboard Cite

Ordered and Sequential Binding of DnaA Protein to , the Chromosomal Origin of

Cited by 92 publications

References 34 publications

Formation of an ATP-DnaA-specific Initiation Complex Requires DnaA Arginine 285, a Conserved Motif in the AAA+ Protein Family

Formation of an ATP-DnaA-specific Initiation Complex Requires DnaA Arginine 285, a Conserved Motif in the AAA+ Protein Family

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

Box

Contact Info

Product

Resources

About