Novel heat shock protein HspQ stimulates the degradation of mutant DnaA protein in <i>Escherichia coli</i>

Shimuta, Toh Ru; Nakano, Kiyotaka; Yamaguchi, Yoko; Ozaki, Shogo; Fujimitsu, Kazuyuki; Matsunaga, Chika; Noguchi, Kenji; Emoto, Akiko; Katayama, Tsutomu

doi:10.1111/j.1365-2443.2004.00800.x

Cited by 23 publications

(29 citation statements)

References 51 publications

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“…The DnaA46 protein is unstable at 41°C with a half-life of about 25 min in vivo (43). When plasmids bearing either the dnaA R281A or dnaA R285A allele were transformed into the dnaA46 mutant (KA413), colonies were formed at 30°C with a similar efficiency.…”

Section: Resultsmentioning

confidence: 86%

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

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137

283

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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.

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Section: Resultsmentioning

confidence: 86%

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

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137

283

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“…Strains, Plasmids, and Oligonucleotides-E. coli strains KH5402-1 (wild type), KA450 (⌬oriC1071::Tn10 rnhA199(Am) dnaA17(Am)), KA451 (dnaA::Tn10 rnhA::cat), and KA413 (dnaA46) were previously described (26,33). pKA234, M13KEW101, pTHMA-1, pOZ14, and pOZ18 were also previously described (26,34).…”

Section: Methodsmentioning

confidence: 99%

“…First, we used as a host a dnaA46 mutant strain, KA413, that is defective in colony formation at 42°C. The initiation activity of the DnaA46 protein is temperature-sensitive, and the protein is labile at 42°C, with a shortened half-life in vivo (33,38). The first series of DnaAproducing plasmids bearing wild-type or mutant dnaA alleles was introduced into KA413 cells, and the transformant cells were incubated overnight at 30°C or 42°C (Table 3, experiment A).…”

Section: E Coli Dnaa Mutants Inactive In In Vivomentioning

confidence: 99%

A Common Mechanism for the ATP-DnaA-dependent Formation of Open Complexes at the Replication Origin

Ozaki

Kawakami

Nakamura

et al. 2008

Journal of Biological Chemistry

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131

257

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Initiation of chromosomal replication and its cell cycle-coordinated regulation bear crucial and fundamental mechanisms in most cellular organisms. Escherichia coli DnaA protein forms a homomultimeric complex with the replication origin (oriC). ATPDnaA multimers unwind the duplex within the oriC unwinding element (DUE). In this study, structural analyses suggested that several residues exposed in the central pore of the putative structure of DnaA multimers could be important for unwinding. Using mutation analyses, we found that, of these candidate residues, DnaA Val-211 and Arg-245 are prerequisites for initiation in vivo and in vitro. Whereas DnaA V211A and R245A proteins retained normal affinities for ATP/ADP and DNA and activity for the ATPspecific conformational change of the initiation complex in vitro, oriC complexes of these mutant proteins were inactive in DUE unwinding and in binding to the single-stranded DUE. Unlike oriC complexes including ADP-DnaA or the mutant DnaA, ATPDnaA-oriC complexes specifically bound the upper strand of single-stranded DUE. Specific T-rich sequences within the strand were required for binding. The corresponding conserved residues of the DnaA ortholog in Thermotoga maritima, an ancient eubacterium, were also required for DUE unwinding, consistent with the idea that the mechanism and regulation for DUE unwinding can be evolutionarily conserved. These findings provide novel insights into mechanisms for pore-mediated origin unwinding, ATP/ADP-dependent regulation, and helicase loading of the initiation complex.Initiation of chromosomal replication and its cell cycle-coordinated regulation bear crucial and fundamental mechanisms in most cellular organisms. In Escherichia coli, DnaA forms a stable complex with ATP or ADP and binds to 9-mer sequences called DnaA boxes within the replication origin oriC, resulting in the formation of homomultimeric complexes (1-4). A DnaA-binding protein, DiaA, directly stimulates formation of ATP-DnaA multimers on oriC (5, 6). ATP-DnaA multimers, but not ADP-DnaA multimers, promote specific inter-DnaA interactions on oriC, resulting in the adoption of an activated conformation as the initiation complexes, which interact with ATP-DnaA-specific low affinity sites within oriC (7-9). This conformational change triggers duplex unwinding of the ATrich 13-mer repeats (DNA unwinding element (DUE) 4 ) within oriC with the aid of the superhelicity of DNA and heat energy, creating open complexes (10, 11). The mechanisms and functional structures within DnaA directly responsible for the ATPDnaA-specific duplex unwinding remain unexplored.Open complex formation is a critical regulatory point for determining whether replicational initiation will occur during the cell cycle (1, 2). DnaB helicase is loaded onto the singlestranded (ss) region in open complexes in a manner depending on a DnaA-DnaB interaction and the DnaC helicase loader. The loaded helicase expands the ssDNA region, which leads to the assembly of replication machineries, including DnaG primase and ...

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“…Transcription of the dnaA gene is autoregulated, and ATP-DnaA represses about twofold more tightly dnaA transcription than ADP-DnaA in vitro (Speck et al 1999). The cellular DnaA molecules are present at a level of 1000-2000 molecules per cell and they are very stable (Torheim et al 2000;Shimuta et al 2004). Thus, fluctuations in dnaA transcription should not necessarily largely affect the total cellular DnaA levels.…”

Section: Dars1 or Dars2 Multicopies Promote Replication Initiationmentioning

confidence: 99%

Specific genomic sequences of E. coli promote replicational initiation by directly reactivating ADP-DnaA

Fujimitsu¹,

Senriuchi²,

Katayama³

2009

Genes Dev.

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130

247

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In Escherichia coli, ATP-DnaA, unlike ADP-DnaA, can initiate chromosomal replication at oriC. The level of cellular ATP-DnaA fluctuates, peaking at around the time of replication initiation. However, it remains unknown how the ATP-DnaA level increases coordinately with the replication cycle. In this study, we show that two chromosomal intergenic regions, herein termed DnaA-reactivating sequence 1 (DARS1) and DnaA-reactivating sequence 2 (DARS2), directly promote regeneration of ATP-DnaA from ADP-DnaA by nucleotide exchange, resulting in the promotion of replication initiation in vitro and in vivo. Coordination of initiation with the cell cycle requires DARS activity and its regulation. Oversupply of DARSs results in increase in the ATP-DnaA level and enhancement of replication initiation, which can inhibit cell growth in an oriC-dependent manner. Deletion of DARSs results in decrease in the ATP-DnaA level and inhibition of replication initiation, which can cause synthetic lethality with a temperature-sensitive mutant dnaA and suppression of overinitiation by the lack of seqA or datA, negative regulators for initiation. DARSs bear a cluster of DnaA-binding sites. DnaA molecules form specific homomultimers on DARS1, which causes specific interactions among the protomers, reducing their affinity for ADP. Our findings reveal a novel regulatory pathway that promotes the initiation of chromosomal replication via DnaA reactivation.[Keywords: DnaA reactivation; nucleotide exchange; initiation regulation; functional DNA sequence; protein complex; replication cycle] Supplemental material is available at http://www.genesdev.org.

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Novel heat shock protein HspQ stimulates the degradation of mutant DnaA protein in Escherichia coli

Cited by 23 publications

References 51 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

A Common Mechanism for the ATP-DnaA-dependent Formation of Open Complexes at the Replication Origin

Specific genomic sequences of E. coli promote replicational initiation by directly reactivating ADP-DnaA

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