Isolation and characterization of novel cold-sensitive dnaA mutants of Escherichia coli

Guo, Lei

doi:10.1016/s0378-1097(99)00261-x

Cited by 13 publications

(23 citation statements)

References 21 publications

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“…The mutagenic PCR method was used as previously described, with minor modifications (Guo et al 1999). Briefly, diaA (0.56-kb fragment) was amplified by PCR using pTKM601 as the template, and the primers TAKU15 and TAKU4, in the presence of 0.05 mM or 0.1 mM manganese.…”

Section: Introduction Of Mutations and Selection Of Dysfunctional Diamentioning

confidence: 99%

The interaction of DiaA and DnaA regulates the replication cycle in E. coli by directly promoting ATP–DnaA-specific initiation complexes

Keyamura¹,

Fujikawa

Ishida³

et al. 2007

Genes Dev.

118

192

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Escherichia coli DiaA is a DnaA-binding protein that is required for the timely initiation of chromosomal replication during the cell cycle. In this study, we determined the crystal structure of DiaA at 1.8 Å resolution. DiaA forms a homotetramer consisting of a symmetrical pair of homodimers. Mutational analysis revealed that the DnaA-binding activity and formation of homotetramers are required for the stimulation of initiation by DiaA. DiaA tetramers can bind multiple DnaA molecules simultaneously. DiaA stimulated the assembly of multiple DnaA molecules on oriC, conformational changes in ATP-DnaA-specific initiation complexes, and unwinding of oriC duplex DNA. The mutant DiaA proteins are defective in these stimulations. DiaA associated also with ADP-DnaA, and stimulated the assembly of inactive ADP-DnaAoriC complexes. Specific residues in the putative phosphosugar-binding motif of DiaA were required for the stimulation of initiation and formation of ATP-DnaA-specific-oriC complexes. Our data indicate that DiaA regulates initiation by a novel mechanism, in which DiaA tetramers most likely bind to multiple DnaA molecules and stimulate the assembly of specific ATP-DnaA-oriC complexes. These results suggest an essential role for DiaA in the promotion of replication initiation in a cell cycle coordinated manner.[Keywords: initiation of replication; protein complex; complex structure; DNA dynamics; cell cycle regulation; initiator regulation] Supplemental material is available at http://www.genesdev.org.

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Section: Introduction Of Mutations and Selection Of Dysfunctional Diamentioning

confidence: 99%

The interaction of DiaA and DnaA regulates the replication cycle in E. coli by directly promoting ATP–DnaA-specific initiation complexes

Keyamura¹,

Fujikawa

Ishida³

et al. 2007

Genes Dev.

118

192

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“…The results were similar (Table 1). The colony sizes and growth rates obtained with dnaA435 were indistinguishable from those obtained with the wild-type gene (pKP2206) [25]. Thus, DnaA435, which shows a decreased interaction with CL and with ATP in itro, is able to initiate DNA replication in i o.…”

Section: Replication Activity Of Mutant Dnaa Proteins In Vivomentioning

confidence: 82%

“…To construct plasmids for complementation analysis, we ligated the coding regions of the mutant dnaA gene (BamHI-HindIII fragment) into pKP2204 [25], which contains the wild-type promoter of the dnaA gene.…”

Section: Site-directed Mutagenesis and Plasmid Constructionmentioning

confidence: 99%

“…Temperature-sensitive dnaA mutant cells were transformed with pKP2204 (a derivative from mini-R plasmid) [25] containing either the wild-type dnaA or each mutant dnaA gene. Cultures were diluted appropriately and spread on Luria-Bertani agar plates containing 20 µg\ml chloramphenicol.…”

Section: Plasmid Complementation Analysismentioning

confidence: 99%

“…The coding regions of these mutant and the wild-type dnaA genes were conjugated with the wild-type dnaA promoter in pKP2204 [25]. Each resultant plasmid was introduced into E. coli KS1003, in which DnaA protein has the temperature-sensitive dnaA46 mutations in its ATP-binding site [36].…”

Section: Replication Activity Of Mutant Dnaa Proteins In Vivomentioning

confidence: 99%

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Biochemical analysis of DnaA protein with mutations in both Arg328 and Lys372

et al. 2002

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The Plasmodium HU homolog, which binds the plastid DNA sequence‐independent manner, is essential for the parasite's survival

et al. 2009

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Edited by Michael IbbaKeywords: DNA binding protein HU Knock-out Plastid Plasmodium berghei Plasmodium falciparum a b s t r a c tThe nuclear genome of the human malaria parasite Plasmodium falciparum encodes a homolog of the bacterial HU protein (PfHU). In this study, we characterised PfHU's physiological function. PfHU, which is targeted exclusively to the parasite's plastid, bound its natural target -the plastid DNAsequence-independently and complemented lack of HU in Escherichia coli. The HU gene could not be knocked-out from the genome of Plasmodium berghei, implying that HU is important for the parasite's survival. As the human cell lacks the HU homolog, PfHU is a potential target for drugs to control malaria.

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Isolation and characterization of novel cold-sensitive dnaA mutants of Escherichia coli

Cited by 13 publications

References 21 publications

The interaction of DiaA and DnaA regulates the replication cycle in E. coli by directly promoting ATP–DnaA-specific initiation complexes

The interaction of DiaA and DnaA regulates the replication cycle in E. coli by directly promoting ATP–DnaA-specific initiation complexes

Biochemical analysis of DnaA protein with mutations in both Arg328 and Lys372

The Plasmodium HU homolog, which binds the plastid DNA sequence‐independent manner, is essential for the parasite's survival

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