Cloning and nucleotide sequence determination of twelve mutant dnaA genes of Escherichia coli

Hansen, Flemming; Koefoed, S.; Atlung, Tove

doi:10.1007/bf00272340

Cited by 65 publications

(58 citation statements)

References 42 publications

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“…Among the three cold-sensitive dnaA mutants isolated as extra-cistronic suppressors of a temperature-sensitive dnaX mutant [12], one carried a mutation in domain III and two in the DNA binding domain consistent with our results. It is to be noted here that sites responsible for the thermosensitive replication-defective phenotype of the dnaA mutants so far reported [10,13,23] are located either in the upstream region (domain II and the N-terminal one-third of domain III) from the region where cold-sensitive mutations were mapped, or in the region necessary for binding oriC DNA, except the T301I mutation reported by Sutton and Kaguni [13].…”

Section: Discussionmentioning

confidence: 77%

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

Guo

1999

FEMS Microbiology Letters

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We developed an efficient method for isolation of novel dnaA mutations based on PCR mutagenesis in the presence of manganese ion and shuffling of dnaA-carrying plasmids in a dnaA deletion host bacterium. Using this system, we obtained 30 cold-sensitive mutants from 4000 clones carrying plasmids with a mutagenized dnaA gene. All 27 cold-sensitive mutants analyzed were defective in DNA replication; none had a DnaAcos (over-initiation) phenotype. Nucleotide sequencing revealed that novel 15 alleles (mutations in 14 amino acid residues) are responsible for the cold-sensitive phenotype and are all located in the carboxy-terminal half of the DnaA protein. ß

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Section: Discussionmentioning

confidence: 77%

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

Guo

1999

FEMS Microbiology Letters

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“…Several of the 'classical' dnaA mutants, e.g. dnaA5 and dnaA46, carry a mutation A184V close to the ATP-binding site in the Walker A motif [65]. These mutants are temperature-sensitive and do not bind ATP or ADP at any temperature [66^68], but can be activated to ATP binding by DnaK and GrpE proteins [68].…”

Section: The Role Of Atp Binding On Dnaa Structure and Functionmentioning

confidence: 99%

“…These mutants are temperature-sensitive and do not bind ATP or ADP at any temperature [66^68], but can be activated to ATP binding by DnaK and GrpE proteins [68]. All A184V mutants carry secondary mutations in dnaA [65,69]. However, when the A184V mutation is separated from the secondary mutations, it confers similar temperature sensitivity [68].…”

Section: The Role Of Atp Binding On Dnaa Structure and Functionmentioning

confidence: 99%

“…However, when the A184V mutation is separated from the secondary mutations, it confers similar temperature sensitivity [68]. When A184V is present on multicopy plasmids, such plasmids confer cold sensitivity to dnaA þ or dnaA46 hosts [65,68,70]. Intragenic suppressors of dnaA46 have been isolated, dnaAcos [71] which carries two additional mutations (Q156L and Y271H) [69], and dnaA219 with a R342C mutation [46].…”

Section: The Role Of Atp Binding On Dnaa Structure and Functionmentioning

confidence: 99%

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The bacterial replication initiator DnaA. DnaA and , the bacterial mode to initiate DNA replication

MESSER¹

2002

FEMS Microbiology Reviews

183

298

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The initiation of replication is the central event in the bacterial cell cycle. Cells control the rate of DNA synthesis by modulating the frequency with which new chains are initiated, like all macromolecular synthesis. The end of the replication cycle provides a checkpoint that must be executed for cell division to occur. This review summarizes recent insight into the biochemistry, genetics and control of the initiation of replication in bacteria, and the central role of the initiator protein DnaA.

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“…The 94 carboxy amino acids of DnaA protein appear to be necessary and sufficient to bind origin DNA. Sequence analysis and examination of isolated mutant forms of DnaA protein suggest that a segment which includes Alal84 is involved in the high affinity binding of ADP and ATP (Hwang and Kaguni, 1988a,b;Hansen et al, 1992;Skarstad and Boye, 1994), although this has not been demonstrated directly. Thus, other than the domain for DNA binding, little is known about the location of sites on DnaA protein responsible for critical initiation functions.…”

Section: Introductionmentioning

confidence: 99%

Membrane regulation of the chromosomal replication activity of E.coli DnaA requires a discrete site on the protein.

Garner

Crooke

1996

The EMBO Journal

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The capacity of DnaA protein to initiate DNA synthesis at the chromosomal origin is influenced profoundly by the tightly bound nucleotides ATP and ADP. Acidic phospholipids can catalyze the conversion of inactive ADP‐DnaK protein into the active ATP form. Proteolytic fragments of the nucleotide form of DnaA protein were examined to determine regions of the protein critical for functional interaction with membranes. A 35 kDa chymotryptic and 29 kDa tryptic fragment retained the tightly bound nucleotide. The fragments, whose amino‐termini are within three residues of each other, but differ at their carboxyl ends, showed strikingly different behavior when treated with acidic phospholipids. The larger chymotryptic fragment released the bound nucleotide in the presence of acidic, but not neutral phospholipids. In contrast, the smaller tryptic fragment was inert to both forms of phospholipids. Acidic membranes, but not those composed of neutral phospholipids, protect from tryptic digestion a small portion of the segment that constitutes the difference between the 29 and 35 kDa fragments. The resulting 30 kDa tryptic fragment, which possesses this protected region, interacts functionally with acidic membranes to release the bound effector nucleotide. Inasmuch as the anionic ganglioside GM1, a compound structurally dissimilar to acidic glycerophospholipids, efficiently releases the nucleotide from DnaA protein, an acidic surface associated with a hydrophobic environment is the characteristic of the membrane that appears crucial for regulatory interaction with DnaA protein.

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Cloning and nucleotide sequence determination of twelve mutant dnaA genes of Escherichia coli

Cited by 65 publications

References 42 publications

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

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

The bacterial replication initiator DnaA. DnaA and , the bacterial mode to initiate DNA replication

Membrane regulation of the chromosomal replication activity of E.coli DnaA requires a discrete site on the protein.

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