Electrostatic Interactions during Acidic Phospholipid Reactivation of DnaA Protein, the <i>Escherichia coli</i> Initiator of Chromosomal Replication

Kitchen, Jennifer L.; Li, Zhen‐Ya; Crooke, Elliott

doi:10.1021/bi982733q

Cited by 24 publications

(22 citation statements)

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“…Given the affinity DnaA protein has for anionic phospholipids (30), there is the possibility that DnaA protein may reside at these lipid-enriched domains. However, here DnaA protein was found to be evenly distributed about the cell membrane.…”

Section: Discussionmentioning

confidence: 99%

“…Recent biophysical studies suggest that anionic lipid headgroups are necessary for the reactivation of ADP-DnaA in that they participate in stabilizing electrostatic interactions between DnaA protein and lipid bilayers (30). The physiological relevance of acidic phospholipids being involved in initiation of replication from oriC is strongly supported by the observation that cells unable to synthesize acidic phospholipids are unable to grow once their membrane content of acidic phospholipids drops below a critical level (21,22).…”

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confidence: 99%

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DnaA, the Initiator of Escherichia coli Chromosomal Replication, Is Located at the Cell Membrane

Newman

Crooke

2000

J Bacteriol

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Given the lack of a nucleus in prokaryotic cells, the significance of spatial organization in bacterial chromosome replication is only beginning to be fully appreciated. DnaA protein, the initiator of chromosomal replication in Escherichia coli, is purified as a soluble protein, and in vitro it efficiently initiates replication of minichromosomes in membrane-free DNA synthesis reactions. However, its conversion from a replicatively inactive to an active form in vitro occurs through its association with acidic phospholipids in a lipid bilayer. To determine whether the in situ residence of DnaA protein is cytoplasmic, membrane associated, or both, we examined the cellular location of DnaA using immunogold cryothin-section electron microscopy and immunofluorescence. Both of these methods revealed that DnaA is localized at the cell membrane, further suggesting that initiation of chromosomal replication in E. coli is a membrane-affiliated event.Initiation of Escherichia coli chromosomal replication occurs at the chromosomal origin, oriC, a unique sequence that contains all necessary cis-acting elements (42, 47). To initiate a round of replication, approximately 20 molecules of DnaA protein bind to the DnaA boxes in oriC (10,13,14,38,41,62), promote strand opening of the AT-rich 13-mers (2,3,17), and facilitate the loading of DnaB helicase (14,40). The remaining components of the replisome are subsequently recruited to the sites of the newly formed replication forks (33,39).Given the central role of DnaA protein in the initiation process, its regulation is most likely crucial for normal, cellcycle-controlled chromosomal replication. While dnaA mRNA levels fluctuate throughout the cell cycle, DnaA protein levels remain relatively constant (51). Therefore, other mechanisms must regulate the protein's activity. Prominent among these in vitro is the influence on the replicative action of DnaA protein by the tight binding of ATP and ADP (52,65). Both the ADP and ATP forms of DnaA protein are capable of binding oriC (52). However, only the binding of ATP-DnaA leads to duplex melting and the ensuing initiation events (52,53,65). The ATP bound to DnaA protein is slowly hydrolyzed in a DNA-dependent manner, with the resulting ADP remaining tightly bound, thus producing inactive ADP-DnaA protein. A partially purified factor, termed IdaB, and ␤-subunit, the processivity factor of DNA polymerase III, stimulate the conversion of ATPDnaA to 29).Treatment of ADP-DnaA with anionic phospholipids causes the release of the bound nucleotide (8, 54). When exposed to anionic phospholipids in the presence of oriC DNA and excess ATP, replicatively inactive ADP-DnaA is rejuvenated into the active ATP form (9, 54). An examination of DnaA proteolytic fragments demonstrated that a distinct segment of DnaA is necessary for productive interaction with lipid bilayers (15). Amino acid sequence analysis of this region suggests the existence of an amphipathic helix (57), a motif commonly found in peripherally associated membrane proteins. Chemical cro...

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Section: Discussionmentioning

confidence: 99%

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DnaA, the Initiator of Escherichia coli Chromosomal Replication, Is Located at the Cell Membrane

Newman

Crooke

2000

J Bacteriol

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“…The initiation activity of DnaA protein, the initiator of bacterial DNA replication, is regulated by its adeninenucleotide binding (Sekimizu et al, 1987;Katayama et al, 1998;Kurokawa et al, 1999;Nishida et al, 2002). The affinity of DnaA protein for adenine nucleotides is affected by acidic phospholipids (Sekimizu & Kornberg, 1988;Castuma et al, 1993;Xia & Dowhan, 1995;Mizushima et al, 1996;Kitchen et al, 1999). LPG inhibits the interaction between DnaA protein and acidic phospholipids, PG and CL, and the S. aureus mprF mutant exhibits an increased amount of replication origins (Ichihashi et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

Characterization of the Staphylococcus aureus mprF gene, involved in lysinylation of phosphatidylglycerol

et al. 2004

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Lysylphosphatidylglycerol (LPG) is a basic phospholipid in which L-lysine from lysyl-tRNA is transferred to phosphatidylglycerol (PG). This study examined whether the Staphylococcus aureus mprF gene encodes LPG synthetase. A crude membrane fraction prepared from wild-type S. aureus cells had LPG synthetase activity that depended on PG and lysyl-tRNA, whereas the membrane fraction from an mprF deletion mutant did not. When S. aureus MprF protein was trans-expressed in wild-type Escherichia coli cells, LPG synthesis was induced, whereas it was not observed in E. coli pgsA3 mutant cells in which the amount of PG is significantly reduced. In addition, LPG synthetase activity and a 93 kDa protein whose molecular size corresponded to that of MprF protein were co-induced in the crude membrane fraction prepared from E. coli cells expressing MprF protein. The K m values of the LPG synthetase activity for PG and for lysyl-tRNA were 56 mM and 6?9 mM, respectively, consistent with those of S. aureus membranes. These results suggest that the MprF protein is LPG synthetase.

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“…Acidic phospholipids in a fluid membrane were shown to be responsible for the acceleration of the nucleotide exchange in vitro (11) and are involved in the DnaA-dependent initiation in vivo (12). Further investigations shed light on the mode of DnaA-membrane interaction (13,14) and pointed to a specific region of the protein responsible for this interaction (15-18; and for a review see Refs. 19 and 20).…”

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“…Data on changes in the level of individual phospholipids during the cell cycle are controversial (24,25). Such changes could provide the activation signal in view of the stimulation of nucleotide dissociation from DnaA by raising the fraction of acidic phospholipids in mixed liposomes (13). However, there is an indication that clustering, rather than the concentration of acidic phospholipids, in such a membrane is essential for nucleotide dissociation (26).…”

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Membrane-catalyzed Nucleotide Exchange on DnaA

Aranovich

Gdalevsky

Cohen‐Luria

et al. 2006

Journal of Biological Chemistry

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DnaA is the initiator protein for chromosomal replication in bacteria; its activity plays a central role in the timing of the primary initiations within the Escherichia coli cell cycle. A controlled, reversible conversion between the active ATP-DnaA and the inactive ADP forms modulates this activity. In a DNA-dependent manner, bound ATP is hydrolyzed to ADP. Acidic phospholipids with unsaturated fatty acids are capable of reactivating ADP-DnaA by promoting the release of the tightly bound ADP. The nucleotide dissociation kinetics, measured in the present study with the fluorescent derivative 3-O-(N-methylantraniloyl)-5-adenosine triphosphate, was dependent on the density of DnaA on the membrane in a cooperative manner: it increased 5-fold with decreased protein density. At all surface densities the nucleotide was completely released, presumably due to protein exchange on the membrane. Distinct temperature dependences and the effect of the crowding agent Ficoll suggest that two functional states of DnaA exist at high and low membrane occupancy, ascribed to local macromolecular crowding on the membrane surface. These novel phenomena are thought to play a major role in the mechanism regulating the initiation of chromosomal replication in bacteria.

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Electrostatic Interactions during Acidic Phospholipid Reactivation of DnaA Protein, the Escherichia coli Initiator of Chromosomal Replication

Cited by 24 publications

References 35 publications

DnaA, the Initiator of Escherichia coli Chromosomal Replication, Is Located at the Cell Membrane

DnaA, the Initiator of Escherichia coli Chromosomal Replication, Is Located at the Cell Membrane

Characterization of the Staphylococcus aureus mprF gene, involved in lysinylation of phosphatidylglycerol

Membrane-catalyzed Nucleotide Exchange on DnaA

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