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• W2045574547 abstract "In the absence of adequate levels of cellular acidic phospholipids, Escherichia coli remain viable but are arrested for growth. Expression of a DnaA protein that contains a single amino acid substitution in the membrane-binding domain, DnaA(L366K), in concert with expression of wild-type DnaA protein, restores growth. DnaA protein has high affinity for ATP and ADP, and in vitro lipid bilayers that are fluid and contain acidic phospholipids reactivate inert ADP-DnaA by promoting an exchange of ATP for ADP. Here, nucleotide and lipid interactions and replication activity of purified DnaA(L366K) were examined to gain insight into the mechanism of how it restores growth to cells lacking acidic phospholipids. DnaA(L366K) behaved like wild-type DnaA with respect to nucleotide binding affinities and hydrolysis properties, specificity of acidic phospholipids for nucleotide release, and origin binding. Yet, DnaA(L366K) was feeble at initiating replication from oriC unless augmented with a limiting quantity of wild-type DnaA, reflecting the in vivo requirement that both wild-type and a mutant form of DnaA must be expressed and act together to restore growth to acidic phospholipid deficient cells. In the absence of adequate levels of cellular acidic phospholipids, Escherichia coli remain viable but are arrested for growth. Expression of a DnaA protein that contains a single amino acid substitution in the membrane-binding domain, DnaA(L366K), in concert with expression of wild-type DnaA protein, restores growth. DnaA protein has high affinity for ATP and ADP, and in vitro lipid bilayers that are fluid and contain acidic phospholipids reactivate inert ADP-DnaA by promoting an exchange of ATP for ADP. Here, nucleotide and lipid interactions and replication activity of purified DnaA(L366K) were examined to gain insight into the mechanism of how it restores growth to cells lacking acidic phospholipids. DnaA(L366K) behaved like wild-type DnaA with respect to nucleotide binding affinities and hydrolysis properties, specificity of acidic phospholipids for nucleotide release, and origin binding. Yet, DnaA(L366K) was feeble at initiating replication from oriC unless augmented with a limiting quantity of wild-type DnaA, reflecting the in vivo requirement that both wild-type and a mutant form of DnaA must be expressed and act together to restore growth to acidic phospholipid deficient cells. The cycle of chromosomal DNA replication in Escherichia coli is largely controlled at the initiation stage. A cluster of multiple DnaA protein molecules binds to DnaA recognition sites in the unique origin (oriC) (1Fuller R. Funnell B. Kornberg A. Cell. 1984; 38: 889-900Abstract Full Text PDF PubMed Scopus (464) Google Scholar, 2Funnell B. Baker T.A. Kornberg A. J. Biol. Chem. 1987; 262: 10327-10334Abstract Full Text PDF PubMed Google Scholar, 3Crooke E. Thresher R. Hwang D.S. Griffith J. Kornberg A. J. Mol. Biol. 1993; 233: 16-24Crossref PubMed Scopus (87) Google Scholar, 4Carr K.M. Kaguni J.M. J. Biol. Chem. 2001; 276: 44919-44925Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Aided by architectural proteins HU and integration host factor (IHF), DnaA protein promotes strand opening of the AT-rich 13-mer repeats of oriC and mediates the delivery of DnaB helicase from DnaB·DnaC complexes onto the exposed single strands. The complementary strands are subsequently synthesized by replisomes that have been assembled at the two DNA forks (5Messer W. FEMS Microbiol. Rev. 2002; 26: 355-374PubMed Google Scholar). The transition of the origin from duplex to melted DNA is influenced by the high affinity DnaA protein has for ATP and ADP; although both forms of DnaA protein are capable of producing very similar nucleoprotein complexes at oriC, only ATP-DnaA is active for promoting strand opening and subsequent replication steps (6Sekimizu K. Bramhill D. Kornberg A. Cell. 1987; 50: 259-265Abstract Full Text PDF PubMed Scopus (348) Google Scholar). Regulatory inactivation of DnaA (RIDA), 1The abbreviations used are: RIDA, regulatory inactivation of DnaA; PIPES, 1,4-piperazinediethanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. one of the mechanisms to ensure that a round of chromosomal replication happens only once per cell cycle, stimulates the hydrolysis of DnaA-bound ATP, generating ADP-DnaA that is feeble at initiating replication (7Katayama T. Crooke E. J. Biol. Chem. 1995; 270: 9265-9271Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 8Katayama T. Kubota T. Kurokawa K. Crooke E. Sekimizu K. Cell. 1998; 94: 61-71Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 9Kurokawa K. Nishida S. Emoto A. Sekimizu K. Katayama T. EMBO J. 1999; 18: 6642-6652Crossref PubMed Scopus (191) Google Scholar). RIDA activity is dependent on the β-subunit of DNA polymerase III loaded as a sliding clamp onto template DNA and Hda, a protein important for controlled replication of the E. coli chromosome and plasmid RK2 (10Kato J. Katayama T. EMBO J. 2001; 20: 4253-4262Crossref PubMed Scopus (217) Google Scholar, 11Kim D.K. Banack T. Lerman D.M. Tracy J.E. Camara J.E. Crooke E. Oliver D. Firshein W. J. Bacteriol. 2003; 185: 1817-1824Crossref PubMed Scopus (20) Google Scholar, 12Camara J.E. Skarstad K. Crooke E. J. Bacteriol. 2003; 185: 3244-3248Crossref PubMed Scopus (48) Google Scholar). Acidic phospholipids in a fluid bilayer promote the release of bound nucleotide from DnaA in vitro (13Sekimizu K. Kornberg A. J. Biol. Chem. 1988; 263: 7131-7135Abstract Full Text PDF PubMed Google Scholar, 14Castuma C.E. Crooke E. Kornberg A. J. Biol. Chem. 1993; 268: 24665-24668Abstract Full Text PDF PubMed Google Scholar). When ADP-DnaA is stabilized by being bound to oriC, acidic phospholipids facilitate an exchange of ATP for ADP and thus can restore initiation activity to DnaA protein (15Crooke E. Castuma C.E. Kornberg A. J. Biol. Chem. 1992; 267: 16779-16782Abstract Full Text PDF PubMed Google Scholar). Analysis of functional DnaA proteolytic fragments identified a domain of DnaA protein that is necessary for membrane reactivation of the initiation activity of the protein (16Garner J. Crooke E. EMBO J. 1996; 15: 3477-3485Crossref PubMed Scopus (54) Google Scholar). Cross-linking studies that employed a radiolabeled, photoactivatable phospholipid analog revealed that this essential domain of DnaA protein inserts into the hydrophobic portion of the lipid bilayer during the nucleotide release process (17Garner J. Durrer P. Kitchen J. Brunner J. Crooke E. J. Biol. Chem. 1998; 273: 5167-5173Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Analysis of the amino acid sequence of the DnaA protein suggests that this membrane-interacting segment of DnaA protein forms an amphiphatic helix (16Garner J. Crooke E. EMBO J. 1996; 15: 3477-3485Crossref PubMed Scopus (54) Google Scholar), a structural motif that can act as a membrane surface-seeking domain for peripherally associated membrane proteins. Homology modeling of the E. coli DnaA sequence onto the recently solved crystal structure of a truncated version of Aquifex aeolicus DnaA supports this region of E. coli DnaA being such a structure (18Erzberger J.P. Pirruccello M.M. Berger J.M. EMBO J. 2002; 21: 4763-4773Crossref PubMed Scopus (193) Google Scholar). In general, DnaA protein is composed of an amino-terminal domain I involved in helicase recruitment (19Sutton M.D. Carr K.M. Vicente M. Kaguni J.M. J. Biol. Chem. 1998; 273: 34255-34262Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 20Weigel C. Schmidt A. Seitz H. Tungler D. Welzeck M. Messer W. Mol. Microbiol. 1999; 34: 53-66Crossref PubMed Scopus (89) Google Scholar) and with specific residues needed for oligomerization at oriC (21Simmons L.A. Felczak M. Kaguni J.M. Mol. Microbiol. 2003; 49: 849-858Crossref PubMed Scopus (67) Google Scholar), a variable domain II, a core domain III that contains the nucleotide binding site, and a carboxyl-terminal DNA binding domain (22Roth A. Messer W. EMBO J. 1995; 14: 2106-2111Crossref PubMed Scopus (122) Google Scholar, 23Blaesing F. Weigel C. Welzeck M. Messer W. Mol. Microbiol. 2000; 36: 557-569Crossref PubMed Scopus (75) Google Scholar). Domain III and the helix-turn-helix motif of domain IV are connected by the membrane-binding long amphiphatic helix mentioned above. In vivo, a close link between DnaA protein function and the state of the cellular membrane is likely. When the synthesis of acidic phospholipids is impeded via repressed transcription of the gene for phosphatidylglycerophosphate synthase, cells become arrested for growth after a few generations but remain viable (24Heacock P. Dowhan W. J. Biol. Chem. 1989; 264: 14972-14977Abstract Full Text PDF PubMed Google Scholar). The growth arrest can be relieved by expression of certain plasmid-borne mutant forms of DnaA protein, including DnaA(L366K), with initiation of replication occurring at or near oriC (25Zheng W. Li Z. Skarstad K. Crooke E. EMBO J. 2001; 20: 1164-1172Crossref PubMed Scopus (41) Google Scholar). In support of the idea that the arrested growth in acidic phospholipid-deficient cells is associated with impaired chromosomal replication from oriC, the growth arrest can also be alleviated if the cells are permitted to undergo constitutive stable DNA replication, a process that allows DnaA-independent initiation of replication at sites other than oriC (26Xia W. Dowhan W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 783-787Crossref PubMed Scopus (121) Google Scholar). The mechanism of how DnaA(L366K) restores growth to acidic-phospholipid deficient cells has been speculated (25Zheng W. Li Z. Skarstad K. Crooke E. EMBO J. 2001; 20: 1164-1172Crossref PubMed Scopus (41) Google Scholar) to be related to its altered nucleotide state or reactivation by membranes. Possibilities include: (i) the ability of ADP-DnaA(L366K) to be rejuvenated by neutral, zwitterionic membranes, (ii) DnaA(L366K) never being in its ADP form because it fails to bind ATP in the first place, thus negating the need for acidic membrane-mediated reactivation, (iii) DnaA(L366K) binds ATP but fails to hydrolyze it to produce ADP-DnaA or is inert to RIDA, therefore bypassing the necessity to be reactivated by acidic phospholipids, and (iv) ADP-DnaA(L366K) is active at initiating chromosomal replication. A biochemical study of these possibilities is presented here. The membrane reactivation characteristics of another mutant form of DnaA protein that possesses other amino acid substitutions in the membrane-binding region are also presented. Materials—Unless otherwise stated, reagent grade chemicals were from J. T. Baker, Sigma Chemicals, Fisher Scientific, or Roche Applied Science. Growth media components were from BD Biosciences. Phospholipids 1-stearoyl-2-oleoyl-sn-glycero-3-[phospho-rac-[1-choline]], 1-stearoyl-2-oleoyl-sn-glycero-3-[phospho-rac-[1-glycerol]], E. coli phosphatidylethanolamine, and bovine heart cardiolipin were from Avanti Polar Lipids. [α-32P]ATP (3000 Ci/mmol), [2,8-3H]ADP (25–40 Ci/mmol), and [α-32P]TTP (800 Ci/mmol) were from PerkinElmer Life Sciences. Bacterial Strains and Plasmids—Histidine-tagged wild-type and mutant DnaA proteins were expressed from pZL411 (27Li Z. Crooke E. Protein Expression Purif. 1999; 17: 41-48Crossref PubMed Scopus (27) Google Scholar) and its derivative plasmids in strain BLR(DE3)pLysS. Total E. coli lipids were extracted from exponentially growing W3110 cells that were harvested, resuspended in 50 mm Tris·HCl (pH 8.0) and 10% sucrose to an optical density (A600 nm) of 300, and rapidly frozen in liquid nitrogen. Nucleotide Binding and Membrane-mediated Release—ATP and ADP binding were assessed as described previously (6Sekimizu K. Bramhill D. Kornberg A. Cell. 1987; 50: 259-265Abstract Full Text PDF PubMed Scopus (348) Google Scholar). Briefly, polyhistidine-tagged DnaA proteins in buffer HD (50 mm PIPES·KOH (pH 6.8 at 1 m), 10 mm magnesium acetate, 200 mm ammonium sulfate, 20% (w/v) sucrose, 0.1 mm EDTA, and 2 mm dithiothreitol) were mixed with 1 μm [α-32P]ATP or [2,8-3H]ADP in 25 μl of buffer pp60 (50 mm HEPES·KOH (pH 7.6 at 1 m), 2.5 mm magnesium acetate, 20% (v/v) glycerol, 0.007% (v/v) Triton X-100, 0.3 mm EDTA, and 7 mm dithiothreitol) and incubated at 0 °C for 15 min. For samples subjected to membrane-mediated nucleotide release, phospholipids in the form of small unilamellar vesicles were added, and mixtures were incubated at 38 °C for 10 min. Samples were filtered through nitrocellulose filters (Type HA, Millipore) presoaked in buffer G (50 mm Tricine·KOH (pH 8.25 at 1 m), 0.5 mm magnesium acetate, 10 mm ammonium sulfate, 17% (v/v) glycerol, 0.005% (v/v) Triton X-100, 0.3 mm EDTA, and 5 mm dithiothreitol). Filters were washed with 5 ml of ice-cold buffer G, dried, and retained nucleotide was measured by liquid scintillation counting. Nucleotide Hydrolysis—Histidine-tagged wild-type or mutant DnaA proteins (2 pmol) were incubated (0 °C, 15 min) with 1 μm [α-32P]ATP in buffer pp60 (16 μl). The resulting ATP-DnaA was incubated (38 °C) with pBSoriC (0.1 μg) for various times. Samples were filtered through nitrocellulose membranes. DnaA-bound ATP and ADP were extracted from the filters with 1 m formic acid (100 μl). Nucleotide extracts (5 μl) were spotted onto thin layer PEI Cellulose plates (EM Science), ADP and ATP were separated using a solvent of 1 m HCOOH and 0.4 m LiCl, the plates were dried, and radioactivity was visualized and measured using a Molecular Dynamics Storm 840 imaging system. Internal ATP and ADP standards were visualized by UV absorption. RIDA Sensitivity—Histidine-tagged wild-type DnaA and DnaA(L366K) were incubated with a crude cell extract or bovine serum albumin in the presence of DNA and ATP. A portion of the mixture was then used as a source of DnaA for in vitro replication in a crude cell extract as described previously (7Katayama T. Crooke E. J. Biol. Chem. 1995; 270: 9265-9271Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). oriC Binding—[3H]pBSoriC was prepared by in vitro methylation with HhaI methylase as described (28Yung B.Y. Kornberg A. J. Biol. Chem. 1989; 264: 6146-6150Abstract Full Text PDF PubMed Google Scholar). Histidine-tagged wild-type and mutant DnaA proteins were incubated (25 °C, 10 min) with radiolabeled DNA in buffer DB (25 mm HEPES·KOH (pH 7.5 at 1 m), 0.5 mm magnesium acetate, 17% (v/v) glycerol, 0.005% (v/v) Triton X-100, 0.3 mm EDTA, and 2 mm dithiothreitol). Samples were filtered through nitrocellulose filters (Type HA, Millipore) presoaked in buffer DB. Filters were washed with 5 ml of ice-cold buffer DB, dried, and retained DNA was measured by liquid scintillation counting. DNA Replication in a Crude Cell-free Extract—In vitro replication of oriC-plasmid M13mpRE85 in a crude enzyme fraction (Fraction II) was performed basically as described (29Fuller R.S. Kaguni J.M. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 7370-7374Crossref PubMed Scopus (240) Google Scholar, 30Crooke E. Methods Enzymol. 1995; 262: 500-506Crossref PubMed Scopus (20) Google Scholar). Briefly, a crude protein fraction was prepared from WM433 (dnaA204(Ts)) by a freeze-thaw lysis, isolation of the soluble extract, and ammonium sulfate fractionation. DNA synthesis reactions (25 μl) contained: 40 mm HEPES·KOH (pH 7.6 at 1 m); 10 mm magnesium acetate; 40 mm phosphocreatine; 2.5 μg of creatine kinase; 7% (w/v) polyvinyl alcohol (average molecular weight 30,000–70,000); 2 mm ATP; 0.5 mm each GTP, CTP, and UTP; 0.1 mm each dATP, dGTP, dCTP, and [α-32P]TTP (∼250 mCi/mmol); 200 μg of the crude protein extract prepared from WM433; and 200 ng (600 pmol as nucleotide) of M13mpRE85 replicative form I DNA. Histidine-tagged wild-type or mutant DnaA protein was added to the reaction, and the preparation was incubated at 30 °C for 20 min. Nucleotides incorporated into acid-insoluble materials were retained on GF/C filters (Millipore) and measured by liquid scintillation counting. DNA Replication with Defined Components—In vitro replication of pBSoriC, dependent on eight purified replication proteins, was largely performed as described (30Crooke E. Methods Enzymol. 1995; 262: 500-506Crossref PubMed Scopus (20) Google Scholar). Reactions (25 μl) contained: 30 mm Tricine·KOH (pH 8.25); 7 mm magnesium acetate; 2 mm ATP; 0.5 mm each GTP, CTP, and UTP; 0.1 mm each dATP, dGTP, dCTP, and [α-32P]TTP (∼250 mCi/mmol); 200 ng (600 pmol as nucleotide) pBSoriC; 450 ng of single strand binding protein (SSB); 10 ng of HU; 400 ng of gyrase A subunit; 180 ng of gyrase B subunit; 150 ng of DnaB·DnaC complex; 12 ng of DnaG primase; 500 ng of DNA polymerase III*; 25 ng of DNA polymerase III holoenzyme β-subunit; and the indicated amounts of DnaA proteins. The mixtures were assembled at 0 °C and incubated at 30 °C for 20 min. Nucleotides incorporated into acid-insoluble materials were retained on GF/C filters (Millipore) and measured by liquid scintillation counting. Extraction of E. coli Phospholipids—A frozen suspension (5 g) of W3110 cells was thawed, mixed with a solution (20 ml) of 0.12 m sodium acetate (pH 4.8), and extracted with chloroform (25 ml) and methanol (55 ml). Cell debris was removed by centrifugation (5 min, 7,000 × g, 20 °C), and the supernatant was made biphasic by the addition of chloroform and water to a final chloroform:methanol:water ratio of 2:2:1.8. The aqueous phase was removed, and the organic layer was dried under a stream of nitrogen gas. The residue was dissolved in diethyl ether (0.5 ml), mixed with nitrogen gas-purged acetone (25 ml) that contained β-mercaptoethanol (1 mm), and stirred for 3 h. The insoluble lipids were collected by centrifugation, dried under nitrogen gas, and dissolved in a minimum volume of chloroform. The amount of phosphorous was determined (31Ames B.V. Dubin D.T. J. Biol. Chem. 1960; 235: 769-775Abstract Full Text PDF PubMed Google Scholar), and the concentration of phospholipids calculated assuming an average molecular weight of 700. Preparation of Lipid Vesicles—To prepare small unilamellar vesicles, phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, or E. coli phospholipids in CHCl3 were dried under a stream of nitrogen gas and suspended in water by sonication (15 min, 0 °C) with 30% bursts with a microtip probe sonicator (Branson). Sonicated lipids were centrifuged (10 min, 140,000 × g, 4 °C) and the supernatants, which contained small unilamellar vesicles, were collected. Phospholipid concentrations were measured by a phosphomolybdate colorimetric assay (31Ames B.V. Dubin D.T. J. Biol. Chem. 1960; 235: 769-775Abstract Full Text PDF PubMed Google Scholar). Other Methods—Protein concentrations were determined by the Bradford dye-binding method using bovine serum albumin as a standard. Nucleotide Binding by DnaA(L366K) and Wild-type DnaA Is Similar—DnaA(L366K) possesses high affinities for ATP and ADP with dissociation constants of 135 and 58 nm, respectively (Table I), values similar to those observed for wild-type DnaA (Table I and Ref. 6Sekimizu K. Bramhill D. Kornberg A. Cell. 1987; 50: 259-265Abstract Full Text PDF PubMed Scopus (348) Google Scholar). In addition to similar binding affinities, the molar ratios of bound nucleotide to protein were indistinguishable between DnaA and DnaA(L366K), whether for the binding of ATP or ADP (Table I).Table INucleotide-binding properties of DnaA and DnaA(L366K)DnaADnaA(L366K)ATP bindingKD (μm)0.033 ± 0.0050.058 ± 0.013ATP/DnaA0.30 ± 0.090.36 ± 0.03ADP bindingKD (μm)0.095 ± 0.0230.135 ± 0.03ADP/DnaA0.43 ± 0.060.59 ± 0.32 Open table in a new tab DnaA(L366K) Retains Specificity of Acidic Phospholipids for Nucleotide Release—The leucine to lysine amino acid substitution in DnaA does not alter the requirement of the protein that phospholipid bilayers must contain anionic species to promote release of bound nucleotide from DnaA. DnaA and DnaA(L366K) respond identically in the release of bound ADP when treated with increasing amounts of phosphatidylglycerol vesicles (Fig. 1A). Likewise, both the wild-type and mutant forms of DnaA retain the bound nucleotide when exposed to lipid bilayers containing only phosphatidylcholine (Fig. 1B), a zwitterionic phospholipid previously shown to be as equally poor as phosphatidylethanolamine in promoting nucleotide release (14Castuma C.E. Crooke E. Kornberg A. J. Biol. Chem. 1993; 268: 24665-24668Abstract Full Text PDF PubMed Google Scholar). In addition, both forms of the protein responded equally well to phospholipids extracted from E. coli cells (Fig. 1C), which have a membrane phospholipid composition of phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin at an approximate ratio of 70:22:8. Thus, DnaA(L366K) does not appear to facilitate growth in acidic phospholipid-deficient cells through a mechanism of protein rejuvenation by neutral membranes. DnaA(L366K) Has Intrinsic ATPase Activity—DnaA(L366K) retains the intrinsic ATPase activity of wild-type DnaA. Bound ATP is slowly hydrolyzed with half being consumed in ∼45 min (Fig. 2), kinetics comparable with what has previously been seen for purified DnaA under similar conditions (6Sekimizu K. Bramhill D. Kornberg A. Cell. 1987; 50: 259-265Abstract Full Text PDF PubMed Scopus (348) Google Scholar, 27Li Z. Crooke E. Protein Expression Purif. 1999; 17: 41-48Crossref PubMed Scopus (27) Google Scholar). And as with the wild-type protein, the resulting ADP from ATP hydrolysis remains bound to DnaA(L366K) (Fig. 2). Therefore, the point mutation in DnaA(L366K) does not affect the protein conversion from the active ATP form to inactive ADP-DnaA. DnaA(L366K) Is Sensitive to RIDA—DnaA(L366K) treated with crude cell extracts showed diminished replication activity (data not shown), indicating that it is sensitive to RIDA. Furthermore, in vivo experiments have shown that deletion of the hda gene (12Camara J.E. Skarstad K. Crooke E. J. Bacteriol. 2003; 185: 3244-3248Crossref PubMed Scopus (48) Google Scholar), an essential component of RIDA (10Kato J. Katayama T. EMBO J. 2001; 20: 4253-4262Crossref PubMed Scopus (217) Google Scholar), failed to alleviate the growth arrest in cells lacking adequate levels of acidic phospholipids (data not shown). Both of these findings support the conclusion that the growth rescuing phenomenon of DnaA(L366K) is not caused by its resistance to Hda-mediated inactivation. DnaA(L366K) Is Feeble at Initiating Replication from oriC in Vitro—DnaA(L366K), although indistinguishable from the wild-type protein in its nucleotide binding and hydrolysis and requirement for acidic phospholipids to promote nucleotide release, is poor at directing replication from oriC in a system reconstituted with purified proteins (Fig. 3A). Wild-type DnaA produced maximal DNA synthesis when levels of protein that represent DnaA to oriC ratios of ∼10–12 DnaA molecules per oriC were used, similar to previous data. However, when DnaA(L366K) was used, even at a level of 18 mutant protein molecules per oriC, only a very low amount of DNA synthesis resulted (Fig. 3A). Moreover, DnaA(L366K) was poor at initiating replication in a crude cell extract that lacked DnaA activity because it was prepared from a dnaA temperature-sensitive strain. Again, maximal synthesis was seen when the reactions contained ∼10 wild-type DnaA molecules per oriC.In contrast, DnaA(L366K) was completely inactive despite being present at up to 20 protein molecules per oriC (Fig. 3B). oriC Binding Activity of DnaA(L366K) Is Comparable with That of DnaA—The inability of DnaA(L366K) to initiate replication is not due its failure to bind oriC. The wild-type and the mutant forms of DnaA were equally effective at binding to supercoiled oriC plasmids as determined by a filter-retention assay (Fig. 4). In agreement with the in vitro DNA replication results, DNA binding reached a plateau of about 130 fmol of template bound, corresponding to ∼15 DnaA or DnaA(L366K) molecules per origin. ATP-DnaA(L366K) Augments Limiting Levels of Wild-type DnaA in Initiating Replication from oriC in Vitro—The mutant form of the protein, although feeble at initiating replication, is able to participate in efficient replication when coupled with minimal levels of wild-type DnaA. Only 53 pmol of DNA synthesis occurred when ATP-DnaA was added at the level of 2 DnaA molecules per oriC (Fig. 5A). When that limiting level of DnaA was combined with either additional wild-type DnaA or DnaA(L366K) protein, robust DNA synthesis ensued with only slightly more DnaA(L366K) needed to obtain the same level of synthesis possible with wild-type DnaA (Fig. 5A). Like wild-type DnaA, DnaA(L366K) needs to be in its ATP form to actively initiate replication from oriC. As expected, the limiting amount of 2 ADP-DnaA molecules per oriC completely failed to initiate replication, being indistinguishable from reactions lacking any DnaA protein (Fig. 5B). However, when supplemented with ATP-DnaA(L366K), over 500 pmol of DNA synthesis was obtained with a total of 13 DnaA molecules per oriC (Fig. 5B). On the other hand, supplementing the ADP-DnaA with ADP-DnaA(L366K) resulted in poor DNA synthesis. Thus, the nucleotide requirements for DnaA(L366K) to initiate chromosomal replications are similar to those previously seen for the wild-type protein (6Sekimizu K. Bramhill D. Kornberg A. Cell. 1987; 50: 259-265Abstract Full Text PDF PubMed Scopus (348) Google Scholar, 15Crooke E. Castuma C.E. Kornberg A. J. Biol. Chem. 1992; 267: 16779-16782Abstract Full Text PDF PubMed Google Scholar). DnaA(L366K) Must Act in Conjunction with DnaA in Vivo— Replacement of the chromosomal copy of dnaA with the gene for DnaA(L366K) by homologous recombination (32Slater S. Maurer R. J. Bacteriol. 1993; 175: 4260-4262Crossref PubMed Google Scholar) occurred efficiently when cells carried a low copy plasmid expressing dnaA under its own promoter. Specifically, following the second cross-over event, 18 of 20 candidates screened had replaced their chromosomal dnaA with the gene encoding the mutant protein. When cells arising from successful allelic replacement were cured of the expression plasmid harboring wild-type dnaA, 10 of 10 candidates examined had reverted to the wild-type gene at the dnaA locus. Thus, cells cannot tolerate dnaA(L366K) as the sole allele for dnaA. In agreement, in the absence of a plasmid-encoded dnaA, allelic replacement completely failed, with 0 of 100 candidates having exchanged wild-type dnaA for the mutant allele following the second cross-over event. Similar results were seen when cells were depleted of their acidic phospholipids prior to selecting for the second crossover event. Another DnaA with Membrane-binding Domain Mutations Behaves Like DnaA(L366K)—Mizushima and co-workers (33Hase M. Yoshimi T. Ishikawa Y. Ohba A. Guo L. Mima S. Makise M. Yamaguchi Y. Tsuchiya T. Mizushima T. J. Biol. Chem. 1998; 273: 28651-28656Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) have also constructed DnaA proteins that contain alterations in the membrane-binding domain. Specifically, basic lysine and arginine residues, singularly or in tandem, were substituted with the acidic amino acid glutamate. The purified mutant proteins retained their capacity to bind adenine nucleotide, and it was reported that DnaA proteins that had the lysine at position 372 substituted with glutamate (K372E) had ∼4-fold decreased rates for the release of bound nucleotide when compared with wild-type DnaA (33Hase M. Yoshimi T. Ishikawa Y. Ohba A. Guo L. Mima S. Makise M. Yamaguchi Y. Tsuchiya T. Mizushima T. J. Biol. Chem. 1998; 273: 28651-28656Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). These kinetic measurements were performed with a low molar ratio of cardiolipin to protein. Working with DnaA(R360E, R364E, K372E) (termed DnaA431 in Ref. 33Hase M. Yoshimi T. Ishikawa Y. Ohba A. Guo L. Mima S. Makise M. Yamaguchi Y. Tsuchiya T. Mizushima T. J. Biol. Chem. 1998; 273: 28651-28656Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) that we purified, as well as some kindly provided by T. Mizushima, we obtained a similar 3- to 4-fold decreased rate in nucleotide release from DnaA431 compared with wild-type DnaA when examined at the same low cardiolipin to DnaA ratio (Fig. 6A, squares). However, when cardiolipin levels were increased just 2-fold, the difference seen between DnaA431 and wild-type decreased (Fig. 6A, triangles), and with the concentration of cardiolipin increased 4-fold, the rate of release of nucleotide from DnaA431 approached that for wild-type DnaA (Fig. 6A, circles). The similar behavior of DnaA and DnaA431 when exposed to acidic phospholipids is further illustrated by their comparable response to treatment with cardiolipin vesicles over a 10-fold concentration range (Fig. 6B). We further examined DnaA431 using vesicles composed of lipids other than 100% cardiolipin. When treated with acidic liposomes composed of 80% phosphatidylglycerol and 20% phosphatidylcholine nucleotide release from DnaA431 and wild-type DnaA were indistinguishable, and DnaA431, like wild-type DnaA, was inert to 100% phosphatidylcholine vesicles (Fig. 6C). Moreover, DnaA431 and wild-type DnaA bound equally well to the acidic and not neutral liposomes (100% phosphatidylcholine) as analyzed by isopynic centrifugation as previously described (34Kitchen J.L. Li Z. Crooke E. Biochemistry. 1999; 38: 6213-6221Crossref PubMed Scopus (25) Google Scholar) (data not shown). Furthermore, DnaA431 protein responded like wild-type DnaA when treated with vesicles composed of phospholipids extracted from E. coli cells (Fig. 6D). Thus, DnaA431 and wild-type DnaA behave in a largely similar manner when treated with acidic phospholipid-containing membranes other than the specific case of low levels of liposomes composed solely of cardiolipin. Like DnaA(L366K) (25Zheng W. Li Z. Skarstad K. Crooke E. EMBO J. 2001; 20: 1164-1172Crossref PubMed Scopus (41) Google Scholar), DnaA431 restored growth to acidic phospholipid-deficient cells but cannot be the sole source of cellular DnaA (data not shown). The biochemical properties of two different DnaA proteins that possess mutations in the membrane-binding region are similar, and both proteins permit growth in cells lacking acidic phospholipids, perhaps through a common mechanism. A genetic and mutational analysis provided the most direct evidence that there is an in vivo link between DnaA protein function and the lipid composition of the cellular membrane (25Zheng W. Li Z. Skarstad K. Crooke E. EMBO J. 2001; 20: 1164-1172Crossref PubMed Scopus (41) Google Scholar). Specifically, cells that have depleted levels of acidic phospholipids, which normally would be arrested for growth, are able to keep growing when DnaA(L366K) is expressed. That a single amino acid substitution in the initiator of chromosomal replication, DnaA protein, can restore growth to cells that have stopped growing because of an altered membrane lipid composition argues for a physiologically relevant relationship between DnaA and the cell membrane. Previous biochemical studies suggest anionic lipids participate in the regulation of DnaA protein. Fluid bilayers possessing adequate levels of acidic phospholipids promote the release of ADP from DnaA, and when the protein is bound to DNA an exchange of ATP for ADP can occur, thereby rejuvenating inactive ADP-DnaA into active ATP-DnaA (13Sekimizu K. Kornberg A. J. Biol. Chem. 1988; 263: 7131-7135Abstract Full Text PDF PubMed Google Scholar, 14Castuma C.E. Crooke E. Kornberg A. J. Biol. Chem. 1993; 268: 24665-24668Abstract Full Text PDF PubMed Google Scholar, 15Crooke E. Castuma C.E. Kornberg A. J. Biol. Chem. 1992; 267: 16779-16782Abstract Full Text PDF PubMed Google Scholar). Recently, it was observed that basic and neutral phospholipids block acidic phospholipids from promoting nucleotide release from Staphylococcus aureus DnaA (35Ichihashi N. Kurokawa K. Matsuo M. Kaito C. Sekimizu K. J. Biol. Chem. 2003; 278: 28778-28786Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). In view of these biochemical findings, purified DnaA(L366K) was examined here to determine whether the molecular mechanism of how the mutant protein supports growth of cells lacking acidic phospholipids is based on the protein being altered in its adenine nucleotide binding and hydrolysis properties or its response to treatment with fluid lipid bilayers. For each of these aspects studied, DnaA(L366K) was largely similar to wild-type DnaA, thereby suggesting that the function of wild-type DnaA adversely affected in acidic phospholipid-deficient cells is something other than nucleotide binding, hydrolysis, and exchange. Although DnaA(L366K) has origin binding activity, the mutant protein is unable to initiate replication from oriC in vitro (Fig. 3) or serve as the sole source of DnaA activity in vivo (25Zheng W. Li Z. Skarstad K. Crooke E. EMBO J. 2001; 20: 1164-1172Crossref PubMed Scopus (41) Google Scholar). Thus, based upon known initiation functions of DnaA protein, DnaA(L366K) acting on its own may not possess the ability to properly oligomerize at oriC and promote strand opening of the AT-rich 13-mers or to successfully recruit the replicative helicase, DnaB, to the opened origin DNA. Analogously, wild-type DnaA is no longer able to carry out all of its necessary chromosomal replication functions in cells lacking sufficient levels of acidic phospholipids (25Zheng W. Li Z. Skarstad K. Crooke E. EMBO J. 2001; 20: 1164-1172Crossref PubMed Scopus (41) Google Scholar). The absence of acidic phospholipids does not appear to cause growth arrest simply by depleting the concentration of available DnaA because high level expression of additional wild-type DnaA failed to restore growth (25Zheng W. Li Z. Skarstad K. Crooke E. EMBO J. 2001; 20: 1164-1172Crossref PubMed Scopus (41) Google Scholar). Instead, an essential DnaA function is most likely perturbed in acidic phospholipid-deficient cells, and growth only occurs when DnaA(L366K) provides this missing function. In such a case, the function of wild-type DnaA affected in acidic phospholipid-deficient cells must be distinct from the missing function of DnaA(L366K). Because both proteins have similar nucleotide binding, hydrolysis, and exchange characteristics in vitro as shown here, none of these functions seem to be the DnaA function adversely affected by the absence of acidic phospholipids, as originally speculated (25Zheng W. Li Z. Skarstad K. Crooke E. EMBO J. 2001; 20: 1164-1172Crossref PubMed Scopus (41) Google Scholar). The results from the in vitro studies presented here support the argument that DnaA(L366K) acts in conjunction with wild-type DnaA, with DnaA and DnaA(L366K) forming functional mixed oligomers at oriC. Specifically, DnaA(L366K) on its own is unable to support DNA replication (Fig. 3) as is an insufficient concentration of wild-type DnaA (Fig. 5). Yet, origins with limiting amounts of wild-type DnaA can be activated for replication when augmented with DnaA(L366K) (Fig. 5). With neither form of DnaA able to initiate replication from oriC on its own under these conditions (Figs. 3 and 5), the most likely possibility is that DnaA and DnaA(L366K) work together at oriC as a mixed oligomer, facilitating DNA replication (Fig. 5). In agreement with the in vivo and in vitro data that DnaA and DnaA(L366K) cooperatively initiate replication at oriC, Kaguni and co-workers (21Simmons L.A. Felczak M. Kaguni J.M. Mol. Microbiol. 2003; 49: 849-858Crossref PubMed Scopus (67) Google Scholar) recently demonstrated that two inactive mutant DnaA proteins can complement each other for DNA replication from oriC in vivo even when one of the DnaA proteins is defective at DNA binding. In support, molecular modeling based on the crystal structure of a truncated form of A. aeolicus DnaA (18Erzberger J.P. Pirruccello M.M. Berger J.M. EMBO J. 2002; 21: 4763-4773Crossref PubMed Scopus (193) Google Scholar) suggests that productive DnaA protomer-protomer association can occur between domain III regions of neighboring DnaA molecules without participation of the DNA binding region. The domain III putative protomer-protomer interactions sites are distinct from residue 366 as well as residues 360, 364, and 372 (the amino acids substituted in DnaA431), all of which lie within a long amphipathic helix that connects domains III and IV (18Erzberger J.P. Pirruccello M.M. Berger J.M. EMBO J. 2002; 21: 4763-4773Crossref PubMed Scopus (193) Google Scholar). According to gel filtration and sedimentation data, purified DnaA exists as a monomer in solution (3Crooke E. Thresher R. Hwang D.S. Griffith J. Kornberg A. J. Mol. Biol. 1993; 233: 16-24Crossref PubMed Scopus (87) Google Scholar, 27Li Z. Crooke E. Protein Expression Purif. 1999; 17: 41-48Crossref PubMed Scopus (27) Google Scholar, 36Hwang D.S. Crooke E. Kornberg A. J. Biol. Chem. 1990; 265: 19244-19248Abstract Full Text PDF PubMed Google Scholar, 37Fuller R.S. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 5817-5821Crossref PubMed Scopus (190) Google Scholar), as does DnaA(L366K). Moreover, DnaA(L366K), which is isolated from cells that also express wild-type DnaA, emerges from gel filtration as a monomer during its purification, indicating that DnaA(L366K) and wild-type DnaA do not oligomerize in solution. In the presence of oriC, multiple copies of DnaA protein cluster on the origin, as detected by electron microscopy (1Fuller R. Funnell B. Kornberg A. Cell. 1984; 38: 889-900Abstract Full Text PDF PubMed Scopus (464) Google Scholar, 2Funnell B. Baker T.A. Kornberg A. J. Biol. Chem. 1987; 262: 10327-10334Abstract Full Text PDF PubMed Google Scholar, 3Crooke E. Thresher R. Hwang D.S. Griffith J. Kornberg A. J. Mol. Biol. 1993; 233: 16-24Crossref PubMed Scopus (87) Google Scholar), filter retention (27Li Z. Crooke E. Protein Expression Purif. 1999; 17: 41-48Crossref PubMed Scopus (27) Google Scholar, 37Fuller R.S. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 5817-5821Crossref PubMed Scopus (190) Google Scholar), footprinting (1Fuller R. Funnell B. Kornberg A. Cell. 1984; 38: 889-900Abstract Full Text PDF PubMed Scopus (464) Google Scholar, 3Crooke E. Thresher R. Hwang D.S. Griffith J. Kornberg A. J. Mol. Biol. 1993; 233: 16-24Crossref PubMed Scopus (87) Google Scholar, 38Hwang D.S. Kornberg A. J. Biol. Chem. 1992; 267: 23083-23086Abstract Full Text PDF PubMed Google Scholar, 39Cassler M.R. Grimwade J.E. Leonard A.C. EMBO J. 1995; 14: 5833-5841Crossref PubMed Scopus (110) Google Scholar, 40Cassler M.R. Grimwade J.E. McGarry K.C. Mott R.T. Leonard A.C. Nucleic Acids Res. 1999; 27: 4570-4576Crossref PubMed Scopus (12) Google Scholar, 41Ryan V.T. Grimwade J.E. Camara J.E. Crooke E. Leonard A.C. Mol. Microbiol. 2004; 51: 1347-1359Crossref PubMed Scopus (99) Google Scholar), and immunoblotting (4Carr K.M. Kaguni J.M. J. Biol. Chem. 2001; 276: 44919-44925Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 21Simmons L.A. Felczak M. Kaguni J.M. Mol. Microbiol. 2003; 49: 849-858Crossref PubMed Scopus (67) Google Scholar). The filter retention stoichiometry data presented here (Fig. 4) indicate that DnaA(L366K) oligomerizes at oriC, and the observed efficient DNA replication (Fig. 5) provides evidence that DnaA and DnaA(L366K) form functional mixed oligomers at oriC in vitro. Beyond permitting growth in cells lacking acidic phospholipids, DnaA(L366K) affects replication in cells with a normal membrane composition. Expression of plasmid-borne DnaA(L366K) in cells possessing chromosomally encoded wild-type dnaA and carrying out ordinary phospholipid synthesis results in reduced initiation events (25Zheng W. Li Z. Skarstad K. Crooke E. EMBO J. 2001; 20: 1164-1172Crossref PubMed Scopus (41) Google Scholar) consistent with the concept that the mutant and wild-type DnaA proteins affect the same origin, acting as a mixed oligomer. The results presented here suggest that the mechanism by which DnaA(L366K) supports growth in cells with low levels of acidic phospholipids is independent of the nucleotide binding and hydrolysis characteristics of the protein as well as how it responds to acidic versus neutral lipids with respect to nucleotide release. Supporting this argument, another DnaA protein with amino acid substitutions in the membrane-binding domain, DnaA431, also rescues acidic phospholipid-deficient cells in vivo and behaves like DnaA(L366K) when exposed to phospholipids in vitro. Defining what domains of DnaA(L366K) are essential versus dispensable for growth to occur in acidic phospholipid-deficient cells will likely give insight into the essential function of DnaA that is perturbed in acidic phospholipid-deficient cells and may reveal a novel acidic phospholipid-dependent function of DnaA in the chromosomal replication cycle." @default.
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• W2045574547 title "Restoration of Growth to Acidic Phospholipid-deficient Cells by DnaA(L366K) Is Independent of Its Capacity for Nucleotide Binding and Exchange and Requires DnaA" @default.
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