©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
DnaA Protein Is Sensitive to a Soluble Factor and Is Specifically Inactivated for Initiation of in Vitro Replication of the Escherichia coli Minichromosome (*)

Tsutomu Katayama (1) (2)(§), Elliott Crooke (2)

From the (1) Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305 and the (2) Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, Washington, D. C. 20007

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

DnaA protein loses the capacity to initiate chromosomal replication when treated with a soluble cell extract. This inactivation depends upon DNA and hydrolyzable ribonucleoside triphosphate. The extract does not affect the activities of other replicative proteins or the ability of DnaA to initiate replication of single-stranded DNA that contains a DnaA-binding hairpin, indicating that the inhibitory effect is specific for the action of DnaA at oriC. Gel filtration experiments implicate a 150-kDa factor as being responsible. Mutant DnaAcos protein, which causes overinitiation in vivo, is insensitive to the inactivating factor, suggesting a requirement for this negative control in vivo. We propose that a soluble factor controls initiation through down-regulation of DnaA protein.


INTRODUCTION

Initiation of genomic replication in Escherichia coli occurs at a unique origin ( oriC) on the chromosome and is responsible for coupling replication to the cell cycle (1, 2) . DnaA protein, the key initiation factor, binds four 9-mer DnaA boxes in the minimal oriC origin (245 base pairs). This complex promotes the opening of 13-mer AT-rich regions in the minimal origin and directs the DnaC-mediated delivery of DnaB helicase to the exposed single strands. The complementary strands are subsequently synthesized by a replisome that includes DnaB, DnaG primase, and DNA polymerase III holoenzyme.

The tight binding of ATP and ADP to DnaA protein ( K= 0.03 and 0.1 µ M, respectively) has been shown to have a profound influence on the protein's activity for initiation in vitro (3, 4, 5) . The ATP-bound form of DnaA protein is active for initiating replication; the ADP form, while capable of binding oriC, is inefficient for catalyzing strand separation and thus feeble for initiation. However, the cellular presence of the nucleotide-bound form is still unknown.

DnaA protein is present at a level of >1000 molecules/cell (6, 7) , although only 20 molecules of the protein seem to be involved in the oriC complex (8, 9) . The cellular amount and synthesis of DnaA protein do not oscillate during the cell division cycle, and the protein is stable for at least 90 min in growing cells (10) . Since initiation occurs properly in a strain in which the dnaA gene is expressed constitutively using the lac promoter (11) , modulation of DnaA protein activity may be responsible for regulating initiation rather than cyclic expression of the dnaA gene.

In vivo roles of DnaA protein include an apparent regulation of the ``on-off switch'' of chromosomal initiation. Overproduction of DnaA protein in vivo can shift the timing of initiation to a point earlier in the cell cycle (11) . Certain dnaA mutants can cause excessive initiation (7, 10, 12, 13, 14, 15, 16) .

The dnaAcos mutant is cold-sensitive (30 °C) for growth and overinitiates the replication of its chromosome at the nonpermissive temperature (14) . Excessive initiation in dnaAcos cells appears to be due to alterations in the regulation of DnaA function rather than to an oversupply of the mutant form of the protein (7) . Whereas de novo protein synthesis is absolutely necessary for new rounds of initiation in wild-type dnaA cells, hyperinitiation in the dnaAcos mutant can occur without concomitant protein synthesis (14) .

The specific activities of purified DnaAcos protein and wild-type DnaA protein for catalyzing minichromosome ( oriC plasmid) replication in a crude in vitro system at 30 °C are very similar (17) . However, there are significant differences between DnaAcos protein and the wild-type form; DnaAcos protein is unable to bind ATP and ADP, and as such, these nucleotides are unable to influence the activity of the mutant protein. Strikingly, DnaAcos protein also has the capacity to initiate replication for longer periods in vitro; whereas initiation by wild-type DnaA protein (0.40 pmol) ceases after 15 min in a crude replication system, that by DnaAcos protein continues for at least 45 min (17) . These findings suggest the possibility that a considerable fraction of wild-type DnaA protein in cells is inactivated, whereas DnaAcos protein is not (7) .

Here we present evidence that an activity in the soluble fraction of a cell lysate is capable of inactivating wild-type DnaA protein specifically for minichromosome replication. The initiation potential of DnaAcos protein is not affected by exposure to the soluble fraction. Thus, we propose that a soluble factor may be an important regulator controlling initiation frequency. The insensitivity of DnaAcos protein to the negative factor coincides with the nonattenuated activity in vitro and the overinitiation trait in vivo.


EXPERIMENTAL PROCEDURES

Materials

Sources were as follows: deoxyribonucleoside triphosphates and Sephacryl S-300 HR, Pharmacia Biotech Inc.; ribonucleoside di- and triphosphates, HEPES, phosphocreatine, and rifampicin, Sigma; [-P]TTP (800 Ci/mmol), Amersham Corp.; creatine kinase and ATPS,() Boehringer Mannheim; X174 RF I DNA, Life Technologies, Inc.; and DEAE-cellulose (DE52), Whatman.

In Vitro DNA Synthesis Complementation Reaction

Standard reactions (25 µl) contained 40 m M HEPES/KOH (pH 7.6 at 1 M); 2 m M ATP; 0.5 m M each CTP, GTP, and UTP; 100 µ M each dATP, dCTP, dGTP, and [-P]TTP (70-130 cpm/pmol); 11 m M magnesium acetate; 7% (w/v) polyvinyl alcohol ( M30,000-70,000); 40 m M phosphocreatine; 100 µg/ml creatine kinase; crude fraction II cell extract (18) (200-250 µg of protein) prepared from WM433 ( dnaA204) cells (15) ; and M13mpRE85 (19) oriC plasmid template DNA (600 pmol as nucleotide; 38 fmol as circle). The reactions were complemented with purified monomeric DnaA (52 kDa; 0.96 10units/mg) or DnaAcos (0.84 10units/mg) protein and incubated for 20 min at 30 °C. Nucleotides incorporated into acid-insoluble material were trapped on GF/C filters and measured by liquid scintillation counting.

Inactivation of DnaA Protein (First-stage Reaction)

Reactions were assembled at 0 °C and incubated at 30 °C for the indicated times. Typically, reactions (25 µl) contained 40 m M HEPES/KOH (pH 7.6 at 1 M), 2 m M ATP, 11 m M magnesium acetate, 7% (w/v) polyvinyl alcohol ( M30,000-70,000), 40 m M phosphocreatine, 100 µg/ml creatine kinase; 1.0 µg of M13mpRE85 or X174 RF I DNA (3.0 nmol as nucleotide), 3.8 pmol of wild-type DnaA or DnaAcos protein, and 200 µg (unless indicated otherwise) of a WM433 extract (fraction II noted above). Portions (5 µl) of the mixtures were added to in vitro DNA synthesis reactions (second-stage reaction) to measure DnaA protein activity remaining after the inactivation procedure (``residual activity''). One unit of inactivation corresponds to a 1-unit decrease in the specific activity of DnaA protein/min at 30 °C.

Formation of ATP-DnaA Protein

DnaA protein (11.5 pmol) was added to mixtures (15 µl) containing 50 m M HEPES/KOH (pH 7.6 at 1 M), 2.5 m M magnesium acetate, 0.3 m M EDTA, 20% (v/v) glycerol, 0.007% Triton X-100, 7 m M dithiothreitol, and 1 µ M ATP and incubated for 15 min at 0 °C (3, 20) . For nucleotide-free DnaA protein, the same conditions were used, except that ATP was omitted.

ABC Primosome System for ssDNA Replication

Reaction conditions were the same as those described for replication of X174 ssDNA (21) , except for the addition of rifampicin (40 µg/ml), phosphocreatine (50 m M), and creatine kinase (100 µg/ml). Each reaction (20 µl) included WM433 fraction II crude extract (100 µg of protein) and M13 A-site ssDNA (300 pmol as nucleotide; 41 fmol as circle) (22) as template. DnaA protein (provided as a component of the first-stage reaction) was added, and the samples were incubated at 30 °C for 10 min. Nucleotides incorporated into acid-insoluble material were trapped on GF/C filters and measured by liquid scintillation counting.

Other Methods

Immunoblot analysis (7) , density centrifugation (23) , and agarose (7%) gel electrophoresis (24) were performed as described. Commercially obtained X174 RF I DNA was further purified by treatment in ammonium acetate (2.6 M) and ethidium bromide (0.4 mg/ml), extracted twice with phenol/chloroform (1:1), and precipitated in ethanol as described (25) . Protein concentrations were measured with the Bradford method (26) using bovine serum albumin as a standard.


RESULTS

DnaA Protein Is Inactivated in a Soluble Cell Extract; DnaAcos Protein Is Not

Unlike wild-type DnaA protein, DnaAcos protein is capable of catalyzing multiple rounds of replication in vivo without de novo protein synthesis (14) . In a crude in vitro replication system, DnaAcos protein can initiate replication of oriC plasmids for a longer time than the wild-type form (17) . Since an unknown secondary factor may modulate DnaA protein activity, we have examined whether the activities of wild-type DnaA and DnaAcos proteins are altered when incubated with a crude cell extract (Fig. 1).


Figure 1: Wild-type DnaA protein, but not DnaAcos protein, loses activity following exposure to a soluble cell extract. DnaA and DnaAcos proteins (7.7 pmol) were added to standard first-stage reactions (50 µl) that contained M13mpRE85 (2.0 µg) and were incubated at 30 °C (see ``Experimental Procedures''). A, at the indicated times, 5-µl portions were transferred to in vitro DNA synthesis complementation reactions (25 µl; 20 min, 30 °C) (see ``Experimental Procedures''). DnaA protein activities remaining after the first-stage periods ( Residual activity) were compared with those obtained without the first-stage incubation (time = 0 min). Activities of 100% correspond to 464 and 390 pmol of DNA synthesized for wild-type DnaA protein (DnaA) and DnaAcos protein, respectively. B, at the indicated times, 5-µl portions of the first-stage reaction including wild-type DnaA protein were mixed with trichloroacetic acid (100 µl of a 5% solution) and kept on ice for at least 20 min. Precipitated proteins were dissolved in SDS-polyacrylamide gel electrophoresis sample buffer, and levels of DnaA protein were determined by immunoblotting with anti-DnaA protein antiserum (7). The arrow identifies the band for DnaA protein. The background control, which included all components except for DnaA protein, was similarly treated. C, at indicated times, 5-µl portions were treated with SDS (150 µl of a 0.5% solution), and M13mpRE85 DNA (7.9 kilobases) was extracted with phenol and precipitated in ethanol (27). Recovered DNA was analyzed by agarose (7%) gel electrophoresis. DNA was visualized with ethidium bromide (0.5 µg/ml). The sizes of molecular size markers ( HindIII-digested DNA) are indicated in kilobases ( kb). F I, F II, and F III indicate the migration of supercoiled, nicked circular, and linearized forms, respectively, of M13mpRE85 DNA.



While DnaAcos protein remained active after exposure to a soluble protein extract, the activity of wild-type DnaA protein decreased to background levels (Fig. 1 A). Immunoblot analysis with anti-DnaA serum detected no significant decrease in either the level of DnaA protein or its apparent molecular mass during treatment with the protein extract (Fig. 1 B). Thus, the inactivation of DnaA protein does not seem to be due to overall proteolytic degradation, a result consistent with the stability of DnaA protein in vivo (10) .

The oriC plasmid (M13mpRE85) was included with the cell extract for this inactivation. Inasmuch as adequate superhelicity is required for oriC plasmids to serve as templates for replication in vitro (27) , nicking of template DNA by endonucleases in the protein extract might be responsible for the observed inefficient replication. However, agarose gel electrophoresis did not reveal any significant changes in the amount or form of template DNA present in the mixture prior to replication (Fig. 1 C). Thus, such changes are not the cause of the loss of replication activity (also see below).

Inactivation Depends on a Soluble Cell Extract

The inactivation we observed here (Fig. 1 A) required the inclusion of a cell extract; mixtures in which the extract was replaced with bovine serum albumin in buffer (Fig. 2 A) or HO (data not shown) were unable to inactivate DnaA protein. Furthermore, DnaA protein must be exposed to the extract for its inactivation to occur. Replication did not decrease when the extract was first incubated without DnaA protein and then mixed with DnaA protein and added to replication reactions (Fig. 2 B). The protein extract seems to act directly upon DnaA protein during the first incubation and does not generate an inhibitor for the subsequent replication reaction.


Figure 2: The soluble extract is responsible for the inactivation of DnaA protein. A, DnaA protein (3.8 pmol) was added to first-stage reactions (50 µl) that contained M13mpRE85 (2.0 µg) and either a soluble extract () or bovine serum albumin (400 µg; ) and was incubated at 30 °C. At the indicated times, 5-µl portions of the mixtures were transferred to in vitro DNA synthesis complementation reactions (25 µl; 20 min, 30 °C). DnaA protein activities remaining after the first-stage incubation ( Residual activity) were compared with those obtained without inactivation (time = 0 min). Activities of 100% correspond to 249 and 220 pmol of DNA synthesized for mixtures containing extract and bovine serum albumin, respectively. B, the first-stage reactions (25 µl) with () and without () DnaA protein (3.8 pmol) were incubated at 30 °C. At the indicated times, 5-µl portions were transferred to in vitro DNA synthesis complementation reactions (25 µl; 20 min, 30 °C); DnaA protein (0.77 pmol) was added to samples that lacked DnaA protein during the first stage. Activities of 100% correspond to 411 and 452 pmol of DNA synthesized for inactivation reactions with and without DnaA protein, respectively. C, DnaA protein (7.7 pmol) was added to first-stage reactions (50 µl) containing varied amounts of a soluble extract and was incubated at 30 °C for 40 min. Samples (5 µl, with the indicated amounts of extract protein) were transferred to in vitro DNA synthesis complementation reactions (25 µl; 20 min, 30 °C). DnaA protein activities remaining after the first stage (residual activity) were compared with those obtained without extract. A residual activity of 100% corresponds to 515 pmol of DNA synthesized.



The extent of inactivation was proportional to the amount of extract added (Fig. 2 C), further suggesting that a factor(s) in the extract is responsible for the inactivation of DnaA protein. The inactivation activity of the extract is heat-labile in that heating the extract at 55 °C for 5 min prevented the extract from being able to act upon DnaA protein (data not shown).

Inactivation of DnaA Protein Is DNA-dependent

The inclusion of DNA during the treatment of DnaA protein with the cell extract is necessary for inactivation to occur. When DnaA protein was incubated with the extract (40 min) in the presence of oriC template DNA (M13mpRE85) and then the mixture was added to a replication reaction, 80% of the replication activity was lost (Fig. 1 A and ). In contrast, significant replication was observed when DnaA protein was incubated with the extract (40 min) in the absence of DNA and then assayed in a replication reaction in which template DNA was provided (). However, the need for specific DNA sequences, such as a DnaA box, was not found in that M13mp19 RF I, X174 RF I, and X174 ssDNA supported the inactivation of DnaA protein (). The ability of nontemplate DNA to promote the inactivation process confirms the finding that the loss of replication activity is not caused by changes in template DNA (Fig. 1 C).

Inactivation of DnaA Protein Is ATP-dependent

ATP was essential for the inactivation of DnaA protein (Fig. 3 A), with maximal inactivation occurring at concentrations of 100 µ M or higher (Fig. 3 B). The requirement for ATP was similar when either the nucleotide-free or ATP-bound form of DnaA protein was used (Fig. 3, A and B). Thus, the exogenous ATP appeared not to be necessary for the formation of the ATP form of DnaA protein and may be needed for the inactivating factor.


Figure 3: ATP requirement for the inactivation of DnaA protein. A, nucleotide-free DnaA protein (7.7 pmol; and ) or ATP-bound DnaA protein (7.7 pmol; and ) (see ``Experimental Procedures'') was added to first-stage reactions (25 µl) with ( and ) and without ( and ) ATP (2 m M) and was incubated at 30 °C. At the indicated times, 5-µl portions were transferred to in vitro DNA synthesis complementation reactions. Residual DnaA protein activities were calculated as described in the legend to Fig. 1. A residual activity of 100% corresponds to 350-450 pmol of DNA synthesized. B, nucleotide-free DnaA protein (0.77 pmol; ) and ATP-bound DnaA protein (0.77 pmol; ) were added to first-stage reactions (5 µl; 30 min, 30 °C) with the indicated levels of ATP. DnaA protein activities were measured in the complementation reaction (25 µl; 20 min. 30 °C), and the residual activities were calculated. Residual activities of 100% for nucleotide-free and ATP-bound forms correspond to 464 and 412 pmol of DNA synthesized, respectively.



Compounds related to ATP were used to examine the nucleotide specificity for DnaA protein inactivation. To prevent DnaA protein from forming complexes with the ATP analogs, the ATP-bound form of DnaA protein was used. Clearly, CTP, GTP, and UTP at 0.5 m M sustained inactivation of ATP-DnaA protein, whereas ADP and the nonhydrolyzable analog ATPS could not ().

DnaA Protein Is Inactivated Specifically for Initiation at oriC

DnaA protein participates in the assembly of primosomes at sites other than oriC (22, 28, 29) . The ABC primosome system requires DnaA, DnaB, and DnaC proteins; DnaG primase; and E. coli single strand binding protein-coated ssDNA that contains a hairpin structure with a single DnaA box (22) . DnaA protein binds to its recognition sequence and, with the aid of DnaC protein, loads the DnaB helicase onto the template. DnaG primase then forms a complex with DnaB protein and synthesizes primer RNA on ssDNA.

DnaA protein-dependent replication of ssDNA (template termed M13 A-site) was examined to see whether or not the inactivation of DnaA protein is specific for initiation at oriC. DnaA protein was incubated with the cell extract; portions of the mixture were added to replication reactions; and DNA synthesis initiated at oriC or the hairpin on M13 A-site ssDNA was measured. While DnaA protein was unable to initiate replication at oriC, its action in this ssDNA replication system was indistinguishable from that of DnaA protein that had not been exposed to the cell extract (Fig. 4). This finding supports the observation that DnaA protein is not globally altered (Fig. 1 B) and indicates that a factor in the cell extract inactivates some function of DnaA protein that is required for initiation at oriC but that is dispensable for initiation of ssDNA replication.

Other Replication Components in the Cell Extract Are Unaffected by Prolonged Incubation

The rate of DNA synthesis in a crude cell extract complemented with DnaA protein (0.19 pmol) decreased after 10 min (Fig. 5). With the addition of fresh DnaA protein, replication was revived and continued for an additional 20 min (Fig. 5). The extent of the additional DNA replication was proportional to the amount of DnaA protein added. This finding indicates that DnaA protein in a crude cell extract loses much of its initiation capacity, while the other factors needed for DNA synthesis retain their activities.


Figure 5: DNA synthesis arrested in a crude extract is reinstated with additional DnaA protein. DNA synthesis complementation reactions (25 µl) containing DnaA protein (0.19 pmol) were incubated (30 °C) for the indicated times (see ``Experimental Procedures''). Additional DnaA protein was added to some reactions ( and ) after 20 min.



Fractionation of the Cell Extract by Gel Filtration

The crude cell extract (12 mg, 3.9 10units) was fractionated through a gel filtration column to determine if the DnaA protein-inactivating factor behaved as a single entity. Column fractions were incubated with X174 RF I DNA, ATP, and DnaA protein, and then replication activities were measured. A peak decrease in DnaA protein activity was observed for fraction 33, which corresponds to an apparent molecular mass of 150 kDa (Fig. 6). The yield of the inactivating factor activity in a pool of fractions 31-39 was 62% of the loaded activity. The loss of DnaA protein activity required incubation of DnaA protein with the column fractions (Fig. 6), and as expected, the inclusion of ATP and DNA in the inactivating step was necessary (data not shown).


Figure 6: Size-exclusion chromatography of the soluble extract. The soluble extract (fraction II; see ``Experimental Procedures'') was applied to a Sephacryl HR-300 column (1.3 28 cm, 37 ml) equilibrated with 25 m M HEPES/KOH (pH 7.6 at 1 M), 0.1 m M EDTA, 2 m M dithiothreitol, 50 m M KCl, and 20% sucrose. Proteins were eluted with the same buffer (flow rate of 0.3 ml/min), and fractions (0.33 ml) were collected. Portions (3.9 µl) of column fractions were added to first-stage reactions (11 µl; containing 600 ng of X174 RF I DNA) and incubated with DnaA protein (2.3 pmol). Portions (5 µl) of the mixtures before () and after () incubation (30 min, 30 °C) were assayed for replication activity in in vitro DNA synthesis complementation reactions as described in the legend to Fig. 1. Markers are catalase ( cata; 232 kDa), bovine serum albumin ( BSA; 68 kDa), and cytochrome c ( cyto; 12 kDa).



Fractionation Profile through a DEAE-cellulose Column

While the inactivating factor failed to bind to several chromatographic resins (data not shown), it had affinity for DEAE-cellulose (DE52). Fractions eluted from a DE52 column by a linear gradient of sodium chloride were assayed for the inactivation of DnaA protein (Fig. 7); one major peak of DnaA-inactivating activity was observed. The yield of activity in the pool of fractions 27-43 was 54%. This pooled fraction had a 4.5-fold increase in specific activity over fraction II and required ATP and X174 RF I DNA for inactivation (data not shown).


Figure 7: Anion-exchange chromatography of the soluble extract. The soluble extract was prepared as described under ``Experimental Procedures,'' except that proteins were precipitated with 0.235 mg/ml ammonium sulfate. Fraction II proteins (24 mg, 4.1 10units) were loaded onto a DE52 column (1.3 2.3 cm, 3 ml) equilibrated with 25 m M BisTris/HCl (pH 6.1), 0.1 m M EDTA, 2 m M dithiothreitol, 15% glycerol, and 100 m M sodium chloride. Activity was eluted in the same buffer by a linear gradient (30 ml) from 100 to 250 m M sodium chloride. Portions (5 µl) of column fractions were added to first-stage reactions (10 µl; containing 600 ng of X174 RF I DNA) and incubated with DnaA protein (1.0 pmol). Portions (5 µl) of these reactions after incubation (40 min, 30 °C) were assayed for replication activity in in vitro DNA synthesis complementation reactions.




DISCUSSION

Chromosomal replication is regulated at the initiation stage, most likely through a balance of positive- and negative-acting factors. Here we have indicated that a soluble cellular factor inactivates DnaA protein for initiating replication at oriC in vitro and propose that such a negative factor plays an important role in controlling initiation in vivo. A mutant form of DnaA protein, DnaAcos, which overinitiates replication at oriC, is not affected by exposure to the factor; the inability to respond to such a negative-acting factor may be deeply involved in the overinitiation observed for dnaAcos mutants. Unlike wild-type DnaA protein activity, DnaAcos protein activity for initiation in vitro is thermosensitive, and its specific activity at 42 °C is only 25% of that at 30 °C (17) . Since the dnaAcos mutant can grow normally at 42 °C, these facts also support the importance of down-regulation of initiation activity.

Considerable portions of DnaA and DnaAcos proteins are found as aggregates during their purification from overproducing cells (6, 17, 18) . Aggregates of wild-type DnaA protein include phospholipids with a composition similar to that of cell membranes (23) . The initiation activity of the aggregate form is about half of that of monomeric DnaA protein in a crude replication system and is completely absent in a system reconstituted with purified proteins (23) . The inactivated DnaA protein observed here, however, differs from the aggregated form; constant velocity centrifugation through sucrose indicated that a large majority (>80%) of DnaA protein was monomeric following treatment with the crude extract either in the presence or absence of X174 RF I DNA and ATP (data not shown).

Genetic analyses and reconstitution studies with purified components have identified >10 different proteins that are necessary for replication from oriC (1) . Among these numerous participants, DnaA protein seems to be the only factor that is inactivated by the soluble crude extract (Fig. 5). This finding supports the hypothesis that the inactivating factor has a specific role in the control of chromosome initiation during cell growth.

We found that the inactivation of DnaA protein is specific for initiation at oriC; initiation of A-site ssDNA replication is not affected (Fig. 4). This finding suggests that the DNA-binding and DnaB helicase-loading activities of DnaA protein are not affected. Perhaps DnaA protein's additional function of strand separation of the AT-rich region of oriC is what is lost.


Figure 4: Replication activity of DnaA protein treated with a soluble extract for synthesis of M13 A-site ssDNA and minichromosomes. DnaA protein (8.7 pmol) was added to a first-stage reaction (75 µl). Before ( and ) and after ( and ) a period of incubation (40 min, 30 °C), the indicated volumes of the reaction were transferred into complementation reactions (20 min, 30 °C) for minichromosome replication (600 pmol (as nucleotide) of M13mpRE85 template DNA; and ) and for the ABC primosome system for single-stranded DNA synthesis (300 pmol (as nucleotide) of M13 A-site ssDNA template DNA; and ; see ``Experimental Procedures'').



A possible molecular mechanism for the inactivation of DnaA protein may include enhanced hydrolysis of the tightly bound ATP. Wild-type DnaA protein has a weak intrinsic ATPase activity; tightly bound ATP is very slowly hydrolyzed to ADP at 37 °C in the presence of DNA, thereby generating replicatively inefficient ADP-DnaA protein (3) . The requirement of sequence-independent DNA for the hydrolysis of the bound nucleotide (3) is similar to that described here for the inactivation of DnaA protein ().

Although the slow conversion of ATP-DnaA protein to the ADP form can occur independently of other cellular components (3) , the inactivating factor may stimulate the feeble intrinsic ATPase. In the regulation of eukaryotic cell growth, a similar example is seen in the oncogenic Ras GTPase; the very weak GTPase of Ras protein is stimulated by interaction with GTPase-activating protein, and the GTP-bound form active for signaling cell proliferation is rendered to the GDP-bound inactive form (30) . DnaA protein inactivated by the 150-kDa factor appears to resemble the ADP form that is inert for initiation at oriC (3) , but is active for initiation of A-site ssDNA (22) .() The requisite hydrolysis of a ribonucleotide to inactivate DnaA protein () suggests that the inactivation process may either be energy-consuming or involve a phosphorylation event. However, phosphorylated forms of DnaA protein have not been detected (data not shown).

A protein termed IciA ( inhibitor of chromosomal initiation) was previously shown to prevent the strand opening at oriC and to act as a specific inhibitor of initiation in vitro (31) . The action of the factor we describe here differs from that of IciA protein in that (i) IciA protein must be bound to the 13-mer region of oriC to inhibit the initiation activity of DnaA protein, while the proposed inactivating factor can restrain the action of DnaA protein in the absence of oriC (); (ii) IciA protein is a homodimer of 33-kDa subunits, as opposed to the apparent native molecular mass of 150 kDa observed for the DnaA protein-inactivating factor (Fig. 6); and (iii) there is an absolute requirement for a nucleoside triphosphate for the inactivation of DnaA protein ( Fig. 3and Table II). None is required for IciA protein to function.

A rapidly growing bacterium (doubling time of <60 min) must initiate the next round of chromosomal replication before ongoing replication has been completed and the cell divides and thus will bear multiple copies of oriC (32) . Initiation of the multiple origins within a cell is synchronous, occurring at the same time during the cell cycle. Modification of adenine residues in the sequence GATC by DNA-adenine methylase (the dam gene product) (33) is essential for this synchrony (2) ; damcells with either point or null mutations are viable (34, 35) , but exhibit an asynchronous phenotype (36) . In wild-type cells, semiconservative replication of fully methylated origins produces hemimethylated oriC DNA that binds to the cell membrane (37) . The hemimethylated state of oriC seems to be maintained for 10 min (38) ; and then Dam methylase modifies the GATC sites in the oriC region, and the resultant fully methylated DNA is released from the membrane. This process, termed sequestration, plays a role in establishing the synchrony of initiations of multiple chromosomes and preventing secondary initiation at newly replicated origins.

The recently identified seqA gene encodes a protein that appears to negatively modulate replication by sequestration (39) . seqA-deficient cells are viable, but cannot sequester oriC DNA or synchronize initiations. The action of SeqA protein in controlling initiation is independent of the proposed negative regulation through the inactivation of DnaA protein. An extract prepared from seqA null cells had the same specific activity for inactivating DnaA protein as wild-type cells and did not affect DnaAcos activity (data not shown). Thus, SeqA protein and the DnaA-inactivating factor appear to act independently to affect initiation at oriC.

Sequestration of oriC to the membrane is transient during the cell cycle, with the duration being much shorter than the time between initiations (38) . As such, it is likely that an as yet unidentified factor functions to determine the occurrence of initiation during the cell cycle. The DnaA-inactivating factor may play a role in this regulation. DnaAcos protein is insensitive to the factor and thus may retain an initiation-competent state for an extended period and cause repeated initiations. A system for inactivating DnaA protein may be necessary for controlling the on-off switch of initiation during the cell cycle. Purification of the inactivating factor is in progress.

  
Table: DNA requirements for the inactivation of DnaA protein

Wild-type DnaA or DnaAcos protein (3.8 pmol) was added to a standard first-stage reaction (25 µl) that lacked or included DNA (1.0 µg for double-stranded DNA; 0.5 µg for ssDNA). Portions (5 µl) of the mixtures, either before or after an incubation period (40 min, 30 °C), were added to an in vitro DNA synthesis complementation reaction (20 min, 30°C) that contained M13mpRE85 as template DNA (see ``Experimental Procedures''). When M13mpRE85 was present in the first-stage reaction, template DNA was not added to the replication reaction. DnaA protein activity remaining after the first-stage incubation (residual activity) was compared with that obtained before and is indicated in percent (in each case, an activity of 100% corresponds to 400-600 pmol of DNA synthesized).


  
Table: Nucleotide requirements for the inactivation of DnaA protein

DnaA protein was incubated with ATP (5 µ M; 15 min, 0 °C) to form the nucleotide-bound form. The resulting ATP-DnaA protein (3.8 pmol) was added to a standard first-stage reaction (25 µl) that contained the indicated nucleotides. Portions (5 µl) of the mixtures, either before or after an incubation period (40 min, 30 °C), were added to an in vitro DNA synthesis complementation reaction (20 min, 30 °C; see ``Experimental Procedures''). DnaA protein activity remaining after the first-stage period (residual activity) was compared with that obtained before and is indicated in percent (in each case, an activity of 100% corresponds to 300-400 pmol of DNA synthesized).



FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant GM 49700 and National Science Foundation Grant MCB 9408830 (to E. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a long-term fellowship from the International Human Frontier Science Program during the work performed in the laboratory of Dr. Arthur Kornberg (Stanford University). To whom correspondence should be addressed: Dept. of Microbiology, Kyushu University School of Pharmaceutical Sciences, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812, Japan. Tel.: 81-92-641-1151 (ext. 6188); Fax: 81-92-632-6648.

The abbreviations used are: ATPS, adenosine 5`- O-(thiotriphosphate); RF, replicative form; ssDNA, single-stranded DNA; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol.

J. Garner and E. Crooke, unpublished data.


ACKNOWLEDGEMENTS

We are grateful to Drs. Nicholas E. Dixon and Hisao Masai for the gift of M13 A-site ssDNA. We also thank Drs. Kazuhisa Sekimizu, Hisaji Maki, and Erik Boye for suggestions.


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