the of vol. no. of 25, 1992 biology, inc. printed in u. s.a ... replication activity of a...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 267, No. 27. Issue of September 25, pp. 19334-19340,1992 Printed in U. S.A.

Defective Replication Activity of a Dominant-Lethal dnuB Gene Product from Escherichia coli*

(Received for publication, May 6, 1992)

Jaroslaw MarszalekS and Jon M. Kaguni4 From the Department of Biochemistry, Michigan State university, East Lansing, Michigan 48824.1319

dnaB protein of Escherichia coli is an essential replication protein. A missense mutant has been obtained which results in replacement of an arginine residue with cysteine at position 231 of the protein (P. Shri- mankar, L. Shortle, and R. Maurer, unpublished data). This mutant displays a dominant-lethal phenotype in strains that are heterodiploid for dnaB. Biochemical analysis of the altered form of dnaB protein revealed that it was inactive in replication in several purified enzyme systems which involve specific and nonspecific primer formation on single-stranded DNAs, and in replication of plasmids containing the E. coli chromosomal origin. Inactivity in replication appeared to be due to its inability to bind to single-stranded DNA. The altered dnaB protein was inhibitory to the activity of wild type dnaB protein in replication by sequestering dnaC protein which is also required for replication. By contrast, it was not inhibitory to dnaB protein in priming of single-stranded DNA by primase in the absence of single-stranded DNA binding protein. Sequestering of dnaC protein into inactive complexes may relate to the dominant-lethal phenotype of this dnaB mutant.

dnaB protein of Escherichia coli is essential for replication of the bacterial chromosome (1-3) and of plasmids and bac- teriophages such as ColEl (4-6), 6x174, and X (7-11) which use this organism as its host. Biochemical studies of dnaB protein from E. coli were first based on its requirement for the conversion of 6x174 single-stranded DNA (ssDNA)’ to duplex replicative form in which dnaB protein is a component of the 4x174 primosome, a multiprotein complex which as- sembles at a unique site on the 4x174 ssDNA (12-14). Move- ment of the primosome on the template strand accounts for primer formation at multiple sites (15).

dnaB protein is a hexamer of identical 52.3-kDa subunits (16-18). In the presence of dnaC protein (27.9 kDa) and ATP (or other ribonucleotides), it forms a stable complex with a stoichiometry of 1:l:l (19-22). Each ATP molecule in this complex is thought to be bound to each dnaC monomer (22).

* This research was supported by Grant GM33992 from the Na- tional Institutes of Health, Research Excellence Funds, and the Michigan Agricultural Experiment Station. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

University of Gdansk, Kladki 24, 80-822 Gdansk, Poland. Present address: Div. of Biophysics, Dept. of Molecular Biology,

To whom correspondence should be addressed: Dept. of Biochem- istry, Michigan State University, East Lansing, MI 48824-1319. ’ The abbreviations used are: ssDNA, single-stranded DNA; SSB, single-stranded DNA-binding protein; RC231, dnaB protein encoded by the RC231 allele; rNTPs, ribonucleotides; dNTPs, deoxyribonucleotides; ATPrS, adenosine 5’-0-(3’-thiotriphosphate).

As a hexamer in the absence of dnaC protein, the ability of dnaB protein to hydrolyze ATP is greatly stimulated by ssDNA (23, 24). The ATPase activity appears to relate to its role as a helicase in which dnaB protein, driven by ATP hydrolysis, moves processively in the 5‘+=3‘ direction along the ssDNA on partially duplex DNA substrates with a 3’- terminal single-stranded end (25, 26). dnaC protein in the dnaB-dnaC protein complex inhibits its ATPase activity (23, 27).

In the identification of genes involved in DNA replication in E. coli, the isolation of conditional mutants defective in DNA synthesis revealed a number of mutants which mapped in the dnaB locus (1). Most of these mutants display a quick- stop phenotype consistent with the role of dnaB protein in propagation of the replication fork of the E. coli chromosome. Given its activity as a helicase (25, 26), the phenotype of dnaB mutants as quick-stop mutants is consistent with its function at the replication fork to facilitate unwinding of the parental duplex. One dnuB allele displays a phenotype consistent with a defect in initiation from the E. coli chromosomal origin, oriC (28).

A class of dnaB mutants represented by the dnuB720 allele have been obtained which displays a dominant-lethal phenotype? The presence of both mutant and wild type alleles in the bacterial cell cannot be tolerated and results in lethality. One possible mechanism for this phenotype is that the mutant protein interferes with replication of the bacterial chromosome by formation of inactive complexes with the replication machinery at the replication fork. Alternatively, sequestering of replication proteins into inactive complexes may occur to prevent their assembly at the fork. In uitro studies on the replication of oriC-containing plas-

mids indicate that the complex of the ATP bound form of dnaA protein to oriC induces a localized unwinding of oriC to allow the subsequent binding of the dnaB-dnaC protein complex (30-33). Formation of a complex of dnaA, dnaB, and dnaC proteins is stimulated by HU protein (31) and, under some conditions, RNA polymerase (34, 35). It is proposed that the release of dnaC protein from the complex is necessary for dnaB protein to unwind the duplex bidirectionally from oriC for primer formation by primase, and DNA synthesis by DNA polymerase I11 holoenzyme (33).

A novel priming mechanism has been discovered which requires dnaA, dnaB, and dnaC proteins on a ssDNA coated with single-stranded DNA-binding protein (SSB) (36). The ssDNA must contain a hairpin structure whose stem contains a dnaA protein recognition sequence. The sequence in the stem is recognized and bound by dnaA protein which then presumably allows the binding of the dnaB-dnaC protein complex (ABC prepriming). Upon addition of primase, synthesis of primers at multiple sites is inferred to occur by the

P. Shrimankar, L. Shortle, and R. Maurer, unpublished data.

19334

dnaB Protein in Initiation of Replication 19335

mobility of dnaB protein (25,26). This novel priming reaction (ABC priming) is distinct from the general priming reaction mediated by dnaB protein and primase on a ssDNA in the absence of SSB protein (37). dnaB is proposed to induce a structure in the ssDNA which is recognized by primase for primer formation.

The study reported here compares the replication activities of a mutant dnaB protein to its wild type complement. The altered form of dnaB protein, herein referred to as RC231, is encoded by the dnaB720 allele which displays a dominant- lethal phenotype, and contains a specific amino acid substitution of cysteine in place of arginine at position 231 of the protein (29).

MATERIALS AND METHODS

Proteins, DNAs, and Reagents-Highly purified replication proteins were purified as described (38). RC231 protein was a gift from Paresh Shrimankar and Russell Maurer, Department of Molecular Biology and Microbiology, Case Western Reserve University. M13-A site ssDNA (36) was a gift from Hisao Masai, Department of Molec- ular Biology, Institute of Medical Science, University of Tokyo. This DNA contains dnaA protein recognition sequences in the stem of a hairpin structure formed by base pairing of an inverted repeat from the R6K y-origin region. M13oriC2LB5 contains the E. coli chromosomal origin inserted into the vector M13AE101 (3). Commercial proteins and chemicals were: bovine serum albumin (fraction V), creatine phosphate, creatine kinase, and rNTPs, from Sigma; HEPES from Behring Diagnostics; dNTPs and Sepharose CL-4B from Phar- macia P-L Biochemicals; [meth~l-~H]dTTP from ICN Radiochemi- cals; horseradish peroxidase-conjugated goat anti-rabbit IgG from Bio-Rad.

ABC Priming-Reaction mixtures (25 pl) were assembled essen- tially as described (36) and contained HEPES-KOH, pH 8.0 (measured at 1 M and 20 'c) , 40 mM; potassium glutamate, 40 mM; magnesium acetate, 10 mM; dithiothreitol, 4 mM; bovine serum albumin, 0.1 mg/ml; sucrose, 4% (w/v); ATP, 2 mM; CTP, GTP, and UTP, 0.25 mM each; dATP, dCTP, dGTP, and [methyL3H]dTTP (25-30 cpm/pmol), 100 p~ each; M13-A site ssDNA, 0.1 pg; SSB, 1 pg; dnaA protein, 28 ng; dnaB protein, 50 ng unless otherwise indicated; dnaC protein, 24 ng unless otherwise indicated; primase, 10 ng; and DNA polymerase I11 holoenzyme, 80 ng. Reaction mixtures were assembled at 0 "C then incubated for 15 min at 30 "C for DNA synthesis. Total nucleotide incorporation (in picomoles) was measured by liquid scin- tillation counting after trichloroacetic acid precipitation onto glass fiber filters (Whatman GF/C).

DNA Replication of oriC Phmids-Reactions (25 p1) were similar t o those described (2, 3) and were comparable to those for ABC priming except for the following: CTP, GTP, and UTP were at 0.5 mM each; phosphocreatine, 6 mM; creatine kinase, 100 pg/ml; M13oriC2LB5 supercoiled DNA instead of M13-A site ssDNA, 200 ng; SSB, 160 ng; HU, 25 ng; gyrase A subunit, 470 ng; gyrase B subunit, 600 ng; dnaA protein, 56 ng; dnaB protein, 55 ng unless otherwise indicated dnaC protein, 24 ng unless otherwise indicated; primase, 10 ng; and DNA polymerase I11 holoenzyme, 80 ng. Reactions were assembled at 0 "C and incubated for 30 min at 30 "C to measure DNA synthesis. Total nucleotide incorporation was measured as in the ABC priming reaction.

General Priming of ssDNAs by dnaB Protein and Primae-Reac- tion conditions (37) (25-pl reaction volume) were similar to those for ABC priming except for the following: dnaB protein, 125 ng unless otherwise indicated; primase, 200 ng; and DNA polymerase I11 holoenzyme, 80 ng. SSB, dnaA, and dnaC proteins were omitted. Reac- tions were assembled on ice and incubations to measure DNA synthesis were for 20 min at 30 "C. Total nucleotide incorporation was measured as described above.

Glutaraldehyde Cross-linking-Incubation of either RC231 or dnaB protein (2 pg each) with M13 ssDNA (1.4 pg) in 100 p1 for 15 min at 30 "C was performed as described for ABC priming but in the absence of other replication proteins and deoxyribonucleotides. Glutaralde- hyde was added to 0.1% followed by incubation at 30 'C for 15 min. Cross-linking was stopped by addition of Tris-HC1 to 67 mM followed by addition of 150 p1 of 80% metrizamide in 40 mM HEPES-KOH, pH 7.6, 20 mM KCI, 0.1 mM EDTA, 5 mM dithiothreitol, 11 mM magnesium acetate, and 1 mM ATP. After mixing, samples were placed at the bottom of Beckman SW 60.1 centrifuge tubes. 40, 35,

and 30% metrizamide in the above buffer (200 pl each) were layered over each sample. Paraffin oil was layered on top and samples were centrifuged for 3 h at 55,000 rpm and 2 "C. Detection of DNA was by ethidium bromide staining of portions of each fraction electrophoresed on an agarose gel. dnaB or RC231 was detected by immunoblot analysis. Alternatively, M13 ssDNA (770 ng) was incubated with dnaB (125 ng) or RC231 (250 ng) followed by cross-linking with glutaraldehyde as described above. Samples were applied to a 0.8% agarose gel in buffer containing 90 mM Tris borate and 2.5 mM EDTA. After ethidium bromide staining to visualize the DNA, the gel was transferred to a nitrocellulose membrane in buffer containing 25 mM Tris and 192 mM glycine at 20 volts for 20 h. RC231 and dnaB protein were detected as described below by use of rabbit antiserum specific for dnaB protein.

Isolation of a Prepriming Protein-DNA Complex by Gel Filtration- Reaction mixtures (equivalent to 4 standard reactions) for ABC priming or for oriC plasmid replication were assembled as described above with the indicated amounts of dnaB protein, RC231, or both (ABC priming), or with the indicated amounts of dnaC protein (oriC prepriming). CTP, GTP, UTP, dNTPs, primase, and DNA polymerase I11 holoenzyme were omitted. The total reaction volume was 84 pl. After incubation for 15 min at 30 "C, reaction mixtures were chromatographed through a Sepharose CL-4B column (0.5 X 9 cm) equilibrated at room temperature in 40 mM HEPES-KOH, pH 8.0, 40 mM potassium glutamate, 10 mM magnesium acetate, 4% (w/v) sucrose, 4 mM dithiothreitol, 100 pg/ml bovine serum albumin, and 2 mM ATP (buffer A). The flow rate was 1 column volume/h. Fractions (80 pl) were collected at room temperature. The excluded volume (fractions 7 and 8) was determined by the eluted position of M13-A site ssDNA or of M13oriC2LB5 DNA, detected by ethidium bromide staining of agarose gels in which portions of column fractions were electrophoresed.

Replication Activity of the Prepriming Complex Isolated by Gel Filtration-To 21 p1 of eluted fractions obtained by gel filtration and maintained at 20 "C, replication activity of the prepriming complex was measured immediately by addition of CTP, GTP, UTP, dNTPs, SSB, primase, and DNA polymerase 111 holoenzyme at levels included in standard assays of ABC priming or of DNA replication with oriC containing plasmids. Where indicated, some reactions were additionally supplemented with dnaB and dnaC proteins at standard levels described above. Reactions (25 pl total volume) of DNA synthesis were incubated for 30 min at 30 "C. Total nucleotide incorporation was measured as described above.

Immunoblot Analysis of Proteins Contained in the Prepriming Complex-After gel filtration, protein contained in 21 pl of the eluted fractions was precipitated by addition of trichloroacetic acid to a final concentration of 5%. Precipitates were washed with cold acetone, resuspended in 30% (v/v) glycerol, 0.08 M Tris base, 0.2% (w/v) sodium dodecyl sulfate, 10 mM dithiothreitol, and 0.2% bromphenol blue, incubated in a boiling water bath for 2 min, and electrophoresed through 12% sodium dodecyl sulfate-polyacrylamide gels as described by Laemmli (39). dnaA, dnaB, and dnaC proteins were detected by immunoblot analysis with rabbit antisera specific for these replication proteins. Colorimetric detection of the bound rabbit antibody was by use of horseradish peroxidase-conjugated goat anti-rabbit IgG.

RESULTS

RC231 Is Not Active in D N A Replication-In vitro systems of DNA replication have been described which depend on plasmids containing the E. coli chromosomal origin, oriC (2, 3, 34, 35). As in the bacterial cell, DNA replication requires dnaA, dnaB, and dnaC proteins in addition to others. A novel priming system (ABC priming) dependent on dnaA, dnaB, and dnaC proteins has been described for the replication of an M13 derivative (M13-A site) containing the dnaA protein recognition sequences (dnaA box) from the R6K y origin in a hairpin structure in the single-stranded viral DNA (36). In either system, dnaA protein appears to act by binding to dnaA boxes to direct the entry of the dnaB-dnaC protein complex (31, 36). This prepriming complex forms a structure which is recognized by primase for RNA primer formation followed by DNA synthesis by DNA polymerase I11 holoenzyme.

Replication activity of dnaB protein was compared to RC231 in these replication systems. Whereas dnaB protein

19336 dnaB Protein in Initiation of Replication

was active, RC231 was inactive at the levels added to replication reactions (Fig. 1). Addition of various amounts of RC231 to reactions containing a fixed amount of dnaB protein was inhibitory in proportion to the amount of mutant protein added. The level of inhibition was dependent on the relative order in which these proteins were added to the reaction. Addition of RC231 protein to reactions of ABC priming which contained dnaB and dnaC proteins prior to incubation to measure DNA synthesis resulted in 50-60% inhibition of DNA synthesis (Fig. lA). By contrast, when dnaB protein was added to ABC priming reactions which contained RC231 protein, substantially more inhibition was observed. The level of inhibition, which was dependent on the relative order of addition of dnaB protein and RC231, was also observed in the oriC replication system (Fig. 1B). One interpretation of these results is that RC231 protein competes with dnaB protein in binding to the complex of dnaA protein bound to the origin to result in formation of an inactive DNA-dnaA-RC231-dnaC complex. Alternatively, RC231 may bind to and sequester, into inactive complexes, other required replication proteins. For example, formation of an inactive RC231-dnaC protein complex may result in an insufficient level of available dnaC protein for replication. Second, formation of mixed hexamers of RC231 and dnaB protein may result in inactive complexes.

RC231 Protein Does Not Form a Complex with M13-A Site ssDNA Bound by dnaA Protein-Incubation of dnaA, dnaB, and dnaC protein with M13-A site DNA bound by single- stranded DNA-binding protein results in formation of a nucleoprotein complex (ABC prepriming complex) (36). The complex, upon isolation, is active in DNA replication upon addition of primase, DNA polymerase I11 holoenzyme, and ribo- and deoxynucleotides. Experiments were performed to determine whether RC231 forms a nonproductive ABC prepriming complex on M13-A site ssDNA. ABC prepriming complexes formed by addition of either RC231 or its wild type counterpart were isolated by chromatography on Sepharose CL-4B. M13-A site ssDNA which eluted in the excluded volume of the column was detected by ethidium bromide staining of agarose gels in which portions of column fractions were electrophoresed. dnaB protein and RC231 were detected in column fractions by immunoblot analysis with antisera against dnaB protein (Fig. 2).

Formation of a prepriming complex with dnaB protein (200 ng) and other required proteins resulted in its coelution with M13-A site ssDNA in the excluded volume (Fig. 2 A ) . The dnaB protein detected in the included volume of the column presumably was due to excess dnaB-dnaC protein complex which did not bind to the SSB-coated DNA. Comparable experiments were performed with a similar amount of RC231.

” ” 2 0 1 0 0 2 0 0 0 1 0 0 2 0 0 0 RC231 (ne)

FIG. 1. RC231 is inactive in DNA replication and inhibits the replication activity of dnaB protein. Reaction mixtures for ABC priming ( A ) or for oriC plasmid replication ( R ) were assembled a s described under “Materials and Methods” with the indicated amounts of RC231 in the absence of dnaB protein (0). Reactions containing dnal? protein (50 ng/reaction) were kept on ice for 4 min before addition of RC231 (0). Alternatively, RC231 was added to reactions lacking dnaB protein which were kept on ice for 4 min before addition of dnaB protein (50 &reaction) (W). DNA replication was measured as described under “Materials and Methods.”

Fraction Number

A dnaB

B RC231

dnaB (-DNA)

4 14 n void volume

-

-

FIG. 2. RC231 is inactive in formation of an ABC prepriming complex. Assembly and isolation of an ABC prepriming complex on M13-A site ssDNA were as described under “Materials and Meth- ods.” Detection of RC231 or dnaB protein in column fractions by immunoblot analysis was as described (see “Materials and Methods”). Purified dnaB protein or RC231 which was electrophoresed in these experiments as controls, and molecular weight markers for immunoblot analysis were comparably reactive with the polyclonal antiserum (data not shown). A , prepriming reactions containing dnaB protein (200 ng) in addition to other required proteins were isolated as described under “Materials and Methods.” B, instead of dnaB protein, prepriming reactions contained RC231 (200 ng) and other required proteins. Column chromatography was as described. C, as a control, proteins required for prepriming including dnaB protein were incubated in the absence of M13-A site ssDNA prior to gel filtration chromatography.

The majority of RC231 was present in the included volume with only trace amounts present in the void volume (Fig. 2B). Even with a 10-fold excess of RC231 relative to dnaB protein in prepriming reactions, only trace amounts of RC231 were detected in fractions corresponding to the void volume of the column (data not shown). By immunoblot analysis, dnaC protein appeared to coelute with RC231 or dnaB protein in the included volume of the column, whereas dnaA protein was detected in both the void and included volumes (data not shown). As a control, proteins required for ABC prepriming complex formation (including dnaB protein) were incubated in the absence of M13-A site ssDNA. By immunoblot analysis, the elution of dnaB protein was similar to that observed with RC231 (Fig. 2C). In these experiments, the polyclonal antiserum was weakly immunoreactive with a protein which mi- grated at a position above dnaB protein or RC231. This protein corresponds to bovine serum albumin present in the column buffer (data not shown).

Portions of fractions from the void volume of the column which resulted from gel filtration of RC231 with other prepriming proteins and the ssDNA (Fig. 2B) were analyzed for replication activity (Fig. 3). DNA replication activity was not detected in these fractions upon supplementation with primase, DNA polymerase I11 holoenzyme, rNTPs, and dNTPs. However, further supplementation of these fractions with dnaB, and dnaC proteins in addition to the above components, resulted in DNA synthesis at levels comparable to control reactions, which were not chromatographed, containing the full complement of required replication proteins (Fig. 3). Given the sample volume applied (84 pl), and the elution of M13-A site DNA in two fractions of the excluded volume (160-pl total volume), the act of chromatography resulted in about a 2-fold dilution of the applied sample. The level of DNA synthesis detected relative to the untreated control is consistent with near total recovery of the replication activity applied. These results suggest that RC231 cannot bind pro- ductively to the ssDNA-dnaA complex in the presence of


x Fraction Number

FIG. 3. Replication activity of the isolated complex formed with RC231 is inactive unless supplemented with dnaB protein. Portions (21 p l ) of the indicated fractions collected by gel filtration of the prepriming reaction containing RC231 (Fig. 2B) were assayed for replication activity after addition of CTP, UTP, GTP, dNTPs, SSB, primase, and DNA polymerase I11 holoenzyme (see “Materials and Methods”) (0). These fractions were additionally supplemented with dnaB and dnaC proteins as described to measure DNA replication activity of the isolated complex (0). As a control, the level of DNA synthesis of a reaction containing dnaB protein and other required proteins for ABC priming which was not chromatographed on Sepharose CL-4B was measured (0).

dnaC protein. The inhibition of DNA replication by RC231 protein in the presence of wild type dnaB protein (Fig. 1) does not appear to result from formation of an inactive ABC prepriming complex on the ssDNA.

The ability of RC231 to form a complex with M13 ssDNA was examined. Complexes formed were stabilized by cross- linking with glutaraldehyde and separated from free protein either by equilibrium sedimentation or by agarose gel electrophoresis (data not shown). By sedimentation, about 60% of dnaB protein was observed in fractions corresponding to the position of the ssDNA. The remaining 40% of dnaB protein was detected in fractions near the bottom of the tube where free protein is expected. RC231 was detected only in fractions corresponding to the position of free protein. By agarose gel electrophoresis, dnaB protein was detected bound to M13 ssDNA but RC231 was not. Glutaraldehyde cross-linking was required to visualize dnaB protein complexed to the ssDNA. These results suggest that the inactivity of RC231 in DNA replication relates to its inability to bind to ssDNA and that the mutation is in a domain which participates in DNA binding.

RC231 Protein Forms a Complex with dnaC Protein and Prevents I t from Acting in DNA Replication-If the mutant dnaB protein forms an inactive complex with dnaC protein to sequester it, addition of excess dnaC protein may restore replication activity to reactions containing RC231 by formation of an active dnaB-dnaC protein complex. To test this possibility, increasing amounts of dnaC protein were added to reactions of ABC priming and of oriC plasmid replication which contained a fixed amount of dnaB protein and varying amounts of RC231 protein. In the absence of mutant dnaB protein, excess levels of dnaC protein were inhibitory in either replication system (Fig. 4). Excess levels of dnaC protein severely inhibit the DNA-dependent ATPase activity of dnaB protein (23, 27). We presume that the inhibition in either replication system by excess levels of dnaC protein is due to this reason. With various levels of RC231, the optimal level of dnaC protein for replication activity was greater in proportion to the amount of RC231 than in the absence of the mutant protein (Fig. 4, A and C). Complete restoration of replication activity was not obtained. The reason for this observation is not known. In reactions containing an optimal level of dnaB protein, and an inhibitory level of dnaC protein, addition of RC231 relieved the inhibition (Fig. 4, B and D). In these experiments, DNA replication activity was restored to a level near that of control reactions lacking RC231 in ABC priming, and to 70% in oriC plasmid replication. These results

RC231 (ng)

C oriC

200

100

0 5 0 100 1 5 0 200

dnaC (ne)

::rr*l 0

0 3 0 0 6 0 0 9 0 0 RC231 (ng)

FIG. 4. RC231 relieves the inhibition of DNA replication caused by excess dnaC protein. A and C, ABC priming or oriC plasmid replication reactions containing dnaB protein (50 ng) and the indicated amounts of dnaC protein were performed as described (see “Materials and Methods”) without (0) or with the indicated levels of RC231. dnaC protein was the last component added to reactions prior to incubation to measure DNA synthesis. B and D, ABC priming or oriC plasmid replication reactions which contained dnaB protein (50 ng) and an inhibitory level of dnaC protein (100 ng) were performed as described (see “Materials and Methods”) with the indicated amounts of RC231. Reactions containing dnaB and dnaC proteins were kept on ice for 10 min prior to the addition of RC231. As a control, DNA synthesis on M13-A site ssDNA (0) or M13oriC2LB5 (W) was measured with optimal levels of dnaB (50 ng) and dnaC proteins (24 ng) as described.

suggest that with excess levels of dnaC protein, the inclusion of RC231 results in formation of inactive RC231-dnaC complexes which relieves the inhibition caused by excess dnaC protein.

In the absence of dnaC protein, it has been established by glycerol gradient sedimentation that RC231 forms a stable hexamer.’ To obtain physical evidence that RC231 forms a physical complex with dnaC protein, the mutant dnaB protein was incubated with dnaC protein in the presence of ATP. With dnaB protein, this condition results in formation of a dnaB-dnaC protein complex near 500 kDa (7, 19, 20, 22). Complexes were isolated by gel permeation chromatography with a column (Superdex 75, Pharmacia) estimated to have an exclusion limit of about 200 kDa. Detection of RC231 and dnaC proteins was by immunoblot analysis. Under these experimental conditions, RC231 eluted broadly with most of the immunoreactive material in and near the excluded volume of the column (Fig. 5A). RC231 protein present in trailing fractions may be due to subassemblies of the hexamer that are resolved by this method of physical separation. dnaB protein eluted in a similar manner under analogous conditions (data not shown). Chromatography of dnaC protein alone under similar conditions resulted in its elution (fractions 47- 50) well separated from the eluted position of the dnaB-dnaC protein complex (Fig. 5B). The absence of immunologically reactive protein in fractions 54 (Fig. 5A), 46, and 51 (Fig. 5B) was due to loss of these samples during handling. Incubation of RC231 with dnaC protein (and ATP) resulted in elution of RC231 and dnaC protein in and near the excluded volume of the column (Fig. 5C). Whereas the elution of RC231 here was similar to that observed upon chromatography of this protein alone, the eluted position of dnaC protein indicates that RC231 protein forms an isolable complex with dnaC protein, probably having the structure of 6 monomers of each.


% 9s m o m a

Fraction Number 32 *55 6 s I void volumei

A RC231

B dnaC

RC231 c +

dnaC

FIG. 5. RC231 forms a complex with dnaC protein. Gel permeation chromatography (Superdex 75 HR 10/30, Pharmacia) was performed with, A, RC231 (750 ng in 25 pl of buffer A (see “Materials and Methods”) incubated for 10 min at 30 “C prior to chromatography; R, dnaC protein (300 ng in 25 pl of buffer A incubated as described above); and C, RC231 and dnaC protein (750 and 300 ng, respectively, in 25 pl of buffer A incubated as described above). The column was equilibrated and run in buffer A at a flow rate of 0.1 pllmin. Fractions of 0.15 pl were collected, concentrated by trichloroacetic acid precipitation, and electrophoresed through 12% sodium dodecyl sulfate- polyacrylamide gels for immunoblot analysis as described under “Materials and Methods.” Included as controls, the extreme right lane of each set contains, A, RC231,60 ng; B, dnaC protein, 50 ng; and C, RC231 and dnaC protein, 30 and 12 ng, respectively.

Inhibition of DNA Synthesis, at High Concentrations of dnaC Protein, Is Not a Result of Stable dnaB-dnaC Protein Interaction-Excess levels of dnaC protein inhibit the ATP- ase activity of dnaB protein and its activity as a DNA helicase (23, 27). The delivery of the dnaB-dnaC protein complex to oriC bound by dnaA protein results in the release of dnaC protein as its presence is not detected in isolated prepriming complexes (31). Since excess levels of dnaC protein were inhibitory to in uitro replication of oriC plasmids (Fig. 4), experiments were performed to address whether the inhibition, in dnaC protein excess, was due to the failure of dnaC protein to dissociate from dnaB protein bound to the DNA template. Prepriming complexes on an oriC plasmid (31) were formed in the presence of a high (inhibitory) level of dnaC protein and then isolated by chromatography on a Sepharose CL-4B column. Fractions from the excluded volume of the column were supplemented with components necessary to measure DNA synthesis (Fig. 6). The observed activity was comparable to the level of DNA replication activity obtained with void volume fractions from a control reaction which contained an optimal level of dnaC protein prior to gel filtration. By contrast, addition of excess dnaC protein to the isolated complex formed in the presence of a high level of dnaC protein was inhibitory. These results suggest that the excess dnaC protein, while inhibitory when not removed or when added subsequently, allows the release of dnaC protein in the dnaB-dnaC protein complex upon gel filtration as inferred by the replication activity of the isolated prepriming complex. Gel filtration yields an active nucleoprotein complex apparently by separation of the excess dnaC protein.

RC231 Does Not Compete with dnaB Protein in General Priming-On any ssDNA in the absence of SSB, dnaB protein is proposed to induce a structure which is recognized by primase for primer formation (general priming) (37). In contrast to other priming reactions, only dnaB protein and primase are necessary for primer formation. RC231 protein interfered with dnaB protein in ABC priming or in oriC replication by sequestering dnaC protein. I t was of interest to determine its influence on the activity of dnaB protein in

A, 1” prepriming

in prepriming

high dnaC in replication

4 5 6 7 0 9 1 0 1 1 1 2

Fraction Number

FIG. 6. Gel filtration chromatography of oriC-prepriming complexes formed with high levels of dnaC protein yields an active intermediate. Prepriming complexes with M13oriC2LB5 DNA were formed with an optimal (92 ng) or high (224 ng) level of dnaC protein, and isolated as described under “Materials and Meth- ods.” Portions (21 pl ) of void volume fractions were assayed for replication activity by supplementation with CTP, UTP, GTP, dNTPs, SSB, primase, and DNA polymerase 111 holoenzyme at levels used in standard replication reactions (see “Materials and Methods”). Where indicated, DNA replication reactions were further supplemented with a high level of dnaC protein (224 ng). DNA synthesis was measured as described.

general priming. By comparison with wild type dnaB protein, RC231 was not active in general priming of DNA synthesis (Fig. 7A). When RC231 and dnaB protein were both present in the reaction mixture, inhibition or stimulation was not observed (Fig. 7 B ) . Thus, RC231 does not inhibit the activity of dnaB protein in general priming. It has not been determined whether RC231 forms mixed hexamers with dnaB protein nor what activities of dnaB protein are altered or preserved in such mixed hexamers.

DISCUSSION

dnaB protein of E. coli appears to function as the major DNA helicase to facilitate unwinding of the parental duplex ahead of the replication fork (25,26). It also may act to direct primer formation by primase for both leading and lagging DNA strands. In the hexameric complex of dnaB, and dnaC proteins bound to DNA, dnaB protein is in a nonfunctional


a z

60

40

20

0- ” 0 5 0 1 0 0 1 5 0 2 0 0 2 5 0

protein (ne) 0 1 0 0 2 0 0 3 0 0 4 0 0 500

AC231 (ng)

FIG. 7. RC231 is not active nor inhibitory to the activity of dnaB protein in general priming. A , reactions of general priming were performed as described under “Materials and Methods” with the indicated amounts of dnaB protein or RC231. B, to general priming reactions containing dnaB protein (125 ng), the indicated amounts of RC231 were added. DNA synthesis was measured as described.

state with regard to ATPase and prepriming activities (27). The hydrolysis of ATP bound to dnaC protein in the complex appears to destabilize it to result in release of dnaC protein, and in function of dnaB protein as a helicase.

Several biochemical deficiencies have been ascribed to RC231, an altered form of dnaB protein encoded by a dnaB allele which displays a dominant-lethal phenotype.‘ The mutation results in replacement of an arginine with cysteine at residue 231 which resides within a consensus sequence found in one class of ATP-binding proteins (40, 41). Although this protein forms a hexamer active in supporting RNA primer formation by primase on poly(dT), and binds to ATP or adenosine 5’-0-(3’thiotriphosphate) (ATP-yS), it is defective in DNA-dependent ATP hydrolysis and consequently is inactive as a helicase.

RC231 Is Inactive in DNA Replication-The study reported here characterizes the properties of RC231 in DNA replication. RC231 was inactive in purified enzyme systems which sustain ABC priming, general priming, and in oriC plasmid replication. RC231 was also inactive in oriC plasmid replication dependent on a crude enzyme fraction (data not shown). As this protein forms an RC231-dnaC protein complex, the inactivity in replication is not due to an inability to interact with dnaC protein.

General priming by dnaB protein on ssDNA requires ATP to confer ssDNA-binding activity (37,42). On 6x174 ssDNA, poly(dT), or poly(dC), dnaB protein stimulates primer formation by primase (37). However, general priming on poly(dC) was greatly reduced relative to poly(dT). The inability of other synthetic ssDNAs to serve as templates for RNA synthesis in general priming has been interpreted to reflect the preference of primase for purine nucleotides as the initiator nucleotide. ATPyS, poorly hydrolyzed by dnaB protein (42) or RC231,’ sustains general priming, a result which indicates that ATP hydrolysis is not required (42,29). RC231 is active in binding to ATP or ATPyS, and in sustaining RNA primer formation with primase on poly(dT).’ By contrast, RC231 was inactive in general priming on ssDNA under conditions in which dnaB protein was active (Fig. 7). Despite the ability of RC231 to form primers on poly(dT), it does not appear to bind to naturally occurring ssDNA. By use of glutaraldehyde cross-linking, dnaB protein was detected bound to M13 ssDNA, whereas RC231 was not. The inactivity in ssDNA binding apparently accounts for its inactivity in general priming and in DNA replication, and indicates that the amino acid substitution of RC231 resides in a domain involved in DNA binding.

RC231 Is Inhibitory to the Action of dnaB Protein in DNA Replication-Addition of RC231 to systems of ABC priming or oriC plasmid replication which also contained dnaB protein

was inhibitory. This inhibition appeared to be due to the sequestering of available dnaC protein based on several lines of evidence.

First, the level of dnaC protein which was optimal for replication activity was higher in proportion with the levels of RC231, compared to reactions which lacked RC231.

Second, the addition of RC231 reversed the inhibition caused by excess levels of dnaC protein. Prepriming complexes formed under these conditions with wild type dnaB protein and isolated away from excess dnaC protein were comparably active relative to control reactions containing optimal levels of dnaB and dnaC proteins. One mechanism is that dnaC protein, in excess, is in dynamic equilibrium with the dnaB-dnaC complex to favor stabilization of the dnaB- dnaC complex, rather than dissociation of dnaC protein. Since dnaC protein in the dnaB-dnaC complex inhibits the ATPase activity of dnaB protein (23,27), the inhibition observed with excess levels of dnaC protein may be due to stabilization of the dnaB-dnaC protein complex.

Third, the relative order in which RC231 and dnaB protein were both added to reactions affects the extent of inhibition by RC231. Addition of RC231 to reactions containing dnaC protein followed by addition of dnaB protein resulted in severe inhibition in comparison to the reverse order of addition of dnaB protein followed by RC231 (Fig. 1). In reactions where dnaC protein was added last, the level of inhibition was proportional to the amount of RC231 present (Fig. 4, data not shown). One interpretation of these results is that dnaB protein and RC231 compete in binding to dnaC protein. Under conditions in which dnaC protein is neither in excess nor inhibitory, the formation of inactive complexes with RC231 and dnaC protein followed by addition of dnaB protein is inhibitory due to the inability of dnaB protein to bind to dnaC protein to form an active complex. Formation of complexes between dnaB and dnaC proteins minimizes the inhibitory effect of RC231 when it is added subsequently. The above mechanism presumes that subunit exchange between hexamers of dnaB protein and RC231 is minimal, or that, if it occurs, mixed hexamers are active. Indeed, the addition of RC231 to assays of general priming which contained dnaB protein was not inhibitory.

Under conditions in which dnaC protein is in excess and inhibitory, the presence of RC231 relieves this inhibition by sequestering excess dnaC protein. dnaC protein may be irre- versibly bound to RC231 since the complex fails to bind to ssDNA to allow the release of dnaC protein upon hydrolysis of ATP. Alternatively, mixed hexamers composed of different proportions of RC231 and dnaB protein may result in pro- portionately lower activity in DNA replication. While this possibility seems unlikely and is not supported by the observation that RC231 does not inhibit DNA synthesis dependent on dnaB protein in general priming, direct evidence on formation of mixed oligomers has not been obtained.

Similar conclusions were reached by Allen and Kornberg (29) where the inhibitory effect of excess dnaC protein was counteracted by elevated levels of dnaB protein. Their study is in agreement with the results presented here indicating that the ratio of dnaB to dnaC protein is critical for replication activity and underscores the importance of dnaC protein levels on replication in uiuo.

The Dominant-Lethal Phenotype of dnaBldnaB720 Heter- odiploids-The inhibitory nature of RC231 on dnaB protein is consistent with the dominant-lethal phenotype of hetero- diploids containing the wild type dnaB allele and the dnaB720 allele on a multicopy plasmid.’ However, simultaneous expres- sion of both alleles in uiuo may result in formation of hexamers


containing both mutant and wild type gene products. The experiments described here, while they do not exclude the possibility of formation of mixed hexamers in uitro, involve the separate addition of dnaB protein and RC231 to reactions. Further experiments to demonstrate the formation of mixed hexamers of RC231 and dnaB protein, and associated biochemical properties are necessary.

Acknowledgments-We thank P. Shrimankar and R. Maurer for their gift of RC231 protein and communication of unpublished results and H. Masai for bacteriophage M13-A site. We especially thank Ted Hupp for helpful discussions and advice.

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