INTRODUCTION

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The dnaX polymerization gene of Escherichia coli encodes two DNA polymerase III components,  and .  is the full-length translational product of the DnaX reading frame. The shorter  is identical to the first 430 residues of , but its C terminus is generated by a programmed 1 ribosomal frameshift which results in the incorporation of a glutamate as the 431st amino acid followed by a stop codon (3, 19, 63).

functions as the replisome organizer, dimerizing the core polymerase (30, 33, 43, 58) and interacting with and stimulating the replicative DnaB helicase and primase (31, 73).  also contributes to processivity by stabilizing the processivity clamp (32) and the holoenzyme (73) on the leading strand.

functions in a five-subunit complex (_2-4--'--) (12, 20, 42, 49, 51, 62) to load and unload the processivity clamp  (2, 5, 25, 26, 45, 47, 59, 67). The binding of two or three ATP molecules by the  subunit of the complex alters the conformation of the complex, allowing  to bind directly to and open the clamp and allowing assembly of a primed DNA-open clamp- complex structure (25, 26, 45). Hydrolysis of the ATP, required for closing the clamp around primed DNA, occurs in two sequential steps. The first might release  from the  complex; the second might then release DNA (enclosed within the clamp). Alternatively, the first hydrolysis might release DNA from the  complex into the open clamp and the second would then release  (encircling the DNA). Another possibility for the second hydrolysis might be resetting of the  complex for the next cycle (26).

The N-terminal region of  is identical to , except for the 431st residue, and is capable of all the known activities of  in vitro, including loading  (with  or -') (59) and assembly in vitro or in vivo (from an artificial -complex operon) into clamp-loading  complexes (_2-4--'--) (13, 49, 54). The  complex is often thought to be the principal clamp-loading machine because it, but not the  complex, has been isolated from cellular extracts and from holoenzyme (42, 51). Moreover, DNA polymerase III (Pol III) lacking  but assembled with ATP-binding mutant  could not load , whereas that assembled with ATP-binding mutant  could (72). On the other hand, another study found that  clamp loading by the  complex was more characteristic of the loading reaction of native holoenzyme than the -complex-dependent reaction (13) and that the  subunit is dispensable in vivo whereas  is essential for growth (4). These data indicate uncertainty as to the individual roles of  and  in clamp loading in vivo.

Clamp-loading proteins of various organisms from phage T4 to prokaryotes to humans have significant homology over much of their lengths, including several highly conserved regions (8, 11, 14, 21, 23, 46). We report here highly conserved residues essential for DnaX⁺-complementing activity in vivo and DNA-dependent ATPase and  clamp loading in vitro.

	MATERIALS AND METHODS

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Strains, plasmids, and growth conditions. The dnaX⁺ strain C600 was from the laboratory collection. Strain AB27a is a dnaX(Ts)2016 zbb::Tn10 derivative of strain C600. It was constructed by P1 transduction (70) from a zbb::Tn10 derivative of dnaX(Ts) strain AX733 (9). Strain AB28 is a zib::Tn10 dnaX⁺ rpsL derivative of strain C600, with Tn10 closely linked to dnaA⁺. Strain BL21(DE3)pLysS was the host for production of  and  proteins. Strain SM10 pir (44a) was used to propagate Pir protein-dependent plasmid pKAS32 (55a) and its derivatives. Phage MO1 consists of a 2,350-bp PstI-SmaI dnaX⁺ fragment cloned into PstI- and SmaI-cut M13mp19. pET X⁺ is a derivative of pET 21a⁺ with the wild-type dnaX gene on a 2.3-kb EcoRI-AvaI fragment from phage MO1 cloned downstream of the T7 promoter. A mutant dnaX allele with the threonine codon 142 changed to alanine (dnaXT142A) was cloned as an amplified 2.35-kb KpnI-HindIII fragment (including some polylinker sequence) from a dnaXT142A derivative of MO1 into similarly cut temperature-sensitive (TS) suicide vector pMAK705 (23a), generating pABMXT142A. The dnaXT142A mutant allele was cloned also as 2.3-kb KpnI-PvuII fragment, including some polylinker, from the dnaXT142A derivative of MO1 into rpsL⁺ Ap^r pKAS32 (55a) cut with KpnI and EcoRV, generating pKXT1. Yeast extract (0.5%)-tryptone (1%) medium containing 0.5% NaCl was supplemented with ampicillin (200 µg/ml), chloramphenicol (30 µg/ml), streptomycin sulfate (500 µg/ml), or tetracycline (25 µg/ml), as needed.

DNA preparations. Plasmids were purified by use of Qiagen Miniprep spin columns and chromosomal DNA was purified by use of Puregene DNA isolation kits (Gentra Systems) according to the manufacturers' directions. For determining the chromosomal dnaX sequence, a 2.2-kb fragment was amplified from chromosomal DNA by the Expand high-fidelity PCR system using Taq and Pwo DNA polymerases (Boehringer Mannheim) and sequenced with an ABI Prism 377 DNA sequencer.

Other sources. -, , ', -', , and single-strand binding protein (SSB) were generous gifts from Mike O'Donnell. Other reagents were ultrapure ammonium sulfate (Baker), FastFlow Q and Sp Sepharose (Pharmacia), phenylmethylsulfonyl fluoride (PMSF) (Sigma), pET 21a⁺ and host strain BL21(DE3)pLysS (Novagen), and molecular mass markers (Bio-Rad). Restriction enzymes were from Promega; T4 DNA ligase was from Ambion, Inc. (Austin, Tex.); oligonucleotides were from Life Sciences or Bio-Synthesis, Inc. (Lewisville, Tex.). All oligonucleotide sequences are available on request.

Mutagenesis. Phage MO1 was mutagenized by the Kunkel technique (35). The dnaX strand complementary to the messenger was present in the single-strand DNA of the hybrid phage. Confirmed mutant alleles were cloned from replicative-form DNA into pET X⁺, replacing an AatII-AvaI fragment.

Gene replacement. Two independent procedures were used to introduce the dnaXT142A allele into the chromosome in the haploid condition. The first procedure used the suicide vector pMAK705 of Hamilton et al. (23a). The T142A mutation in pABMXT142A was verified by sequencing, and the plasmid was electroplated into strain C600 using chloramphenicol resistance at 44°C to select cointegrates. The absence of detectable plasmid DNA was verified in eight transformants, which were then pooled and grown at 30°C for three cycles each of 12 generations in the presence of chloramphenicol to allow resolution. From nine resolution cultures, four plasmids carried the mutant allele and five carried the dnaX⁺ allele. Two of the cultures carrying the wild-type allele on the plasmid were cured by six generations of growth at 44°C in the absence of chloramphenicol. These no longer contained detectable plasmids, and sequencing the chromosomal dnaX over the region containing codon 142 confirmed that the wild-type allele had been replaced by the dnaXT142A mutant allele.

4

Purification of wild-type and mutant  and . Strain BL21(DE3)pLysS was transformed with pET X⁺ or pET X encoding each of the mutations D126N, T142A, and R169A, selecting on yeast extract-tryptone medium containing ampicillin and chloramphenicol (100 and 30 µg/ml, respectively). Six-liter cultures were grown at 30°C to an absorbance of 0.3 and were induced with 0.4 mM IPTG (isopropyl--D-thiogalactopyranoside) for 3 h; the cells were centrifuged, resuspended in lysis solution A (50 mM Tris-HCl [pH 7.5], 10% sucrose, 0.5 mM protease inhibitor PMSF), and lysed by freezing and thawing (56) to produce Fr I. The purification (48) of  and  from pET X encoding R169A, which was typical, will be described. Fr I consisted of 48 ml containing 8 mg of protein/ml. Buffer A consisted of 50 mM Tris-HCl, 10% glycerol, and 0.1 mM EDTA. All solutions used in purification contained 0.5 mM PMSF. Ammonium sulfate precipitation, backwashing (41), and dialysis against buffer A plus 20 mM NaCl yielded Fr II: 40.7 mg of protein in 3.7 ml. A 2-ml Fast Flow Q Sepharose column was equilibrated with buffer A plus 1 mM dithiothreitol (DTT) and loaded with 40 mg of Fr II, washed with 20 ml of the same solution, and eluted with a 200-ml linear NaCl gradient in the same buffer. Both  and  eluted in (2-ml) fractions 48 to 69. After precipitation with 0.488 g of AmSO₄/ml, dissolving in buffer A, and dialysis against 3 liters of buffer A containing DTT, Fr III contained 8.8 mg of protein in 4.6 ml. The mixture of  and  was loaded onto a 4-ml Fast Flow Sp Sepharose column, equilibrated with buffer A plus DTT, and washed with 40 ml of the same solution. Elution with a NaCl gradient in buffer A plus DTT (50 ml of 0 to 100 mM NaCl linear gradient, 50 ml of 100 mM NaCl, and 50 ml of 100 to 400 mM NaCl linear gradient) and collection of 2-ml fractions yielded Fr IV  in fractions 16 to 20 and Fr IV  in fractions 57 to 59. These fractions were dialyzed separately against buffer A plus DTT. The total yield of  was 810 µg in 8.75 ml; the  yield was 370 µg in 5.25 ml.

View larger version (33K): [in this window] [in a new window]   FIG. 1.   Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of wild-type and mutant  and . Two micrograms of each protein was electrophoresed on a 10% polyacrylamide-0.1% SDS gel at 200 V and stained with Coomassie blue. Molecular mass markers are shown on the left.

ATPase assay. The assay of Onrust and O'Donnell (50) was used. Each 20-µl assay mixture contained 840 ng of single-strand M13Gori1 DNA, 0.4 µCi of [-³²P]ATP in 2,000 pmol, and 0.5 to 2.0 pmol of  or  all in 20 mM Tris-HCl, pH 7.5-8 mM MgCl₂-1 mg of bovine serum albumin/ml. If  or ' was included, it was added at the level of 8 pmol. The assay mixtures were mixed on ice, and the assays were started by transferring the mixtures to 37°C; duplicate 1-µl samples were collected at 0, 15, and 30 min and spotted onto polyethyleneimine thin-layer chromatography plates which had been washed with deionized water and dried. The samples were spotted onto areas which had been prespotted with 5 µl of stop buffer (50 mM EDTA [pH 8.0], 1 mM [each] ATP and ADP) and dried. The labeled ATP and ADP were separated by chromatography with 750 mM K₂HPO₄ and quantitated by a Molecular Dynamics PhosphorImager and ImageQuaNT software. For kinetic studies, the ATP concentration was varied as shown in Fig. 3 and five samples were taken at 3-min intervals.

Replication assay. The assay for wild-type and mutant  and  in  clamp loading was based on the Onrust et al. (51) procedure. The 25-µl assay mixtures contained 0.5 or 2.0 pmol of  or , 250 ng of uniquely primed (57) M13Gori1 single-strand DNA, 22.5 ng of - complex (0.14 pmol), 10.5 ng of the  subunit (0.14 pmol as a dimer), 2 ng of -' (0.025 pmol each), 980 ng of SSB (13.6 pmol as a tetramer), 1 µCi/500 pmol of [-³²P]dATP in a solution of 20 mM Tris-HCl (pH 7.5), 8 mM MgCl₂, 500 µM ATP, 30 µM TTP, 60 µM (each) dGTP and dCTP, 18 mM NaCl, 5 mM DTT, 100 µM EDTA, 4% glycerol, and 40 µg of bovine serum albumin/ml. After 30 min at 37°C, 20 µl of the assay mixture was added to 1 ml of cold 5% trichloroacetic acid containing 10 mM sodium pyrophosphate, filtered onto Whatman 2.4-cm-diameter filters, and quantitated with a PhosphorImager as described above.

	RESULTS

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Motifs of  and  homologs. A BLAST search (1) of GenBank release 118 using E. coli  and  residues 1 to 223, with a C substituted for the P-loop (GxxGxGKT) lysine to reduce the number of unrelated ATPases detected, identified 27 prokaryotic  and , 10 prokaryotic ', and 33 eukaryotic clamp loader (replication factor C [RFC] or activator 1) (7, 17, 37, 38, 64, 65, 66) subunits. A hypothetical protein of eukaryotic origin, that of Arabidopsis thaliana (40) (GenBank accession no. AAC18938), is more closely related to prokaryotic  and  than to RFC subunits and has been included with  and  for motif identification. It is 26.5% identical to E. coli  over the first 430 residues and is designated a  and  homolog. The 27 prokaryotic  and  homologs plus the Arabidopsis  and  homolog were aligned by CLUSTAL W (60), which employs the BLOSUM series of weight matrices (24). Nine highly conserved regions were identified and compared to previously defined clamp loader protein motifs. With the rare exceptions noted below, all 28  and  homologs contained all nine motifs.

View this table: [in this window] [in a new window]   TABLE 1.   Clamp loader - and -subunit motifsc

Motif residues essential for DnaX⁺ activity in vivo. Single-base substitutions were introduced into the P loop and the DExx, VIc, and SRC motifs of dnaX, cloned in the pET 21a vector, and the resulting plasmids were transferred into TS dnaX mutant strain AB27a for complementation analysis. The TS mutation in this strain changes codon 118 from a glycine to an aspartate codon (4) in both  and . DNA polymerization stops abruptly when this mutant is shifted to a nonpermissive temperature (18). Specific mutations included the P loop G45A and K51A, DExx motif D126E and D126N, motif VIc T142A, and SRC motif R169A. Transformants selected at 30°C were tested for growth at the nonpermissive 42°C by streaking plates and quantitatively by efficiency of plating (Table 2). Although the wild-type dnaX allele on the plasmid restored growth at high temperature, all the point mutations, except T142A, inactivated complementing activity. (The pET 21a vector is designed to limit uninduced expression by virtue of a lacI gene and the presence of the lac operator upstream of the cloning site, but the basal [i.e., uninduced] level of dnaX expression from this vector was adequate to provide wild-type complementing activity.) The dnaXT142A allele restored recipient growth at 42°C.

View this table: [in this window] [in a new window]   TABLE 2.   and  residues essential for DnaX+ complementation activity in strain AB27a, a dnaX(Ts) host

ATPase activity of wild-type and mutant . Although  has inefficient DNA-dependent ATPase activity, with a k_cat (maximum catalytic rate at saturating substrate) on the order of 3 × 10³ s¹ (25, 50, 51), that low activity is thought to be intrinsic to  rather than the result of a contaminating ATPase.  and ', which are not ATPases themselves, stimulate the intrinsic  ATPase (50, 51). Each of the wild-type and mutant  proteins was assayed for DNA-dependent ATPase activity alone, with , with ', and with -' (Fig. 2A). As individual proteins, wild-type and mutant  had ATPase activities that were essentially background and  had no detectable effect on any of them. ', on the other hand, stimulated the wild-type  ATPase about sixfold and each of the mutants about twofold. The combination of -' was more active than either  or ' alone and stimulated the wild-type  about 22-fold. Addition of both  and ' stimulated the residual activity of mutant  also, the D126N mutant about threefold, the T142A mutant about eightfold, and the R169A mutant about twofold. Interestingly, R169A mutant residual activity was stimulated about twofold by ' alone and by the combination -'. This mutation apparently interferes with  ability to respond to the synergistic effect of  and '.

View larger version (34K): [in this window] [in a new window]   FIG. 2.   ATPase activities of wild-type and mutant  (A) and  (B). Each protein was assayed alone or with , ', or -', as indicated. The assay mixtures all contained single-strand DNA (as described in Materials and Methods), 0.5 to 2.0 pmol of  or , and 8 pmol each of  and '. Error bars, ±1 standard deviation.

1

View larger version (22K): [in this window] [in a new window]   FIG. 3.   Lineweaver-Burk plot of wild-type and mutant  ATPase activities with single-strand DNA and -'. The solid circle on the y axis represents +1 standard deviation from the mean (1.43) of the intercepts of the three lines representing +,  T142A, and  R169A.

View this table: [in this window] [in a new window]   TABLE 3.   Kinetic analysis of wild-type and mutant DNA-dependent, -'-stimulated  ATPase

ATPase activity of wild-type and mutant . The wild-type  has weak, but significant, DNA-dependent ATPase activity, even in the absence of -' (39, 62). With the M13Gori1 single-strand DNA as the effector, wild-type  hydrolyzed about 35 mol of ATP/min/mol of protein (Fig. 2B). All three mutations, D126N, T142A, and R169A, reduced that activity to neligible levels.

Replication activity of wild-type and mutant  and . Replication assays were done as described by Onrust et al. (51) to measure  clamp loading by wild-type and mutant  and . Each was mixed with uniquely primed SSB-coated M13Gori1 single-strand DNA, -, and  and incubated with substrates, including radioactive dATP, with and without -', at 37°C for 30 min. Total incorporation of nucleotides into replicative-form DNA was measured. The wild-type  and , with -', supported the incorporation of 0.4 and 2.2 mol of nucleotide into DNA product/min/mol of protein, respectively. All three mutant forms of both  and  were essentially inactive (Fig. 4). Increasing the mutant  and  concentrations from 0.5 to 2.0 pmol/assay did not significantly change their clamp-loading specific activities (Fig. 4), suggesting that the mutant proteins have defects in intrinsic catalytic activity rather than affinity differences.

View larger version (25K): [in this window] [in a new window]   FIG. 4.   Replication assay of  clamp loading of wild-type and mutant  and . The assay mixtures contained uniquely primed single-strand DNA, -, , single-strand binding protein, and  or , each with and without -'. For each condition,  and  were added at both 0.5 and 2.0 pmol/assay. Because the increasing concentrations did not change specific activities significantly, the averages of all assays are presented. Error bars, ±1 standard deviation. Each assay was done in quadruplicate.

	DISCUSSION

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Identity and functional motifs have previously been described in clamp-loading proteins from phage T4, bacteria, and eukaryotes (8, 11, 21, 23, 46), and the structure of the N-terminal 80% of  has been proposed, based on  homology with ', for which the structure has been solved (23). The N-terminal 330 residues of  are organized into three domains with a "C" shape, and ATP is thought to bind within the inner surface of the "C" (23). In this study, we have used the BLAST program (1) to identify 28 homologs of the E. coli  and  clamp-loading subunits (27 prokaryotic proteins and 1 eukaryotic protein) plus 10 prokaryotic homologs of the ' subunit and 33 eukaryotic RFC subunits. The 28  and  homologs contain nine highly conserved motifs within the N-terminal halves of the molecules (Table 1). Also identified by the BLAST search were seven closely related prokaryotic homologs of a hypothetical E. coli protein (GenBank accession no. P45526) with significant homology to  and  and RuvB (H. R. Neely and J. R. Walker, unpublished).

and  functional motifs include motif III, the P loop or phosphate-binding region (68), DExx motif V, common to helicases and nucleases (6, 22, 27), and a Zn binding motif. Although the Zn binding sequence is atypical,  contains Zn²⁺ (cited by Guenther et al. [23]). Two sensor motifs (Table 1) which would respond to ATP have been proposed by Guenther et al. (23) by analogy to crystal structures for known nucleotide binding proteins complexed with substrates (or their analogs). Sensor 1, proposed to be residues 152 to 157 (FLLATT) of E. coli  and , is highly conserved. It is extended to F&(FL)aTT(ED). This motif is strongly conserved, with three residues being invariant; the second position is always a bulky hydrophobic residue (&), the third position is always F or L, and the (ED) is present in at least 90% of 28 homologs. The second proposed sensor motif, motif 2, is GSLRDA (residues 212 to 217) of  and  (23) and is part of motif VIII described by Cullman et al. (11). The R, invariant in all known  and  proteins, might bind the phosphate groups of ATP (23). The sensor 2 (motif VIII) highly conserved region is extended to include GsxRDx2(ST)Ux(DE)q (Table 1). Four additional motifs (II, IV, VIc, and VII) are identified by high degrees of homology among all the known  and  homologs (Table 1).

P loop residues bind nucleotide - and -phosphates (15, 28, 36, 61). The E. coli  and  P-loop residues are essential in vivo and in vitro. Both G45A and K51A mutations eliminated DnaX activity in vivo (Table 2). Xiao et al. (72) mutated the P-loop lysine to alanine (K51A) and showed that mutant  and  did not bind ATP with high affinity and that mutant  and  complexes assembled with  K51A or  K51A neither hydrolyzed ATP nor loaded  clamps. They used the mutant proteins to provide evidence that the  complex, but not the  complex, loads  in the holoenzyme. Pol III* (holoenzyme without  [44, 69]) assembled with wild-type  and mutant  was inactive in loading  and polymerizing DNA in vitro, but Pol III* assembled with mutant  and wild-type  was active. The  complex assembled with  K51R had reduced ATPase activity and was inactive in clamp loading (25).

DExx aspartates are situated at the carboxy ends of  strands and participate in catalysis by binding magnesium ions, directly or indirectly through water, which bridge the nucleotide - phosphates (see, e.g., references 16, 28, and 52). Pause and Sonenberg (53) mutagenized the DEAD box of the mammalian translation initiation factor eIF-4A. Changing the invariant first aspartate to asparagine eliminated ATPase and helicase activity, but ATP binding was not severely affected. The charge on that aspartate is critical for hydrolysis but not for nucleotide binding. All the prokaryotic  and  homologs contain invariant DExH residues within the DExx motif, and the invariant aspartate in E. coli  and , D126, is similarly situated at the carboxy end of a  strand (23). A D126N mutation reduced both the ATPase k_cat and catalytic efficiency, eliminated clamp loading in vitro, and eliminated DnaX activity in vivo (Fig. 2 to 4; Table 2).  complexes assembled with  D126N should support one round of clamp opening, because ATP binding is adequate to open the clamp (25, 26), but would not be expected to complete the catalytic cycle.

The T142A mutation altered a conserved residue within motif VIc. This motif has three forms; Cullman et al. (11) described VIa and VIb for RFC subunits 1 and 2 to 5, respectively. Motif VIb [s(ML)TxxAQxLRRtmE] is more closely related to VIc (fnaLLKtUEEPP). In all subunits with motif VIb or VIc, the underlined E is invariant. In  and , the naLLKtL comprises alpha helix 5; the invariant glutamate is the first residue in a solvent-exposed loop at the lower exterior of the "C"-shaped molecule (23). Mutation of the conserved T to A reduced the --' ATPase catalytic efficiency to about 14%, and k_cat to about 30%, of the wild-type levels. The  T142A protein residual ATPase responded to -' stimulation.  T142A had greatly reduced ATPase activity, Neither  T142A nor  T142A had significant clamp-loading activity.

Unexpectedly, the dnaXT142A mutant allele retained sufficient activity in vivo to support growth. The mutant allele on a multicopy plasmid complemented a dnaX(Ts) host (although the expression level was basal and not specifically induced) and a haploid dnaXT142A mutant grew normally under laboratory conditions. Perhaps the mutant DnaXT142A activity was restored in vivo by the mass action effect of other DnaXT142A molecules in replication complexes (which might not have assembled in the in vitro reactions) or was stabilized by interaction with other replication factors. Studies with mutant dna alleles in vivo and mutant proteins in vitro have defined another paradox. The principal clamp loader in vitro was concluded to be the  complex, and not the  complex (73), but  is dispensable in vivo, whereas  is indispensable (4).

Motif VII is characterized by the invariant SR followed by C or T. The arginine is located in a solvent-exposed loop leading from  helix 6 to -strand 5, the last structured region of domain 1 (23). Changing the arginine to alanine reduced ATPase activity of  (with -'); catalytic efficiency was reduced to 14% of the wild-type level, and k_cat was reduced to 18% of the wild-type level (Table 3). In , this mutation greatly reduced the ATPase activity of  and eliminated clamp loading by  or . The R169A mutation did not abolish ATPase stimulation of  or  by -', although the overall activity was reduced. This mutation also abolished DnaX activity in vivo.

The degree of wild-type  DNA-dependent ATPase stimulation by  and ' is dependent on the primer template. In all reported cases, the -' combination stimulated more than the sum of  and ' alone, but responses to  or ' individually varied with the identity of the DNA effector. The  ATPase with poly(dA):oligo(dT) was stimulated more by  than by ' (51). With M13mp18 single-strand DNA, ' was more stimulatory than  (50). With M13Gori1 single-strand DNA,  did not respond significantly to  but was stimulated by ' (Fig. 2A). The nature of the , ', and -' stimulation of  ATPase remains unknown.