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J. Biol. Chem., Vol. 280, Issue 18, 18152-18162, May 6, 2005
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¶
From the
Howard Hughes Medical Institute and Rockefeller University, New York, New York 10021
Received for publication, December 2, 2004 , and in revised form, December 22, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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, is smaller than its Gram-negative homolog, yet retains the ability to bind single-stranded DNA and contains DNA-stimulated ATPase activity comparable with E. coli . S. aureus also stimulates PolC, although they do not form as stabile a complex as E. coli polymerase III·. We demonstrate that the extreme C-terminal residues of PolC bind to and function with 
clamps from different bacteria. Hence, this polymerase-clamp interaction is highly conserved. Additionally, the S. aureus 
wrench of the clamp loader binds to E. coli . The S. aureus clamp loader is even capable of loading E. coli and Streptococcus pyogenes clamps onto DNA. Interestingly, S. aureus PolC lacks functionality with heterologous clamps when they are loaded onto DNA by the S. aureus clamp loader, suggesting that the S. aureus clamp loader may have difficulty ejecting from heterologous clamps. Nevertheless, these overall findings underscore the conservation in structure and function of Gram-positive and Gram-negative replicases despite >1 billion years of evolutionary distance between them. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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1 kb/s) and extends DNA by thousands of nucleotides without dissociating from the DNA template (1–3). These special features require accessory proteins, as the polymerase subunit alone is only weakly active in synthesis and lacks high processivity. The accessory proteins act as a clamp loader ATPase complex and a ring-shaped subunit that functions as a DNA sliding clamp (). The clamp encircles the DNA duplex and binds the polymerase, tethering it to DNA for rapid and processive chain extension. The clamp loader uses energy derived from ATP hydrolysis to pry open the clamp and to close it around a primed template.
The E. coli 
/ clamp loader consists of five proteins required for clamp loading (211'1) and also the attached 
and 
ancillary subunits (4–7). In E. coli, the / subunits connect the replicase to single-stranded DNA-binding protein (SSB),1 but are not required for clamp loading (8–10). The / subunits also contribute to the stability of the / complex in vitro (11).
In E. coli, the dnaX gene produces two polypeptides, and . (71 kDa) is the full-length product of the gene, whereas (47 kDa) is a truncated version of , produced by a translational frameshift (12–14). Both and are capable of functioning with and ' in clamp loading action (15). However, the unique C-terminal 24-kDa C-terminal region of provides extra functions relative to . For example, (but not ) binds to the polymerase directly (16). Hence, the presence of multiple subunits within the clamp loader enables it to cross-link two polymerases, thereby coupling the leading and lagging strand polymerases. Furthermore, (but not ) also binds the DnaB helicase and stimulates DNA unwinding by >20-fold (17). Finally, (but not ) binds DNA and separates the polymerase from , but only when DNA synthesis is complete (18). This polymerase dissociation function is important for lagging strand synthesis, as it frees the polymerase to function with a new clamp on a new primed site after it finishes an Okazaki fragment (19).
In some cells, the product is made by transcriptional slippage instead of a translational frameshift (20, 21). However, many cells do not produce at all, as demonstrated for thermophilic Aquifex aeolicus and Gram-positive Streptococcus pyogenes (22, 23). Most bacteria, including the Gram-positive bacteria, have no recognizable and homologs. However, it is possible that orthologs of and or even novel proteins may exist to perform the roles of these ancillary subunits.
The /, , and ' proteins are all members of the AAA+ family of proteins (24). These subunits are arranged in a circular fashion to form the clamp loader (6). Biochemical and structural studies have outlined the mechanism by which these subunits perform the clamp loading operation. In this process, opening of the clamp is performed by (25–28). binds near an interface of the dimer and distorts it, thus acting as a "wrench" to pry open the ring (29, 30). The dimer appears to be spring-loaded and automatically opens and is presumed to remain that way, whereas distorts the interface. The / subunits also bind (31), but the -interactive surfaces of and / are occluded in the complex, thus preventing the / complex from binding (27, 29). ATP, which binds only the / subunits, induces a conformational change that correlates with the ability of the / complex to bind and to open the ring (25, 26). Upon binding primed DNA, the ATP is hydrolyzed, which results in the ejection of the / complex from , allowing the ring to close around DNA (27).
The E. coli polymerase III core is a heterotrimer (
) in which the polymerase (, DnaE, 129 kDa) and proofreading 3'–5' exonuclease (, DnaQ, 28 kDa) are separate subunits (32, 33). The holE gene encoding can be deleted with no consequence (34), and no activity is attributed to the small subunit (8.6 kDa). The PolC chromosomal polymerase of Gram-positive cells is homologous to the E. coli subunit and also contains a region of homology to . Accordingly, PolC (164 kDa) contains both enzymatic activities of and on one polypeptide chain (35–44). The PolC polymerase contains some unique sequence regions not found in the Gram-negative subunit, including a zinc finger and possibly a second nucleotide-binding site (45, 46). Gram-positive cells also contain a second homolog to , the DnaE polymerase, which lacks proofreading activity like E. coli . The Gram-positive DnaE polymerase is essential and is proposed to participate in lagging strand synthesis (43, 47). Unlike E. coli or Gram-positive PolC, the Gram-positive DnaE polymerase efficiently by-passes DNA lesions, suggesting a role in DNA repair (48, 49).
Besides the E. coli DNA polymerase III holoenzyme, we have previously characterized the replicases of Gram-positive S. pyogenes and thermophilic A. aeolicus (22, 23). We have also examined the polymerase and clamp of the Gram-positive pathogen Staphylococcus aureus (50). This work extends these studies on the S. aureus replicase to include the clamp loading subunits leading to a fully reconstituted S. aureus replicase. We also perform subunit mixing studies of three different replicases to determine which components are interchangeable and thus to identify those subunit contacts that are highly conserved. Conserved contacts may be expected to serve as targets of broad-spectrum antibiotic compounds.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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(53); and ' (15); (54); and the ·', , and complexes (5) were purified and reconstituted as described. S. pyogenes PolC, , , , ', and the ·' complex were purified and reconstituted as described (22). DNA restriction enzymes were from New England Biolabs Inc. pET expression vectors and E. coli BL21(
DE3) protein expression strains were from Novagen. S. aureus genomic DNA was isolated as described previously (50). S. aureus strain 4220 was a generous gift of Dr. Pat Schlievert (University of Minnesota). S. aureus PolC and were purified as reported previously (50). Peptides used for fluorescence experiments were synthesized by Bio-Synthesis, Inc. (Lewisville, TX). Buffer A contained 20 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 5 mM dithiothreitol (DTT), and 10% glycerol. Replication buffer was composed of 25 mM Tris-HCl (pH 7.5), 8 mM MgCl2, 40 µg/ml bovine serum albumin (BSA), 4% glycerol, and 0.5 mM EDTA. Stop solution contained 1% SDS and 40 mM EDTA.
Identification of S. aureus dnaX and Purification of the Subunit— The dnaX gene of S. aureus was identified prior to publication of the genome sequence of this organism. The dnaX genes of Bacillus subtilis, E. coli, and Hemophilus influenzae were aligned, and two areas of high homology were used to design PCR primers based on the predicted amino acid sequence of this region of the S. aureus dnaX gene product and the codon usage of S. aureus. The first region of homology corresponded to residues 37–46 (HHAYLFSGTR) of the E. coli dnaX product. The second region corresponded to residues 138–148 (ALLKTLEEPPE) of the E. coli dnaX product. The upstream 38-mer used for PCR was 5'-GCGGATCCCATGCATATTTATTTTCAGGTCCAAGAGG-3' (with the BamHI site underlined). The downstream 39-mer used was 5'-CCGGAATTCTGGTGGTTCTTCTAATGTTTTTAATAATGC-3' (with the EcoRI site underlined). The PCR product (300 bp) was digested with EcoRI and BamHI and purified on a 0.8% agarose gel. The fragment was ligated into pUC18 that had been digested with EcoRI and BamHI and gel-purified on a 0.8% agarose gel. The predicted amino acid sequence within one reading frame of the insert had homology to the E. coli / proteins. This DNA sequence was used to design circular PCR primers. S. aureus chromosomal DNA was digested with HincII and ligated, and circular PCR was performed using the rightward directed primer (5'-TTT GTA AAG GCA TTA CGC AGG GGA CTA ATT CAG ATG TG-3') and the leftward directed primer (5'-TAT GAC ATT CAT TAC AAG GTT CTC CAT CAG TGC-3'). The resulting 1.6-kb PCR product was purified on a 0.8% agarose gel and sequenced directly; it encodes the N terminus of the S. aureus dnaX gene. A stretch of 750 nucleotides was obtained using the rightward directed primer in circular PCR. To obtain the complete C-terminal sequence, other sequencing primers were designed in succession based on the information of each new sequencing run. Once both the N and C termini were identified, new primers were designed for PCR of the entire dnaX gene from S. aureus genomic DNA. The N-terminal primer introduced an NdeI restriction site, and the C-terminal primer introduced a BamHI restriction site. The dnaX gene (63,471 Da, 565 residues) was then cloned into pET11a digested with NdeI/BamHI to produce pET11aSadnaX.
The pET11aSadnaX plasmid was transformed into E. coli BL21(DE3) cells. Cells (24 liters) were grown in LB medium supplemented with 200 µg/ml ampicillin to A = 0.6; harvested by centrifugation; and resuspended in 600 ml of 50 mM Tris-HCl (pH 7.5), 10% sucrose, 1 M NaCl, 30 mM spermidine, 5 mM DTT, and 2 mM EDTA. Cells were lysed by two passages through a French press (20,000 p.s.i.), followed by centrifugation in a Sorvall SLA 1500 rotor at 13,000 rpm for 30 min. Ammonium sulfate (0.3 g/ml) was added to the clarified cell lysate, followed by centrifugation at 13,000 rpm for 20 min in the Sorvall SLA 1500 rotor. The resulting pellet was resuspended in and dialyzed against buffer A. The dialyzed protein (3.5 g in 70 ml) was applied to a 180-ml DEAE-Sepharose column, and the protein was eluted with a 1.5-liter linear gradient of 50–500 mM NaCl in buffer A. 160 fractions were collected and analyzed on an 8% SDS-polyacrylamide gel to locate . The peak fractions containing (fractions 95–145; 600 mg) were combined, and the protein was concentrated by addition of ammonium sulfate to a final concentration of 0.3 g/ml. The precipitated protein was collected by centrifugation, resuspended in 60 ml of buffer A (600 mg), dialyzed against buffer A, and then loaded onto a 20-ml Mono Q column. The column was eluted with a 250-ml linear gradient of 50–500 mM NaCl in buffer A; 80 fractions were collected and analyzed on a 10% SDS-polyacrylamide gel. Peak fractions (fractions 44–58; 620 mg) were pooled, aliquoted, and stored at -80 °C.
Purification of S. aureus '—The S. aureus holB gene was identified by searching the S. aureus data base with the sequences of the E. coli and S. pyogenes ' subunits. The S. aureus holB gene encodes a 248-residue ' protein of 28,973 Da. The holB gene was amplified by PCR using an upstream 69-mer (5'-GGA TAA CAA TTC CCC GCT AGC AAT AAT TTT GTT TAA CTT TAA GAA GGA GAT ATA CCC ATG GAT GAA CAG-3') that contains an NcoI site and a downstream 39-mer (5'-AAT TTT AAA GGA TCC GTG TAT AAT ATT CTA ATT TTC CCG-3') that contains a BamHI site. The PCR product was digested with NcoI and BamHI, purified, and ligated into the NcoI and BamHI sites of pET11a to produce pETSaholB.
The pETSaholB plasmid was transformed into E. coli BL21(DE3)recA cells. A single colony was used to inoculate 12 liters of LB medium supplemented with 200 µg/ml ampicillin. Cells (12 liters) were grown, induced, and lysed as described for purification of the subunit. Ammonium sulfate (0.3 g/ml) was added to the clarified lysate (9.6 g in 32 ml). The pellet was backwashed with 30 ml of buffer A containing 0.1 M NaCl and 0.24 g/ml ammonium sulfate using a Dounce homogenizer, and then the pellet was recovered by centrifugation. The resulting pellet was resuspended in 20 ml of buffer A and dialyzed against buffer A. The dialyzed protein (1.3 g in 30 ml) was applied to a 20-ml Q-Sepharose fast flow column and eluted with a 200-ml linear gradient of 0–500 mM NaCl in buffer A; 80 fractions were collected and analyzed on a 10% SDS-polyacrylamide gel. Peak fractions (fractions 54–75; 72 mg in 200 ml) were combined and dialyzed against buffer A. The ' preparation was aliquoted and stored frozen at -80 °C.
Purification of S. aureus —The S. aureus holA gene was identified by searching the S. aureus data base with the sequences of the E. coli and S. pyogenes subunits. S. aureus holA encodes a 310-residue protein of 35,804 Da. The holA gene was amplified by PCR using an upstream 28-mer (5'-GGG AGT TTG TAA TCC ATG GAT GAA CAG C-3') that contains an NcoI site and a downstream 37-mer (5'-CTG AAC ACC TAT TAC CCT AGG CAT CTA ACT CAC ACC C-3') that contains a BamHI site. The PCR product was digested with NcoI and BamHI, purified, and ligated into the NcoI and BamHI sites of pET11a to produce pETSaholA.
The pETSaholA plasmid was transformed into E. coli NovaBlue cells (recA1 lac [F' proA+B+ lacqZ
M15::Tn10(TcR)]) (Novagen). Cells (12 liters) were grown, induced, and lysed as described for purification of the subunit. Ammonium sulfate (0.3 g/ml) was added to the clarified lysate, followed by centrifugation. The resulting pellet was resuspended in 50 ml of buffer A. The dialyzed protein (2.5 g in 300 ml) was applied to a 50-ml Q-Sepharose fast flow column and eluted with a 500-ml linear gradient of 0–500 mM NaCl in buffer A; 80 fractions were collected and analyzed on an 8% SDS-polyacrylamide gel to identify . Peak fractions (fractions 28–36) were combined (67 mg) and dialyzed against buffer A. The dialyzed protein (67 mg in 75 ml) was applied to a 1-ml Mono S-Sepharose column and eluted with a 30-ml linear gradient of 50–500 mM NaCl in buffer A; 80 fractions were collected. Peak fractions (fractions 14–18; 15 mg) of the preparation were stored frozen at -80 °C.
Purification of S. aureus SSB—The S. aureus ssb gene was identified by searching the S. aureus data base with the sequences of the E. coli and S. pyogenes SSB proteins. The S. aureus ssb gene encodes a 167-residue protein of 18,539 Da. The ssb gene was amplified by PCR using an upstream 41-mer (5'-AGA GGG GGC GTT CAT ATG CTA AAT AGA GTT GTA TTA GTA GG-3') that contains an NdeI site and a downstream 37-mer (5'-CCG CCT CTT CTG GAT CCA CCT GCC ATG ATT GTG TGC C-3') that contains a BamHI site. The PCR product was digested with NdeI and BamHI, purified, and ligated into the NdeI and BamHI sites of pET11a to produce pETSassb.
The pETSassb plasmid was transformed into E. coli BL21(DE3)-recApLysS cells. A single colony was used to inoculate 12 liters of LB medium supplemented with 200 µg/ml ampicillin. Cells (12 liters) were grown, induced, and lysed as described for purification of the subunit. Ammonium sulfate (0.16 g/ml) was added to the clarified lysate. The pellet was backwashed five times with 50 ml of buffer A containing 0.1 M NaCl and 0.13 g/ml ammonium sulfate using a Dounce homogenizer, and the pellet was recovered by centrifugation each time. The final pellet was resuspended in 20 ml of buffer A and dialyzed against buffer A. The dialyzed protein (800 mg in 100 ml) was applied to a 150-ml Q-Sepharose fast flow column and eluted with a 1-liter linear gradient of 0–500 mM NaCl in buffer A; 80 fractions were collected and analyzed on a 14% SDS-polyacrylamide gel to identify SSB. Peak fractions (fractions 39–42; 500 mg) were combined and dialyzed against buffer A. The SSB preparation was aliquoted and stored frozen at -80 °C.
Preparation of S. aureus and S. pyogenes ·' Complexes—S. aureus and S. pyogenes ·' complexes were prepared by the following procedure. Twenty milligrams of each and ' were mixed, and the conductivity of the mixture was measured. The conductivity was lowered to the equivalent of 100 mM NaCl by addition of buffer A. Mixtures were incubated at 24 °C for 5 min and then injected onto an 8-ml Mono Q column equilibrated with buffer A containing 100 mM NaCl. Protein complexes were eluted with a 20-column volume linear gradient (160 ml) of 100–500 mM NaCl in buffer A. Eighty 2-ml fractions were collected. Fractions were analyzed on a 12% SDS-polyacrylamide gel. Peak fractions (fractions 58–64) were combined and frozen at -80 °C. Typically, the yield for the ·' complexes was 50% of the initial protein amount.
PolC Holoenzyme Replication Assays—Reaction mixtures contained the following: 70 ng (25 fmol) of singly primed M13mp18 ssDNA, 0.82 µg of S. aureus SSB, 50 ng of S. aureus PolC, 100 ng of S. aureus , 25 ng of S. aureus ·', and 100 ng of S. aureus in 23.5 µl of replication buffer containing 0.5 mM ATP and 60 µM each dGTP and dCTP. Reactions were incubated at 37 °C for 2 min, and synthesis was initiated upon addition of 1.5 µl of dATP and [-32P]dTTP (2000–4000 cpm/pmol), yielding final concentrations of 60 and 20 µM, respectively. Individual reactions were performed for each time point of the time course. Reactions were quenched with an equal volume of stop solution; one-half was analyzed on a 0.8% alkaline agarose gel, and the other half was spotted onto a DE81 filter for quantitation of DNA synthesis as described (55). Assays lacking one protein subunit (see Fig. 1C) were performed similarly, except that one subunit was omitted; reactions were for 20 s; and 10 ng of either or ' were used in place of the ·' complex. (The control using 20 ng of ·' is also shown in Fig. 1C.)
Stimulation of PolC by —Reactions contained 70 ng (25 fmol) of singly primed M13mp18 ssDNA; 0.82 µg of S. aureus SSB; 10 ng of S. aureus PolC; 0, 30, or 100 ng of S. pyogenes, E. coli, or S. aureus ; 0.5 mM ATP; 60 µM each dGTP, dCTP, and dATP; and 20 µM [32P]dTTP (specific activity of 2000–4000 cpm/pmol) in 25 µl of replication buffer. Time points were removed at the following times: 1.0, 1.5, 2.0, 2.5 5.0, and 10.0 min. Reactions were quenched with an equal volume of stop solution and then analyzed for total DNA synthesis as described (55).
Peptide Inhibition of DNA Synthesis—Replication reactions contained 70 ng (25 fmol) of 60-mer singly primed M13mp18 ssDNA; 1 µg of E. coli SSB; clamp loader (200 ng of , 20 ng of ·') and clamp (40 ng of ) from S. aureus, S. pyogenes,or E. coli; and the indicated amount of 20-mer peptide corresponding to the C-terminal residues of the DNA polymerase in 25 µl of replication buffer containing 0.5 mM ATP, 60 µM dGTP, 60 µM dCTP, 60 µM dATP, and 20 µM [-32P]dTTP (2000–4000 cpm/pmol). Reactions were preincubated at 37 °C for 3 min, and synthesis was initiated upon addition of 50 ng of polymerase subunit (S. aureus PolC, S. pyogenes PolC, or E. coli pol III core). Reactions were allowed to proceed for 3 min prior to being quenched with an equal volume of stop solution. One-half of the reaction was analyzed for total DNA synthesis using DE81 filter paper.
Interchangeability of and ' Subunits—Reaction mixtures contained 70 ng (25 fmol) of singly primed M13mp18 ssDNA; 0.82 µg of E. coli SSB; 0.5 mM ATP; 60 µM each dGTP, dCTP, and dATP; and 20 µM [32P]dTTP (specific activity of 2000–4000 cpm/pmol) in 23 µl of replication buffer. Individual reactions also contained, when present, 30–300 ng of /' mixture, 50 ng of S. aureus PolC, 100 ng of S. aureus , and 40 ng of S. aureus . Proteins were incubated for 3 min at 37 °C. Reactions were allowed to proceed for 2 min before being quenched with an equal volume of stop solution (25 µl of 1% SDS and 40 mM EDTA). /' mixtures were assembled by incubating equimolar amounts of and ' (S. aureus (2.24 µg) and ' (2.0 µg) and S. pyogenes (2.33 µg) and ' (2.0 µg)) in 50 µl of buffer A at 15 °C for 10 min.
Clamp and Clamp Loader Exchange Reactions with S. aureus PolC— Replication reactions contained 70 ng (25 fmol) of singly primed M13mp18 ssDNA; 0.82 µg of E. coli SSB; 0.5 mM ATP; 60 µM each dGTP, dCTP, and dATP; and 20 µM [32P]dTTP (specific activity of 2000–4000 cpm/pmol) in 23 µl of replication buffer. Individual replication reactions contained combinations of the following proteins as indicated: 50 ng of S. aureus PolC, 100 ng of E. coli complex ('), 40 ng of E. coli , 100 ng each of S. pyogenes and ·',40ngof S. pyogenes , 100 ng each of S. aureus and ·', and 40 ng of S. aureus . Proteins (in 2 µl) were added to the reactions on ice, and reactions were shifted to 37 °C. DNA synthesis was allowed to proceed for 5 min before being quenched with an equal volume (25 µl) of stop solution. One-half of the quenched reaction was analyzed for total DNA synthesis using DE81 paper, and the other half was analyzed on a 0.8% native agarose gel.
Gel Filtration Analysis of Protein-Protein Interaction—Analysis of polymerase- interaction was performed in 200 µl of buffer A containing 100 mM NaCl, 1 mg of DNA polymerase subunit (E. coli or S. aureus PolC), and, when present, 1 mg of either E. coli or S. aureus subunit. Reactions were incubated at 24 °C for 15 min and then injected onto a Superose 6 HR 10/30 column equilibrated with buffer A containing 100 mM NaCl. After collecting the void volume, 200-µl fractions were collected and analyzed on a 10% SDS-polyacrylamide gel.
Analysis of -' interaction was performed in reactions that contained 200 µg of S. aureus or ' (or both) in 200 µl of buffer A containing 100 mM NaCl. Reactions were incubated at 24 °C for 15 min and then injected onto a Superose 6 HR 10/30 column equilibrated with buffer A containing 100 mM NaCl. Fractions of 200 µl were collected and analyzed on a 12% SDS-polyacrylamide gel.
Loading Reactions—A 6-residue N-terminal kinase site was introduced immediately behind the initiating methionine of S. aureus by PCR using a primer with a sequence encoding a 6-amino acid recognition site (RRASVP) for cAMP-dependent protein kinase to form PK. The gene encoding S. aureus PK was cloned into the NcoI/BamHI sites of pET16b. S. pyogenes PK and E. coli PK were purified as described (19, 22). S. aureus PK was expressed and purified as described (50). PK proteins were radiolabeled using cAMP-dependent protein kinase and [-32P]ATP as described for E. coli PK (19, 56, 57). Clamp loading reactions contained 2 µg of uniquely nicked pBluescript plasmid DNA (prepared as described previously (19)), 100 ng of 32P-labeled ,70ngof , and 30 ng of ·' (when present) in 75 µl of 20 mM Tris-HCl (pH 7.5), 4% glycerol, 0.1 mM EDTA, 5 mM DTT, 1 mM ATP, 40 µg/ml BSA, and 10 mM MgCl2. Reactions were incubated for 10 min at 37 °C and then applied to 5-ml Bio-Gel A-1.5m columns (Bio-Rad) pre-equilibrated with 20 mM Tris-HCl (pH 7.5), 5% glycerol, 2 mM DTT, 40 µg/ml BSA, 8 mM MgCl2, and 100 mM NaCl. Gel filtration was performed at 4 °C, and 200-µl fractions were collected; 150 µl of each fraction were analyzed by liquid scintillation counting.
Protein-Protein Interaction Analysis Using 96-Well Microtiter Plates—E. coli or S. aureus (20 ng/ml) was incubated in the wells of a vinyl 96-well microtiter plate (Costar Corp.) for 12 h at 4 °C in 100 µl of buffer B (20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 2 mM DTT, 5 mM MgCl2, 100 mM NaCl, and 20% glycerol). Solutions were removed from the wells, and the wells were washed four times with 100 µl of buffer C (0.1 M sodium phosphate (pH 7.5), 0.15 M NaCl, and 0.1% Tween 20). The wells were then blocked with 100 µl of buffer C containing 5% nonfat milk for 3 h at 23 °C. Following the blocking step, the wells were washed four times with buffer C and then incubated with 100 µl of buffer B containing E. coli, S. pyogenes,or S. aureus 32P-labeled PK (20 ng/ml) for 2 h at 23 °C. The solutions were removed, and the wells were washed four times with 100 µl of buffer C, air-dried, and analyzed using a PhosphorImager.
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(as monomer), 1 µM M13Gori1 ssDNA (as nucleotide), and 1 mM [-32P]ATP. Aliquots were removed at various times and quenched with an equal volume of stop solution. Quenched solutions were spotted (0.5 µl) onto polyethyleneimine-cellulose thin layer chromatography sheets and then developed in 0.5 M LiCl and 1 M formic acid. [-32P]ATP and [-32P]ADP were quantitated using the PhosphorImager and ImageQuant software (Amersham Biosciences).
DNA Gel Shift Assay—Gel mobility shift reactions were performed in 20 µlof20mM Tris-HCl (pH 7.5), 4% glycerol, 0.1 mM EDTA, 5 mM DTT, 40 µg/ml BSA, and 10 mM MgCl2. Reactions contained 0.4–16 µM S. aureus (as monomer) and 1 µM 32P-labeled (dT)15 DNA (as 15-mer). Reactions were incubated for 10 min at 37 °C. Products were resolved on a 6% nondenaturing polyacrylamide gel.
Steady-state Fluorescence Measurements—The E. coli subunit was labeled at Cys333 using Oregon Green 488 maleimide (Molecular Probes, Inc., Eugene, OR) to form OG as described previously (58). Peptide titrations contained OG at a concentration of 500 nM, 1 µM, or 2 µM in 60 µl of 20 mM Tris-HCl (pH 7.5), 5 mM DTT, 1 mM EDTA, and 50 mM NaCl. The peptides used in this study were as follows: E. coli C-terminal peptide, NH2-RLLNDLRGLIGSEQVELEFD-COOH; S. pyogenes PolC C-terminal peptide, NH2-GEMGILGNMPDNQLSLFDDFFCOOH; and S. aureus PolC C-terminal peptide, NH2-DELGSLPNLPDKAQLSIFDM-COOH. Reactions were mixed on ice, shifted to 22 °C for 20 min, and transferred into a 3 x 3-mm cuvette. Measurements were taken using a QuantaMaster spectrofluorometer (Photon Technology International, South Brunswick, NJ). Fluorescence emission spectra were recorded from 500 to 630 nm using an excitation wavelength of 490 nm. Fluorescence emission at 517 nm was used for analysis. Data points were fit according to the model A + B 
AB using Origin software (Microcal Software Inc., Northampton, MA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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, , ', , and SSB were identified; cloned into pET-based expression vectors; and sequenced. The S. aureus dnaX gene did not reveal either a transcriptional or translational frameshift. Accordingly, overexpression of S. aureus in E. coli produced a single polypeptide corresponding to the mass of the full-length product (63.4 kDa) (Fig. 1A). Recombinant S. aureus PolC, , , ', , and SSB were purified, and samples of the final preparation of each subunit are shown in the Coomassie Blue-stained SDS-polyacrylamide gel of Fig. 1A.
Properly loaded onto a primed site, the clamp tethers its respective replicase to DNA for exceedingly high processivity and a synthetic rate of 500–700 nucleotides/s (22, 59). In Fig. 1B, we tested the ability of the S. aureus clamp and clamp loading subunits to provide PolC with high primer extension speed. The substrate for this assay was a 7.2-kb circular M13mp18 ssDNA genome coated with SSB and uniquely primed with a 60-mer DNA. The S. aureus PolC, , , , and ' subunits were preincubated with the primed substrate and ATP for 5 min to allow time for the proteins to assemble on the primed site (see scheme in Fig. 1B). Two deoxyribonucleoside triphosphates, dCTP and dGTP, were present during the preincubation to prevent loss of the primer via the 3'–5' exonuclease of PolC. Synchronous primer elongation was initiated upon addition of dATP and [32P]dTTP, and reactions were quenched at the indicated times. The results in Fig. 1B demonstrate that the S. aureus PolC holoenzyme completed the 7.2-kb template within 10 s (700 nucleotides/s), a rate that is comparable with the rates of both the E. coli and S. pyogenes holoenzymes (22, 60, 61).
Next, we determined whether all the subunits are needed to reconstitute the rapid and processive replicase. Reactions were performed as described for Fig. 1B, except that one subunit was omitted from each reaction. Reactions were quenched after 20 s, sufficient time for the S. aureus replicase to complete the DNA substrate. The control reaction (containing PolC, , , ', and ) produced the full-length replicative form two (RF II) product (Fig. 1C, lane 7). However, mixtures in which one of the proteins was omitted failed to generate a full-length RF II product (lanes 1–6). Hence, all five protein subunits are required for this reaction.
The S. aureus Subunit Binds DNA—E. coli binds ssDNA, but does not, indicating that the DNA-binding site in lies in the 24-kDa C-terminal region unique to (62). S. aureus (64 kDa) is intermediate in size between E. coli (47 kDa) and (71 kDa) and thus may lack some of the properties of E. coli . In Fig. 2A, we investigated whether S. aureus could bind a 32P-5'-end-labeled ssDNA oligonucleotide in a native polyacrylamide gel shift assay. The results show that S. aureus retains the capacity to bind ssDNA.
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is also a DNA-stimulated ATPase. In Fig. 2B, the ATPase activities of E. coli and S. aureus are compared, with and without ssDNA. The results show that they have comparable ATPase activities and are stimulated by ssDNA to similar extents.
The S. aureus PolC- Interaction Is Weak Compared with the E. coli pol III- Interaction—In the E. coli system, the subunit binds to the pol III core to form the pol III' complex (63). This interaction is mediated directly by the DNA polymerase subunit of the pol III core (16). In Fig. 2C, we examined the interaction between S. aureus PolC and by gel filtration analysis. The E. coli and subunits are shown as a control (upper panel). The E. coli and subunits formed a complex and co-eluted much earlier (fractions 10–16) compared with the subunit alone (fractions 26–30). Next, we examined S. aureus PolC and . As shown in Fig. 2C (lower panels), S. aureus PolC did not alter its elution position in the presence of and thus does not form a stabile PolC· complex under these conditions.
Gel filtration is a non-equilibrium technique; and thus, only the strongest protein·protein complexes survive. Therefore, S. aureus PolC and lack the stability needed to remain associated during this 30-min procedure. Hence, we examined S. aureus for the ability to stimulate PolC as evidence that these two proteins interact.
We have shown that the subunits of E. coli, S. pyogenes, and A. aeolicus stimulate the distributive DNA synthesis activity of their respective DNA polymerase subunits in the absence of the clamp (23). This stimulation is species-specific and therefore relies on specific amino acid contacts between the subunit and the DNA polymerase (58, 64). may enhance polymerase activity by increasing the affinity of the polymerase for DNA because binds to both DNA and the polymerase.
In Fig. 2D, S. aureus was examined for its effect on PolC-catalyzed DNA synthesis using a singly primed M13mp18 ssDNA template. S. aureus increased the total amount of product synthesized by S. aureus PolC as measured by the total amount of incorporated radionucleotide (Fig. 2D). S. aureus stimulation of S. aureus PolC polymerase activity is species-specific, as neither E. coli nor S. pyogenes was capable of stimulating S. aureus PolC (Fig. 2D). These results indicate that S. aureus binds to S. aureus PolC even though the interaction is not stabile to gel filtration analysis.
Analysis of the S. aureus Clamp Loader—Next, we studied the S. aureus clamp loader subunits for the ability to load onto a circular DNA substrate. To follow the S. aureus clamp on and off DNA, a 6-residue kinase recognition motif was placed on the N terminus of . This PK derivative was treated with cAMP-dependent protein kinase and [-32P]ATP to yield 32P-labeled PK. 32P-Labeled PK was incubated with a singly nicked DNA plasmid in the presence of the S. aureus clamp loading subunits (, , and ') and ATP. After 10 min at 37 °C, the reaction was analyzed on a large pore Bio-Gel A-1.5m gel filtration column. This large pore resin includes proteins such as 32P-labeled PK (in fractions 20–30), but excludes the large DNA plasmid; and thus, 32P-labeled PK bound to the large DNA eluted early (in fractions 10–15). Fig. 3 shows that S. aureus 32P-labeled PK eluted with nicked DNA only in presence of the S. aureus clamp loader subunits. Therefore, the S. aureus clamp loading subunits are active in the assembly of onto DNA. The results also show that the S. aureus ·DNA complex is quite stabile, as this technique is performed at room temperature and requires 20 min.
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and ' have demonstrated that they form a tight ·' complex that is stabile to analysis by gel filtration (15, 22). Next, we studied S. aureus and ' for complex formation by gel filtration analysis (Fig. 4A). A mixture of the and ' subunits co-eluted in earlier fractions (i.e. as a higher molecular mass complex) (upper panel) than either subunit alone (middle and lower panels). Therefore, the S. aureus and ' proteins form a sufficiently tight complex to be isolated on a gel filtration column.
We also analyzed a mixture of the S. aureus , , and ' subunits for a stabile ··' complex, but and the ·' complex did not co-elute (data not shown). Under similar conditions, the E. coli subunits form a stabile ··' complex (15). However, studies of S. pyogenes , , and ' have also demonstrated that they form a less stabile ··' complex compared with E. coli ··' (22). Overall, our observations indicate that the subunit from Gram-positive organisms interacts weakly with PolC and the ·' complex compared with the corresponding subunit from the Gram-negative E. coli bacterium.
We examined the and ' subunits of E. coli, S. pyogenes, and S. aureus for cross-species interaction by gel filtration analysis, but no cross-species ·' complexes were stabile to this technique (data not shown). Likewise, we investigated whether the ·' complexes of these three systems can bind a heterologous subunit by this technique, but again observed no such stabile cross-species complexes. As will be shown below, activity assays detect limited functional cross-species interaction among these proteins.
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Can Bind to and Function with E. coli —The wrench binds the clamp and pries open the ring (27, 28). The crystal structure of the E. coli · complex reveals residues essential for this interaction (6). Specifically, residues Leu73 and Phe74 bind to a hydrophobic pocket on .
The E. coli and S. aureus subunits were examined for an interaction with cognate and non-cognate clamps (Fig. 4B). The subunits were immobilized in the wells of a 96-well microtiter plate. After blocking the wells, either E. coli or S. aureus 32P-labeled clamps were added; the wells were washed; and the plates were analyzed for remaining 32P-labeled . S. aureus 32P-labeled was present in wells containing S. aureus and the evolutionarily distant E. coli subunit. This result suggests that S. aureus can bind E. coli . E. coli 32P-labeled was present only in wells containing E. coli . Hence, E. coli does not appear to bind S. aureus .
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clamps onto DNA using non-cognate clamp loaders. In the experiments of Fig. 5, the clamp loaders from E. coli, S. aureus, and S. pyogenes were examined for their ability to load non-cognate clamps onto DNA. The clamps from E. coli, S. aureus, and S. pyogenes all contain the 6-residue kinase motif at the N terminus, allowing them to be labeled with [-32P]ATP. The different 32P-labeled clamps were incubated with nicked circular double-stranded DNA, ATP, and clamp loading subunits for 10 min, and the reaction was analyzed on a large pore Bio-Gel A-1.5m gel filtration column. The results show that the S. aureus clamp loader is capable of loading clamps from S. pyogenes and E. coli, although with less efficiency compared with S. aureus . In contrast, the E. coli clamp loader is capable of loading only its own clamp. The Gram-positive S. pyogenes clamp loader is also capable of loading non-cognate clamps onto DNA, although it is somewhat less efficient in this regard compared with the S. aureus clamp loader.
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Clamp Provided It Is Placed on DNA by Its Cognate Clamp Loader—Fig. 5 demonstrates that the S. aureus clamp loader can load E. coli and S. pyogenes clamps onto DNA and that the S. pyogenes clamp loader can load E. coli and S. aureus clamps onto DNA. Are these clamps capable of function with S. aureus PolC? In the experiment of Fig. 6, we examined this question using singly primed M13mp18 ssDNA coated with SSB as substrate for mixtures of clamps and clamp loaders of these three bacteria. Under the conditions used here, S. aureus PolC cannot extend the unique primer full circle around this large substrate unless a clamp is assembled on the DNA, and the clamp loader must eject from the clamp so that PolC can bind to the clamp (e.g. see Fig. 1).
S. aureus PolC is quite efficient in the synthesis of the RF II duplex product using the clamp of S. pyogenes or E. coli, provided the clamp is loaded onto DNA using its cognate clamp loader (Fig. 6, lanes 1 and 9, respectively). The S. aureus clamp loader is capable of loading S. pyogenes onto DNA (see Fig. 5), and this combination does not result in an RF II duplex product (Fig. 6, lane 2), although it is reduced relative to clamps placed onto DNA by their cognate clamp loader. The S. aureus clamp loader can also load E. coli onto DNA; however, the result in lane 8 shows that no RF II was produced with PolC. Instead, there was a smear of immature product. Presumably, the S. aureus clamp loader interferes with PolC utilization of E. coli , perhaps having difficulty ejecting from the clamp. This is most pronounced in the case of the S. pyogenes clamp loader, which can load the S. aureus and E. coli clamps onto DNA, but the clamps cannot be used by S. aureus PolC (lanes 4 and 7, respectively).
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Can Tolerate Exchange of or ' with S. pyogenes or '—We next determined whether the and ' subunits of the clamp loader can be exchanged among bacterial species. In the experiments of Fig. 7, we focused on the S. pyogenes and S. aureus systems, as we have obtained negative results in subunit exchanges with only the E. coli system.2 The PolC, , and subunits of either S. aureus or S. pyogenes were mixed with the indicated amounts of and ' from either organism and incubated in the presence of singly primed SSB-coated M13mp18 ssDNA, ATP, dCTP, and dGTP. After 3 min, dATP and [32P]dTTP were added to initiate synthesis.
The results show that the S. aureus system can function when either (Fig. 7, lanes 7–10) or ' (lanes 11–14) is replaced with S. pyogenes or '. At high concentrations of either S. pyogenes or ', DNA synthesis by S. aureus PolC, , and was clearly observed (lanes 10 and 14) with these protein exchanges. In contrast, DNA synthesis was inefficient using S. pyogenes PolC, , and and either S. aureus (lanes 15–18) or S. aureus ' (lanes 3–6). Controls using the complete S. aureus (lane 20) and S. pyogenes (lane 19) systems showed much more efficient synthesis. Lanes 1 and 2 show reactions in which both and ' were derived from the heterologous source. Even using the highest level of S. pyogenes ·' with S. aureus PolC, , and , the resulting synthesis was less than observed in reactions containing only one S. pyogenes subunit.
The C-terminal Residues of PolC Interact with E. coli —The C-terminal 20 residues of E. coli form an essential contact with the E. coli clamp (18, 58, 65, 66). Our previous analysis of this sequence indicates that the Gln, Leu, and Phe side chains within the last 7 residues are important contributors to the strength of the interaction with (66). Sequence alignment of the C-terminal tail of E. coli , S. pyogenes PolC, and S. aureus PolC shows that these residues are conserved across these bacterial species (Fig. 8A).
We previously developed an assay using a fluorescently tagged E. coli subunit to quantify the interaction between and a 20-mer peptide corresponding to the C terminus of the E. coli subunit (58). This procedure takes advantage of the fact that only 1 of the Cys residues in E. coli is accessible to solvent (Cys333) and can be labeled using a maleimide (67). Furthermore, Cys333 is located opposite the side of to which the DNA polymerase binds (54, 68). Hence, labeling of Cys333 does not interfere with the function of the clamp. In Fig. 8B, we labeled E. coli with the Oregon Green fluorophore at Cys333 and used it to study the interaction with 20-mer peptides corresponding to the C terminus of S. aureus and S. pyogenes PolC.
PolC from both S. aureus and S. pyogenes is functional with the E. coli clamp (22, 50). Hence, if PolC binds the clamp via the polymerase C-terminal residues, the 20-mer peptide corresponding to the C-terminal residues of S. aureus and S. pyogenes PolC should also bind to E. coli . The control using the E. coli pol III C-terminal 20-mer peptide resulted in a fluorescence enhancement of OG and yielded a Kd of 2.7 ± 0.4 µM. The results for PolC in Fig. 8B demonstrate an increase in OG fluorescence as 20-mer peptides from both S. aureus and S. pyogenes PolC were titrated into the reaction, thus indicating that they also bind to E. coli OG. The affinity of the S. aureus (7.2 µM) and S. pyogenes (2.5 µM) PolC 20-mer peptides for E. coli is within 3-fold of the Kd obtained using the E. coli pol III 20-mer peptide, underscoring the high degree of conservation in the polymerase clamp interaction across divergent bacterial species.
This highly conserved binding site in may provide an attractive target for a broad-spectrum antibiotic compound. If this is the case, then these polymerase C-terminal 20-mer peptides should also bind to S. pyogenes and S. aureus . The clamps of these Gram-positive organisms have not been studied as intensively as the E. coli clamp; and thus, we do not know if they contain a unique exposed Cys residue. Hence, we investigated whether these polymerase peptides inhibit the function of the S. aureus and S. pyogenes clamps in DNA synthesis assays. If the polymerase peptide binds to S. aureus or S. pyogenes in the polymerase-binding site, it should inhibit replication. Furthermore, studies in the E. coli system have shown that the clamp loader also interacts with at the same site, or an overlapping site, as the polymerase-binding site (68). Hence, the pol III C-terminal 20-mer peptide not only inhibits pol III- interaction, but also inhibits clamp loader function with (66).
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) and absence (•) of ssDNA. S. aureus (Sa) 
) and absence (x) of ssDNA. C, mixtures of 
