JBC Papers in Press. Published on January 12, 2005 as Manuscript M

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1 JBC Papers in Press. Published on January 12, 2005 as Manuscript M CONSERVED INTERACTIONS IN THE STAPHYLOCOCCUS AUREUS DNA POLC CHROMOSOME REPLICATION MACHINE Irina Bruck 2, Roxana E. Georgescu 1,2, and Mike O Donnell 1,2 Howard Hughes Medical Institute 1, Rockefeller University 2, 1230 York Ave, New York, New York 10021,Tel: ; Fax: ; odonnel@mail.rockefeller.edu bruck@mod.rockefeller.edu Running Title: The S. aureus replication machine ABSTRACT The PolC holoenzyme replicase of the gram positive Staphylococcus aureus pathogen has been reconstituted from pure subunits. Individual S. aureus replicase subunits are compared to subunits from the gram negative E. coli Pol III holoenzyme for activity and interchangeability. The central organizing subunit, τ, is smaller than its gram negative homolog, yet retains ability to bind ssdna and contains DNA stimulated ATPase activity comparable to E. coli τ. S. aureus τ also stimulates PolC although they do not form as stabile of a complex as E. coli Pol III-τ. We demonstrate that the extreme C-terminal residues of PolC bind and function with β clamps from different bacteria. Hence, this polymerase-to-clamp interaction is highly conserved. Additionally, the S. aureus δ wrench of the clamp loader binds to E. coli β. The S. aureus clamp loader is even capable of loading E. coli β and Streptococcus pyogenes β clamps onto DNA. Interestingly, S. aureus PolC lacks functionality with heterologous β 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 over one billion years of evolutionary distance between them. INTRODUCTION The E. coli chromosomal replicase, DNA polymerase III holoenzyme, is extremely rapid (-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 (β). The β 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 β-clamp, and close it around a primed template. The E. coli γ/τ clamp loader consists of 5 proteins required for clamp loading (τ 2 γ 1 δ 1 δ 1 ) and also the attached χ and ψ ancillary subunits (4-7). In E. coli the χψ subunits connect the replicase to SSB but are not required for clamp loading (8-10). χψ also contribute to stability of the γ/τ complex in vitro (11). In E. coli the dnax gene produces two polypeptides, τ and γ. τ (71 kda) is the full-length product of the gene while γ (47 kda) is a truncated version of τ, produced by a translational frameshift (12-14). Both τ and γ are capable of functioning with δ and δ in clamp loading action (15). However, the unique C-terminal 24 kda C-terminal region of τ provides extra functions relative to γ. For example, τ but not γ, binds to the polymerase directly (16). Hence, the presence of multiple τ subunits within the clamp loader enables it to cross-link two polymerases, thereby coupling the leading Copyright 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

2 2 and lagging strand polymerases. Furthermore, τ (not γ) also binds the DnaB helicase, and stimulates DNA unwinding over 20-fold (17). Finally, τ (not γ) binds DNA and separates the polymerase from β, 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 β clamp on a new primed site after it finishes an Okazaki fragment (19). In some cells the γ product is made by transcriptional slippage instead of a translation frame-shift (20,21). However, many cells do not produce γ at all, as demonstrated for the thermophile, Aquifex aeolicus, and the gram-positive S. pyogenes (22,23). Most bacteria including the grampositive bacteria, have no recognizable χ and ψ homologs. However, it is possible that orthologs of χ and ψ, or even novel proteins, may exist to perform the roles of these ancillary subunits. The γ/ τ, δ and δ proteins are all members of the AAA+ family of proteins (24). These 5 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 δ (25-28). δ binds near an interface of the β dimer and distorts it, thus acting as a wrench to pry open the ring (29,30). The β dimer appears to be spring loaded, and automatically opens and is presumed to remain that way while δ distorts the interface. The γ/τ subunits also bind β (31) but the β interactive surfaces of δ and γ/ τ are occluded in the complex, thus preventing the γ/τ complex from binding β (27,29). ATP, which binds only the γ/ τ subunits, induces a conformation change that correlates with ability of the γ/ τ complex to bind β and open the ring (25,26). Upon binding primed DNA, the ATP is hydrolyzed which results in the γ/ complex τ ejecting from β, allowing the ring to close around DNA (27). The E. coli core polymerase III is a heterotrimer (αεθ) in which the polymerase (α, DnaE, 129 kda) and proofreading 3-5 exonuclease (ε, DnaQ, 28 kda) are separate subunits (32,33). The hole gene encoding θ can be deleted with no consequence (34), and no activity is attributed to the small θ subunit (8.6 kda). The PolC chromosomal polymerase of gram-positive cells is homologous to E. coli α subunit, and also contains a region of homology to ε. Accordingly, PolC (164 kda) contains both enzymatic activities of α and ε on one polypeptide chain (35-44). PolC polymerase contains some unique sequence regions not found in gram-negative α subunit, including a zinc finger and possibly a second nucleotide-binding site (45,46). Gram-positive cells also contain a second homologue to α, the DnaE polymerase, which lacks proofreading activity like E. coli α. The gram-positive DnaE polymerase is essential and is proposed to participate in lagging strand synthesis (43,47). Unlike E. coli α or grampositive PolC, the gram-positive DnaE polymerase efficiently bypasses 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 the grampositive Streptococcus pyogenes and thermophilic Aquifex aeolicus (22,23). We have also examined the polymerase and clamp of the gram-positive pathogen, Staphylococcus aureus (50). The current report 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 identify those subunit contacts that are highly conserved. Conserved contacts may be expected to serve as targets of broad spectrum antibiotic compounds. EXPERIMENTAL PROCEDURES Materials - Radioactive nucleotides were from New England Nuclear (NEN); unlabeled nucleotides were from Amersham Biosciences. DNA

3 3 oligonucleotides were from Invitrogen. M13mp18 ssdna was purified from phage that was isolated by two successive cesium chloride gradients as described (51). M13mp18 ssdna was primed with a 60 mer DNA oligonucleotide (map position ). E. coli Pol III core (52), τ (53), δ and δ (15), β (54), δδ complex, τ complex and γ complex (5) were purified and complexes were reconstituted as described. S. pyogenes Pol C, τ, β, δ, δ and δδ complex were purified and reconstituted as described (22). DNA restriction enzymes were from New England Biolabs. pet expression vectors and E. coli BL21(λDE3) 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 β were purified as previously reported (50). Peptides used for fluorescence experiments were synthesized by Biosynthesis, Inc (Lewisville, TX). Buffer A is 20 mm Tris-HCl (ph 7.5), 0.5 mm EDTA, 5 mm DTT and 10% glycerol. Replication buffer is 25 mm Tris-HCl (ph 7.5), 8 mm MgCl 2, 40 µg/ml BSA, 4% glycerol and 0.5 mm EDTA. Stop solution is 1% SDS, 40 mm EDTA. Identification of S. aureus dnax and purification of τ subunit - The dnax gene of S. aureus was identified prior to publication of the genome sequence of this organism. The dnax genes of B. subtilis, E. coli and H. 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 amino acid residues (HHAYLFSGTR) of the E. coli dnax product. The second region corresponded to residues (ALLKTLEEPPE) of the E. coli dnax product. For PCR, the upstream 38 mer was 5'-GCGGATCCCATGCATATTTATTTTCA GGTCCAAGAGG-3' (BamHI site is underlined). The downstream 39 mer was 5'-CCGGAATTCTGGTGGTTCTTC TAATGTTTTTAATAATGC-3' (EcoRI site is underlined). The PCR product (~300 bp) was digested with EcoRI and BamHI and purified in a 0.8% agarose gel. The fragment was ligated into puc18 which had been digested with EcoRI and BamHI and gel purified in a 0.8% agarose gel. The predicted amino acid sequence within one reading frame of the insert had homology to the E. coli γ/τ proteins. This DNA sequence was used to design circular PCR primers. S. aureus chromosomal DNA was digested with HincII, ligated and circular PCR was performed using the following primers: the rightward directed primer was 5'-TTT GTA AAG GCA TTA CGC AGG GGA CTA ATT CAG ATG TG-3'; the sequence of the leftward primer was 5'-TAT GAC ATT CAT TAC AAG GTT CTC CAT CAG TGC-3'. The resulting 1.6 kb PCR product was purified from a 0.8% agarose gel and sequenced directly; it encoded the N- terminus of the S. aureus dnax gene. A stretch of approximately 750 nucleotides was obtained using the rightward primer in the circular PCR reaction. 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 to PCR 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 pet11asa dnax plasmid was transformed into E. coli BL21(λDE3). Cells (24L) were grown in LB supplemented with 200 µg/ml ampicillin to OD 0.6, then harvested by centrifugation and resuspended in 600 ml 50 mm TrisHCl (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 psi) followed by centrifugation in a Sorval 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 a Sorval SLA 1500 rotor.

4 4 The resulting pellet was resuspended 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 L linear gradient of mm NaCl in buffer A; 160 fractions were collected and analyzed on an 8% SDSpolyacrylamide gel to locate τ. The peak fractions containing τ (fractions , 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 buffer A (600 mg), dialyzed against buffer A, and then loaded onto a 20 ml MonoQ column. The column was eluted with a 250 ml linear gradient of mm NaCl in buffer A; 80 fractions were collected and analyzed on a 10% SDS-polyacrylamide. Peak fractions were pooled (fractions 44-58, 620 mg), aliquoted, and stored at 80 C. Purification of S. aureus δ - The S. aureus holb gene was identified by searching the S. aureus database with the sequences of E. coli and S. pyogenes δ' subunits. The S. aureus holb gene encodes a 248-residue δ 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(λDE3)recA. A single colony was used to inoculate 12L of LB media supplemented with 200µg/ml ampicillin. Cells (12L) were grown, induced, and lysed as described for purification of τ subunit. Ammonium sulfate (0.3 g/ml) was added to the clarified lysate (32 ml, 9.6 g). The pellet was backwashed in 30 ml buffer A containing 0.1 M NaCl and 0.24 g/ml ammonium sulfate using a Dounce homogenizer, 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 FFQ Sepharose column and eluted with a 200 ml linear gradient of mm NaCl in buffer A; 80 fractions were collected and analyzed on an 10% SDS-polyacrylamide gel. Peak fractions (54-75) were combined (72 mg in 200 ml) and dialyzed against buffer A. The δ preparation was aliquoted and stored frozen at -80 C. Purification of S. aureus δ - The S. aureus hola gene was identified by searching the S. aureus database with the sequences of E. coli and S. pyogenes δ 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 (reca1 lac[f'proa+b+lacqz M15::Tn10(TcR)) (Novagen). Cells (12 L) were grown, induced, and lysed as described for purification of τ 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 FFQ Sepharose column and eluted with a 500 ml linear gradient of mm NaCl in buffer A; 80 fractions were collected and analyzed on an 8% SDS-polyacrylamide gel to identify δ. Peak fractions (fr 28-36, 67 mg) were combined and dialyzed against buffer A. The dialyzed protein (67 mg in 75 ml) was applied to a 1 ml MonoS Sepharose column and eluted with a 30 ml linear gradient of mm NaCl in buffer A; 80 fractions were collected. Peak fractions (fr 14-18, 15 mg) of the δ preparation were stored frozen

5 5 at -80 C. Purification of S. aureus SSB - The S. aureus ssb gene was identified by searching the S. aureus database with the sequences of E. coli and S. pyogenes SSB proteins. The S. aureus ssb 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(λDE3)recApLysS. A single colony was used to inoculate 12L of LB media supplemented with 200µg/ml ampicillin. Cells (12L) were grown, induced, and lysed as described for purification of τ subunit. Ammonium sulfate (0.16 g/ml) was added to the clarified lysate. The pellet was backwashed 5 times in 50 ml 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 FFQ Sepharose column and eluted with a 1L linear gradient of mm NaCl in buffer A; 80 fractions were collected, and analyzed in a 14% SDSpolyacrylamide gel to identify SSB. Peak fractions (39-42) were combined (500 mg) and dialyzed against buffer A. The SSB preparation was aliquoted and stored frozen at -80 C. Preparation of S. aureus and S. pyogenes δδ complexes S. aureus and S. pyogenes δδ complexes were prepared by the following procedure: 20 mg of each δ and δ 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 MonoQ column equilibrated in buffer A containing 100 mm NaCl. Protein complexes were eluted with a 20 column volume linear gradient (160 ml) from 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 δδ complexes is 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 S. aureus PolC, 100 ng S. aureus τ, 25 ng S. aureus δ δ, 100 ng S. aureus β, in 23.5 µl replication buffer containing 0.5 mm ATP, and 60 µm each of dgtp and dctp. Reactions were incubated at 37 C for 2 min, and then synthesis was initiated upon addition of 1.5 µl of datp and [α- 32 P] dttp (~2,000-4,000 cpm/pmol) yielding a final concentration 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 and then one half was analyzed in 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 in (Fig. 1C) were performed similarly except one subunit was omitted, reactions were for 20 s, and either 10 ng δ or 10 ng δ were used in place of δδ complex (the control using 20 ng δδ is also shown in Fig. 1C). Stimulation of PolC by τ - Reactions contained 70 ng (25 fmol) of singly primed M13mp18 ssdna, 0.82 µg of S. aureus SSB, 10 ng S. aureus PolC, and either 0, 30 or 100 ng τ (either S. pyogenes τ, E. coli τ, or S. aureus τ), 0.5 mm ATP, 60 µm each of dgtp, dctp, datp and 20 µm 32 P-dTTP (specific activity 2,000-4,000 cpm/pmol) in 25 µl replication buffer. Time points were removed at the following times: 1.0, 1.5, 2.0, , 10.0 min. Reactions were quenched

6 6 with an equal volume of stop solution, then were 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 τ, 20 ng δδ ) and clamp (40 ng β), 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 each of dgtp, dctp, datp and 20 µm [α- 32 P] dttp (~2,000-4,000 cpm/pmol)). Reactions were pre-incubated at 37 C for 3 min, and then 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 δ and δ 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 of dgtp and dctp, datp and 20 µm 32 P- dttp (specific activity 2,000-4,000 cpm/pmol) in 23 µl replication buffer. Individual reactions also contained, when present, 30 ng-300 ng δδ mixture, 50 ng S. aureus PolC, 100 ng S. aureus τ, 40 ng S. aureus β. 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, 40 µm EDTA). δδ mixtures were assembled by incubating equimolar amounts of δ and δ (S. aureus δ (2.24 µg), δ (2.0 µg); S. pyogenes δ (2.33 µg), δ (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 of dgtp, dctp, datp and 20 µm 32 P-dTTP (specific activity 2,000-4,000 cpm/pmol) in 23 µl replication buffer. Individual replication reactions contained combinations of the following proteins as indicated: 50 ng S. aureus PolC, 100 ng E. coli τ complex (τδδ χψ), 40 ng E. coli β, 100 ng each of S. pyogenes τ and δδ, 40 ng S. pyogenes β, 100 ng each of S. aureus τ and δδ, 40 ng S. aureus β. Proteins (in 2 µl) were added to reactions on ice, then 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 in a 0.8% native agarose gel. Gel filtration analysis of protein-protein interaction - Analysis of polymerase-τ interaction was performed in 200 µl buffer A, containing 100 mm NaCl and 1 mg of DNA polymerase subunit (E. coli α or S. aureus PolC) and when present, 1 mg of either E. coli or S. aureus τ subunit. Reactions were incubated at 24 C for 15 min, and then injected onto a HR 10/30 Superose 6 column equilibrated with buffer A containing 100 mm NaCl. After collecting the void volume, fractions of 200 µl each were collected and analyzed on a 10% SDS-polyacrylamide gel. Analysis of δ-δ interaction was performed in reactions that contained 200 µg of either S. aureus δ or δ (or both) in 200 µl buffer A containing 100 mm NaCl. Reactions were incubated at 24 C for 15 min, and then injected onto a HR 10/30 Superose 6 column equilibrated in buffer A containing 100 mm NaCl. Fractions of 200 µl were collected and analyzed on a 12% SDS-polyacrylamide gel. β loading reactions - A six residue N- terminal kinase site was introduced immediately behind the initiating Met of S. aureus β by PCR using a primer with a sequence encoding a 6-amino acid (RRASVP) recognition site for campdependent protein kinase. The gene encoding S. aureus β PK was cloned into the NcoI/ BamHI sites of pet16b. S. pyogenes β PK and E. coli β PK were purified as described

7 7 (19,22). S. aureus β PK was expressed and purified as described (50). β PK proteins were radiolabeled using camp-dependent protein kinase and [γ- 32 P]ATP as described for E. coli β PK (19,56,57). Clamp loading reactions contained 2 µg of uniquely nicked pbluescript plasmid DNA (prepared as described in (19)), 100 ng of 32 P β, 70 ng τ and 30 ng δδ (when present) in 75 µl 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 MgCl 2. Reactions were incubated for 10 min at 37 C and then applied to 5 ml BioGel A15m columns (BioRad) pre-equilibrated with 20 mm Tris- HCl (ph 7.5), 5% glycerol, 2 mm DTT, 40 µg/ml BSA, 8 mm MgCl 2, and 100 mm NaCl. Gel filtration was performed at 4 C, and fractions of 200 µl were collected; 150 µl of each fraction were analyzed by liquid scintillation counting. Protein-protein interaction analysis using 96 well microtitre plates - Either E. coli or S. aureus δ (20 ng/ml) were incubated in wells of a vinyl 96-well microtitre plate (Costar) for 12 hr at 4 C in 100 µl of buffer B (20 mm Tris-HCL (ph 7.5), 1 mm EDTA, 2 mm DTT, 5 mm MgCl 2, 100 mm NaCl, 20% glycerol). Solutions were removed from wells, and wells were washed 4 times with 100 µl TBS buffer (0.1 M sodium phosphate (ph 7.5), 0.15 M NaCl, 0.1% Tween 20). Wells were then blocked with 100 µl TBS buffer containing 5% non-fat milk for 3 hr at 23 C. Following the blocking step, wells were washed 4 times with TBS buffer, and then incubated with 100 µl of buffer B containing either E. coli, S. pyogenes, or S. aureus 32 P-β PK (20 ng/ml) for 2 hr at 23 C. Solutions were then removed, wells were washed 4 times with 100 µl TBS buffer, airdried, and then analyzed using a PhosphorImager. ATP hydrolysis assay- ATPase reactions were performed at 37 C in 20 µl of 20 mm Tris-HCl (ph 7.5), 4% glycerol, 0.1 mm EDTA, 5 mm DTT, 40 µg/ml BSA, and 10 mm MgCl 2. Reactions contained 0.5 µm of either E. coli τ, or S. aureus τ (as monomer), 1 µm M13Gori1 ssdna (as nucleotide), and 1 mm [α- 32 P]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/1 M formic acid. [α- 32 P]ATP and [α- 32 P]ADP were quantitated using a PhosphoImager and ImageQuant software (Molecular Dynamics). DNA gel-shift assay - Gel mobility-shift reactions were performed in 20 µl of 20 mm Tris-HCl (ph 7.5), 4% glycerol, 0.1 mm EDTA, 5 mm DTT, 40 µg/ml BSA, and 10 mm MgCl 2. Reactions contained µm S. aureus τ (as monomer) and 1 µm 32 P- labeled (dt) 15 DNA (as 15 mer). Reactions were incubated for 10 min at 37 C. Products were resolved in a 6% nondenaturing polyacrylamide gel. Steady-state fluorescence measurements - E. coli β -subunit was labeled at Cys333 using Oregon Green 488 maleimide (Molecular Probes, OR) as previously described (58). Peptide titrations contained β OG at a concentration of either 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. Peptides used in this study were as follows: E. coli C-terminal peptide (NH 2 - RLLNDLRGLIGSEQVELEFD-COOH), S. pyogenes PolC C-terminal peptide (NH 2 - GEMGILGNMPDNQLSLFDDFF-COOH), and S. aureus PolC C-terminal peptide (NH 2 -DELGSLPNLPDKAQLSIFDM- COOH). Reactions were mixed on ice, shifted to 22 C for 20 min, and then transferred into a 3 x 3 mm cuvette. Measurements were taken using a Quantamaster spectrofluorimeter (PTI, South Brunswick, NJ). Fluorescence emission spectra were recorded from 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, MA).

8 8 RESULTS Reconstitution of the rapid S. aureus replicase Genes encoding S. aureus PolC, τ, δ, δ, β 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 τ in E. coli produced a single polypeptide corresponding to the mass of the full-length τ product (63.4 kda, Fig. 1A). The recombinant S. aureus PolC, τ, δ, δ, β and SSB were purified, and samples of the final preparation of each subunit are shown in the Coomassie Blue-stained SDSpolyacrylamide gel of Fig. 1A. The β clamp, properly loaded onto a primed site, tethers its respective replicase to DNA for exceedingly high processivity and a synthetic rate of nucleotides/s (22,59). In Fig. 1B, we test ability of the S. aureus clamp and clamp loading subunits to provide PolC with high primer extension speed. The substrate for this assay is a 7.2 kb circular M13mp18 ssdna genome coated with SSB and uniquely primed with a DNA 60 mer. The S. aureus PolC, β, τ, δ and δ subunits were preincubated with the primed substrate and ATP for 5 minutes to allow time for the proteins to assemble on the primed site (see scheme in Fig. 1B). Two deoxyribonucleoside triphophates, 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 32 P- dttp, and reactions were quenched at the indicated times. The results in Fig. 1B demonstrate that the S. aureus PolC holoenzyme completes the 7.2 kb template within 10 s (~700 nt/s), a rate that is comparable to the rates of both the E. coli and the 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 in Fig. 1B except 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, τ, δ, δ, and β, produced the full-length RFII product (Fig. 1C, lane 7). However, mixtures in which one of the proteins was omitted failed to generate a full-length RFII product (Fig. 1C, lanes 1-6). Hence, all five protein subunits are required for this reaction. S. aureus τ subunit binds DNA: E. coli τ binds ssdna, but γ does not, indicating that the DNA binding site in τ lies in the 24 kda C-terminal region unique to τ (62). S. aureus τ (~64 kda) is intermediate in size between E. coli γ (47 kda) and τ (71 kda), and thus may lack some of the properties of E. coli τ. In Fig. 2A we ask whether S. aureus τ can bind a 32 P 5 end-labeled ssdna oligonucleotide in a native polyacrylamide gel shift assay. The result shows that S. aureus τ retains the capacity to bind ssdna. E. coli τ is also a DNA stimulated ATPase. In Fig. 2B, the ATPase activity of E. coli τ and S. aureus τ are compared, plus and minus ssdna. The result shows that they have comparable ATPase activity and are stimulated by ssdna to similar extents. The S. aureus PolC τ interaction is weak compared to that of E. coli Pol III τ In the E. coli system, τ subunit binds to Pol III core to form the Pol III complex (63). This interaction is mediated directly by the α DNA polymerase subunit of Pol III core (16). In Fig. 2C, we examined the interaction between S. aureus PolC and τ by gel filtration analysis. The E. coli α and τ subunits are shown as a control in the top panel of Fig. 2C. The E. coli α and τ subunits form a complex and coelute much earlier (fractions 10-16) than α subunit alone (fractions 26-30). Next we examined S. aureus PolC and τ. As shown in the bottom panels of Figure 2C, S. aureus PolC does not alter its elution position in the presence of τ, and thus does not form a PolC τ complex that is stabile under these conditions. Gel filtration is a nonequilibrium technique and thus only the strongest protein-protein complexes survive.

9 9 Therefore the S. aureus PolC and τ lack the stability needed to remain associated during this 30 minute procedure. Hence we examined S. aureus τ for ability to stimulate PolC as evidence that these two proteins interact. We have shown that the τ subunits of E. coli, S. pyogenes and A. aeolicus stimulate the distributive DNA synthesis activity of their respective DNA polymerase subunit in the absence of the β clamp (23). This stimulation is species specific and therefore relies on specific amino acid contacts between the τ subunit and the DNA polymerase (58,64). τ may enhance polymerase activity by increasing the affinity of the polymerase for DNA, since τ binds to both DNA and the polymerase. In Fig. 2D S. aureus τ was examined for its effect on PolC-catalyzed DNA synthesis using a singly primed M13mp18 ssdna template. S. aureus τ increased the total amount of product synthesized by S. aureus PolC as measured by the total amount of incorporated radionucleotide (Fig. 2D). S. aureus τ stimulation of S. aureus PolC polymerase activity is species specific, as neither E. coli τ nor S. pyogenes τ were capable of stimulating S. aureus PolC (Fig. 2D). These results indicate that S. aureus τ 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 ability to load β onto a circular DNA substrate. To follow the S. aureus β clamp, on and off DNA, a sixresidue kinase recognition motif was placed on the N-terminus of β. This β PK derivative was treated with camp-dependent protein kinase and [γ- 32 P]-ATP to yield [ 32 P]-β PK. The 32 P-β PK was incubated with a singly nicked DNA plasmid in the presence of the S. aureus clamp loading subunits (τ, δ, δ ) and ATP. After 10 minutes at 37 C, the reaction was analyzed on a large pore BioGel A15m gel filtration column. This large pore resin includes proteins, such as 32 P-β PK (in fractions 20-30), but excludes the large DNA plasmid and thus 32 P-β PK bound to the large DNA elutes early, in fractions The results, in Fig. 3, show that the S. aureus 32 P- β PK elutes with nicked DNA only in presence of the S. aureus clamp loader subunits and thus are active in assembly of β onto DNA. The result also shows that the S. aureus β-dna complex is quite stabile as this technique is performed at room temperature and requires about 20 minutes. Earlier studies of E. coli and S. pyogenes δ and δ demonstrate that they form a tight δ-δ complex that is stabile to analysis by gel filtration (15,22). Next, we studied S. aureus δ, and δ for complex formation by gel filtration analysis (Fig. 4A). A mixture of the δ and δ subunits coelute in earlier fractions (i.e. as a higher molecular weight complex, (top panel)) than either subunit alone (bottom two panels). Therefore, S. aureus δ and δ proteins form a sufficiently tight complex to be isolated on a gel filtration column. We have also analyzed a mixture of S. aureus τ, δ, and δ subunits for a stabile τδδ complex but the τ and δδ complex do not coelute (not shown). Under similar conditions, the E. coli subunits form a stabile τδδ complex (15). However, studies of S. pyogenes τ, δ and δ (22) also demonstrate that they form a less stabile τδδ complex than the E. coli τδδ. Overall, our observations indicate that τ subunit from gram positive organisms interacts weakly with PolC and the δδ complex compared to the corresponding subunits from the gram negative E. coli bacterium. We have examined the δ and δ subunits of E. coli, S. pyogenes and S. aureus for cross-species interaction by gel filtration analysis, but no cross-species δδ complexes were stabile to this technique (not shown). Likewise, we have asked whether the δδ complexes of these three systems can bind a heterologous τ subunit by this technique, but again observed no such stabile crossspecies complexes. As will be shown below, activity assays detect limited functional cross-species interaction among these proteins. S. aureus δ can bind and function with E. coli β The δ wrench binds the clamp and pries open the β ring (27,28). The crystal

10 10 structure of E. coli β δ complex revealed residues essential for this interaction (6). Specifically, δ residues Leu 73 and Phe 74 bind into a hydrophobic pocket on β. In Fig. 4B, the E. coli δ and S. aureus δ subunits are examined for an interaction with cognate and non-cognate β clamps. The δ subunits were immobilized in wells of a 96-well microtitre plate. After blocking the wells, either E. coli or S. aureus 32 P- β clamps were added, then the wells were washed and the plates were analyzed for remaining 32 P-β. The S. aureus 32 P β is present in wells containing S. aureus δ and the evolutionarily distant E. coli δ. This result suggests that S. aureus δ can bind E. coli β. The E. coli 32 P β is only present in wells containing E. coli δ. Hence, E. coli δ does not appear to bind S. aureus β. Next we tested whether the observed physical cross-species interaction is functional to load β-clamps onto DNA using non-cognate clamp loaders. In the experiment of Fig. 5, the clamp loaders from E. coli, S. aureus, and S. pyogenes were examined for their ability to load noncognate β clamps onto DNA. In this experiment the β clamps from E. coli, S. aureus, and S. pyogenes all contain the sixresidue kinase motif at the N-terminus allowing them to be labeled with [γ 32 P]-ATP. The different 32 P-β clamps were incubated with nicked circular dsdna, ATP and clamp loading subunits for 10 min, then the reaction was analyzed on a large pore BioGel A15m gel filtration column. The results show that the S. aureus clamp loader is capable of loading β clamps from S. pyogenes and E. coli, although with less efficiency than S. aureus β. In contrast, the E. coli clamp loader is only capable of loading its own clamp. The gram positive S. pyogenes clamp loader is also capable of loading noncognate clamps onto DNA, although it is somewhat less efficient in this regard compared to the S. aureus clamp loader. S. aureus PolC can utilize any β clamp provided it is placed on DNA by its cognate clamp loader. The experiment of Fig. 5 demonstrates that the S. aureus clamp loader can load E. coli β and S. pyogenes β clamps on DNA, and that the S. pyogenes clamp loader can load E. coli and S. aureus clamps onto DNA. Are these β clamps capable of function with S. aureus PolC? In the experiment of Fig. 6 we examine this question using singly primed M13mp18 ssdna coated with SSB as a substrate for mixtures of clamps and clamp loaders of these three bacteria. Under the conditions used here, the S. aureus PolC can not 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 in order that the PolC can bind to the clamp (e.g. see Fig. 1). The S. aureus PolC is quite efficient in synthesis of duplex RFII product using the β 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 β onto DNA (see Fig. 5), and this combination does not result in an RFII duplex product (lane 2), albeit reduced relative to clamps placed onto DNA by their cognate clamp loader. The S. aureus clamp loader can also load E. coli β onto DNA, however the result in lane 8 shows no RFII is produced with PolC. Instead, there is a smear of immature product. Presumably the S. aureus clamp loader interferes with PolC utilization of E. coli β, 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 β clamps onto DNA but the clamps can not be used by S. aureus PolC (lane 4 and 7, respectively). S. aureus PolC and τ can tolerate exchange of δ or δ with S. pyogenes δ or δ We next determined whether the δ and δ subunits of the clamp loader can be exchanged among bacterial species. In the experiments of Fig. 7 we focus on the S. pyogenes and S. aureus systems as we have only obtained negative results in subunit exchanges with the E. coli system (Bruck and O Donnell, unpublished). The PolC, τ and β subunits of either S. aureus or S. pyogenes were mixed with the indicated

11 11 amounts of δ and δ from either organism and incubated in the presence of singly primed SSB coated M13mp18 ssdna, ATP, dctp and dgtp. After 3 min, datp and 32 P-dTTP were added to initiate synthesis. The results show that the S. aureus system can function when either δ (Fig. 6, lanes 7-10) or δ (Fig. 6, lanes 11-14) is replaced with S. pyogenes δ or δ. At high concentrations of either S. pyogenes δ or δ, DNA synthesis by S. aureus PolC, τ, and β is clearly observed (lanes 10 and 14) with these protein exchanges. In contrast, DNA synthesis is inefficient using S. pyogenes PolC, τ, β and either S. aureus δ (Fig. 6, lanes 15-18) or S. aureus δ (Fig. 7, lanes 3-6). Controls using the complete S. aureus (lane 20) and S. pyogenes (lane 19) systems show much more efficient synthesis. Lanes 1 and 2 show reactions in which both δ and δ are derived from the heterologous source. Even using the highest level of S. pyogenes δδ with S. aureus PolC, τ, and β the resulting synthesis is less than observed in reactions containing only one S. pyogenes subunit. The C-terminal residues of PolC interact with E. coli β The C-terminal 20 residues of E. coli α form an essential contact to the E. coli β- clamp (18,58,65,66). Our earlier analysis of this sequence indicates that the Q, L and F side chains within the last seven residues are important contributors to the strength of the interaction with β (66). Sequence alignment of the C-terminal tail of E. coli α, S. pyogenes PolC and S. aureus PolC shows that these residues are conserved across these bacterial species (Fig. 8A). We have previously developed an assay using a fluorescently tagged E. coli β subunit to quantify the interaction between β and a 20 mer peptide corresponding to the C-terminus of the E. coli α subunit (58). This procedure takes advantage of the fact that only one of the Cys residues in E. coli β is accessible to solvent (Cys333) and can be labeled using a maleimide (67). Furthermore, this Cys333 is located on the opposite side of β from that to which the DNA polymerase binds (54,68). Hence, labeling of Cys333 does not interfere with the function of the β clamp. In Fig. 8B we labeled E. coli β 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 β clamp (22,50). Hence, if PolC binds the β clamp via the polymerase C-terminal residues, the 20 mer peptide corresponding to the C-terminal residues of S. aureus PolC and S. pyogenes PolC should also bind to E. coli β. The control, using the E. coli Pol III α C-terminal 20 mer peptide results in a fluorescent enhancement of β OG and yields a K d value of 2.7 µm ±0.4 µm. The results for PolC in Fig. 8B demonstrates an increase in β OG fluorescence as 20 mer peptides from both S. aureus PolC and S. pyogenes PolC are titrated into the reaction, thus indicating that they also bind to E. coli β OG. The affinity of S. aureus 20 mer PolC peptide (7.2 µm) and S. pyogenes 20 mer PolC peptide (2.5 µm) for E. coli β is within 3-fold of the K d value obtained using E. coli Pol III α 20 mer peptide, underscoring the high degree of conservation in the polymerase-clamp interaction across divergent bacterial species. This highly conserved binding site in β 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 β and S. aureus β. The β clamps of these gram positive organisms have not been studied as intensively as the E. coli β clamp and thus we do not know if they contain a unique exposed Cys residue. Hence we asked whether these polymerase peptides inhibit the function of the S. aureus and S. pyogenes clamp in DNA synthesis assays. If the polymerase peptide binds to S. aureus β or S. pyogenes β in the polymerase binding site, it should inhibit replication. Furthermore, studies in the E. coli system show that the clamp loader also interacts with β at the same site, or an overlapping site, as the polymerase binding site (68). Hence, the Pol III α C-20 peptide not only inhibits polymerase III-β interaction, but also inhibits clamp loader function with β (66).

12 12 In the experiment of Fig. 8C, singly primed SSB coated M13mp18 ssdna was incubated in the presence of both the clamp and the clamp loader and an increasing amount of the PolC-20 peptide. DNA synthesis was initiated upon addition of the polymerase subunit (E. coli Pol III core, S. aureus PolC, or S. pyogenes PolC). The first panel shows the control using E. coli proteins in which all three polymerase peptides have been shown to bind E. coli β, (i.e. the experiment of Fig. 8B). The results, in Fig. 8C, demonstrate that each of the three polymerase peptides inhibit DNA synthesis. The S. aureus PolC peptide is the least effective, consistent with its weaker interaction with E. coli β. Titration of the Pol III α and PolC peptides into reactions containing the S. aureus β (middle panel) and S. pyogenes β (third panel) replication systems demonstrate that all three peptides inhibit DNA synthesis, indicating that the polymerase binding site within the β clamp is also conserved across gram positive and gram negative bacterial species. DISCUSSION Reconstitution of S. aureus PolC replicase We have cloned, overexpressed, and purified protein subunits of the S. aureus PolC replicative polymerase. The S. aureus clamp loader subunits (τ, δ, and δ ) are competent to load the S. aureus β-clamp onto DNA. Combining all subunits of the S. aureus Pol III replicase (PolC, τ, δ, δ, β, and SSB) yields the rapid polymerase characteristic of bacterial replicases. The reconstituted polymerase complex converts primed ssdna into double stranded RFII product at a speed of about 700 nt/s, similar to the synthetic rates of E. coli Pol III holoenzyme and Streptococcus pyogenes PolC holoenzyme (22). We also examined subunits of the S. aureus replicase for protein-protein interactions that are sufficiently tight to be stabile to gel filtration analysis. S. aureus PolC and τ do not yield a tight complex that is stabile to gel filtration, but τ stimulates PolC in the presence of primed template indicating that they do indeed interact. Our earlier studies of S. pyogenes PolC and τ have demonstrated that the PolC τ complex can be observed by gel filtration, but the proteins need to be at much higher concentration than the E. coli τ and α subunits to observe the complex. The relatively weak affinity between the gram positive PolC and τ subunit may explain why attempts to isolate the PolC holoenzyme from gram-positive bacterial (B. subtilis, S. aureus, and S. pyogenes) cell lysates using conventional chromatographic techniques were unsuccessful (22,36,41). In these previous studies, only PolC was isolated. S. aureus δ and δ form a tight complex, similar to observations in both the E. coli and S. pyogenes systems. However, S. aureus τ does not form a tight complex with δδ. This is also similar to observations with S. pyogenes τ and δδ complex (22). In contrast, E. coli τ and δ δ from a τδδ complex that is stabile to gel filtration (15). The E. coli clamp loader contains two additional subunits, ψ and χ (53). These subunits further stabilize the interaction of τ(γ) with δ and δ (5,11). We were unable to find χ and ψ homologs in gram-positive genomes by sequence comparison with E. coli χ and ψ. However, it is possible that orthologs of ψ and χ, or even novel proteins, contribute to stability of interactions within the clamp loader subunits of gram-positive organisms. Heterologous clamp loading The S. aureus clamp loading subunits are capable of loading β clamps derived from S. pyogenes and E. coli (Fig. 5). The efficiency by which these heterologous clamps are loaded onto DNA is lower relative to the loading of S. aureus β, but is clearly significant. The loading of heterologous clamps is consistent with the ability of S. aureus δ to bind the evolutionary distant E. coli β (Fig. 4). The converse is not true. We could detect no interaction between E. coli δ and S. aureus β, nor could the E. coli clamp loader assemble the S. aureus β clamp onto DNA. Interestingly, the S. pyogenes clamp loader loads the S. aureus β clamp onto DNA, but

13 13 not E. coli β. Hence, the S. aureus system may have retained the most common structural and functional features important to the clamp loading operation. It is interesting to note that when heterologous β clamps are loaded onto DNA by the S. aureus or S. pyogenes clamp loaders they generally lack functionality with S. aureus PolC, even though S. aureus PolC binds and functions with E. coli and S. pyogenes clamps. This result indicates that the S. aureus and S. pyogenes clamp loaders have difficulty in completing the full clamp loading cycle when loading a heterologous clamp. For example, the E. coli clamp loader must eject from the clamp after loading it on DNA before the polymerase can function with it (27). Perhaps the S. aureus clamp loader has difficulty completing this final step when loading a heterologous clamp. We shall like to examine this reaction in greater detail in future studies to determine whether this is the case, or whether some other explanation underlies this observation. PolC binds to a conserved site in β via C- terminal residues Early studies have shown that the δ subunit of the E. coli clamp loader competes with the Pol III α polymerase subunit for binding to β, suggesting that their binding sites on the β clamp overlap (68). Single point mutants of β eliminated or dramatically reduced binding of β to both the δ and α subunits (68). This result suggested that the binding site on β for these two proteins may be one and the same, or least must share some important contacts to β. The crystal structure of δ in ACKNOWLEDGEMENTS complex with β revealed the details of the hydrophobic binding pocket on β for this clamp loading subunit (29). The location of the binding site was anatomatically similar to the position at which the human p21 cell cycle regulatory protein binds to the PCNA clamp (69), and also the position on the gp45 clamp to which the RB69 phage polymerase binds (70). These studies support the idea that the Pol III α subunit binds to β in the same place as the δ subunit. Subsequent studies of the E. coli Pol III α subunit demonstrated that it forms a essential attachment to the β clamp via the extreme seven C-terminal residues of α (66). This connection point to β is essential for polymerase action with the clamp. The Pol III α C-terminal peptide binds directly to β and even displaces δ from β, further supporting a common binding site for these two proteins on the β clamp. The current study demonstrates that the C-terminal residues of the S. aureus PolC are also used as an attachment point to the β clamp. The polymerase binding pocket must be quite conserved during evolution as we demonstrate here that peptides derived from the C-termini of either S. aureus PolC, S. pyogenes PolC or E. coli α are capable of inhibiting the replication function of β in all three of these systems. One may expect from these observations that this conserved and important functional binding site in the clamp may be an attractive target for a broad spectrum antibiotic compound. Thanks to Dr. Daniel Kaplan for critical reading of the manuscript. This project was supported by a grant from the NIH (GM38839) and by HHMI.

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16 Lopez de Saro, F. J., Georgescu, R. E., Goodman, M. F., and O'Donnell, M. (2003) Embo J 22, Griep, M. A., and McHenry, C. S. (1988) Biochemistry 27, Naktinis, V., Turner, J., and O'Donnell, M. (1996) Molecular Cell 84, Gulbis, J. M., Kelman, Z., Hurwitz, J., O'Donnell, M., and Kuriyan, J. (1996) Cell 87, Shamoo, Y., and Steitz, T. A. (1999) Cell 99, FIGURE LEGENDS Fig 1. Reconstitution of the S. aureus replicase. A). Proteins of the S. aureus replication system are shown in the Coomassie Blue-stained SDS-polyacrylamide gel (10%). Proteins were prepared as described in Experimental Procedures. The molecular mass of standards is shown on the left. B). S. aureus PolC holoenzyme is rapid in DNA synthesis. Proteins (PolC, τ, δ, δ, β) were preincubated with primed DNA and ATP, then synthesis was initiated and quenched at the indicated times. Products were separated on an alkaline 0.8% agarose gel. The position of size markers is shown on the right. C). Single subunit omission experiments. Reactions contained all the S. aureus replicase subunits except the one indicated. Reactions used singly primed M13mp18 ssdna as substrate as described in Experimental Procedures. Products were analyzed in a 0.8% alkaline agarose gel. The positions of primed ssdna substrate and full-length circular duplex product (RFII) are indicated to the right. Fig 2. Characterization of S. aureus τ subunit. A). Gel mobility shift assay. S. aureus τ (molarity as monomer) is incubated with a 32 P end-labeled 15 mer ssdna. Reaction products were resolved on a 6% polyacrylamide gel. B). S. aureus τ is a DNA stimulated ATPase. E. coli τ was incubated in presence of ssdna (open circles), and in absence of DNA (closed circles); S. aureus τ was incubated in the presence (open triangles) and absence of ssdna (crosses). C). Mixtures of τ and polymerase were analyzed by gel filtration. Proteins were resolved on Coomassie Bluestained SDS-polyacrylamide gels (10%). The top two panels are a mixture of E. coli α and τ, and E. coli α alone. The bottom two panels are a mixture of S. aureus PolC and τ, and S. aureus PolC alone. D). S. aureus τ stimulates S. aureus PolC in DNA synthesis assays that do not require β. In separate reactions, S. aureus PolC was incubated with either S. aureus τ, S. pyogenes τ, or E. coli τ.

17 17 Fig 3. The S. aureus clamp loader is functional with β. The S. aureus clamp loading proteins load S. aureus 32 P-β onto a circular nicked DNA. S. aureus clamp loading subunits and 32 P-β were incubated in presence of nicked circular DNA and then the 32 P- β DNA complex was resolved from 32 P-β in solution on a BioGel A15 M column. Open circles, reactions lacked clamp loading subunits. Filled circles, reactions contain clamp loading subunits. Fig 4. Cross-species interaction of δ with β. A). A mixture of S. aureus δ and δ was analyzed by gel filtration (top panel). The middle and bottom panels are similar analyses of δ (middle panel) and δ (bottom panel). Proteins were resolved on a 12% Coomassie Blue-stained SDS-polyacrylamide gel. B) β and δ subunits from E. coli, S. pyogenes, and S. aureus were tested for physical interaction using a solid phase plate based protein-protein binding assay. Either S. aureus (Sa) δ or E. coli (Ec) δ was immobilized in the well followed by addition of 32 P-β from either E. coli or S. aureus as described in Experimental Procedures. Wells were washed and the plate was analyzed by autoradiography. Fig 5. The S. aureus clamp loader is capable of loading heterologous clamps onto DNA. Loading of 32 P-β onto the circular nicked DNA using cognate and non-cognate clamp loaders. 32 P-β from either S. aureus (Sa), S. pyogenes (Sp) or E. coli (Ec) were incubated with clamp loading proteins from either S. aureus, S. pyogenes or E. coli in the presence of singly nicked circular duplex DNA. 32 P-β DNA complexes were resolved from free 32 P-β on BioGel A15 M columns. Panel A) Loading of S. aureus β. Panel B) Loading of S. pyogenes β. Panel C) Loading of E. coli β.

18 18 Fig. 6. S. aureus PolC can utilize heterologous β clamps. S. aureus (Sa) PolC was incubated with the indicated clamps and clamp loaders in replication reactions using the primed M13mp18 ssdna substrate as described in Experimental Procedures. Reaction products were resolved in a 0.8% agarose gel. The positions of primed DNA substrate (SS) and full-length duplex product (RFII) are indicated to the right of the autoradiogram. Fig. 7 The S. aureus clamp loader can tolerate exchange of δ and δ subunits. PolC, τ, and β subunits from either S. aureus (Sa) or S. pyogenes (Sp) were incubated with singly primed M13mp18 ssdna either alone (lanes 3, 7, 11, 15) or with mixtures of increasing amounts of δ and δ proteins. Reaction conditions are described under Experimental Procedures. Lanes 19 and 20 represent reactions using PolC, τ, δ, δ and β which were all from S. pyogenes (Sp complete, lane 19) or S. aureus (Sa complete, lane 20). Fig 8. The C-terminus of S. aureus PolC interacts with β clamps. A) Alignment of the C-terminal 20 residues of S. pyogenes PolC, S. aureus PolC, and E. coli Pol III. B) Fluorescent titrations into E. coli β OG, uniquely labeled with Oregon Green at Cys333, using 20 mer peptides corresponding to the C-terminal 20 residues of either E. coli Pol III α subunit (triangles), S. aureus PolC (diamonds) or S. pyogenes PolC (circles). C) Inhibition of DNA synthesis was tested using polymerase C-terminal peptides from S. aureus (Sa) PolC (circles), S. pyogenes (Sp) PolC (squares), and E. coli (Ec) Pol III (diamonds). The polymerase, τ, δδ and β subunits were incubated in the presence or absence of Pol C-20 peptide and singly primed M13mp18 ssdna as described in

19 19 Experimental Procedures. Left panel: E. coli Pol III system. Middle panel: S. aureus PolC system. Right panel: S. pyogenes PolC system.

20 Fig. 1 A) SDS-PAGE analysis PolC τ β δ δ' SSB 3' M13mp18 ssdna B) Replicase Speed PolC β,τ,δ,δ dctp, dgtp time(sec) β PolC 3' P-dTTP datp RFII RFII C) Subunit Requirements Subunit Omitted PolC, β, τ, δ, δ PolC τ δ δ δδ β None RFII RFII ' '

21 A) B) 400 Fig. 2 τ (µμ) Sa τ+ssdna P-15mer + τ C) 32 P-15mer E.coli S.aureus α τ α PolC τ PolC D) [ADP] µm Ec τ+ssdna DNA synthesis (pmol) S. pyogenes τ E. coli τ S. aureus τ S. aureus PolC Ec τ Sa τ time(min)

22 Fig 3 + τ,δ,δ 32 P-β PK 32 P-β PK (fmol) PK 32P-β on DNA + τ,δ,δ' free 32 P-β PK -τ,δ,δ'

23 Fig. 4 A) Gel Filtration δ + δ δ δ δ δ δ δ B) Immobilized δ sa *βpk ec *βpk δ E.coli δ 32 P-β PK P-β PK δ S. aureus δ 3 4

24 A) S. aureus β loading PK P-β on DNA free 32 P-β PK Fig P-β PK (fmol) B) S. pyogenes β loading 32 P-β PK (fmol) 32 P-β PK (fmol) Sa τδδ Sp τδδ Ec τδδ Sp τδδ Sa τδδ 5 Ec τδδ Fraction C) E. coli β loading Ec τδδ Sa τδδ Sp τδδ Fraction Fraction

25 Fig. 6 3' M13mp18 ssdna β clamp clamp loader Sa PolC RFII clamp S. pyogenes S. aureus E. coli clamp loader S. pyogenes S. aureus E. coli RFII Sa PolC alone Sa PolC Ec core ssdna

26 Fig. 7 τ β + PolC (PolCτβ) Sa (PolCτβ) Sp (δ) Sp (δ') Sa (δ) Sa (δ')sp (δδ ) Sa (δδ ) Sp [δδ ] ng RFII ssdna 3' M13mp18 ssdna δ, δ RFII Sp complete Sa complete

27 Fig. 8 A) Sp PolC-C20 Sa PolC-C20 Ec α C20 EMGILGNMPDN-QLSL-FDDFF DELGSLPNLPDKAQLSI-FDM RLLNDLRGLIGSEQVELEFD B) 1.3 E. coli OG β Pol C-20 I / Io C-terminal peptide E. coli α S. pyogenes PolC S. aureus PolC Ec β (Κ d µμ) 2.7 ± 0.4 µm 2.5 ± 0.6 µm 7.2 ± 0.9 µm 1 C) peptides, µ M 3' M13mp18 ssdna PolC,τ,δ,δ,β PolC-20 peptide PolC