Analysis of a Multicomponent Thermostable DNA Polymerase III Replicase from an Extreme Thermophile*

Home , HolB, Thermophile

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 19, Issue of May 10, pp. 17334–17348, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Analysis of a Multicomponent Thermostable DNA Polymerase III Replicase from an Extreme Thermophile*

Received for publication, October 23, 2001, and in revised form, February 18, 2002 Published, JBC Papers in Press, February 21, 2002, DOI 10.1074/jbc.M110198200

Irina Bruck‡, Alexander Yuzhakov§¶, Olga Yurieva§, David Jeruzalmi§, Maija Skangalis‡§, John Kuriyan‡§, and Mike O’Donnell‡§ʈ From §The Rockefeller University and ‡Howard Hughes Medical Institute, New York, New York 10021

This report takes a proteomic/genomic approach to polymerase III (pol III) structure and function has been ob- characterize the DNA polymerase III replication appa- tained from studies of the Escherichia coli replicase, DNA ratus of the extreme thermophile, Aquifex aeolicus. polymerase III holoenzyme (reviewed in Ref. 6). Therefore, a Genes (dnaX, holA, and holB) encoding the subunits re- brief overview of its structure and function is instructive for the ␶ ␦ ␦؅ quired for clamp loading activity ( , , and ) were iden- comparisons to be made in this report. In E. coli, the catalytic Downloaded from tified. The dnaX gene produces only the full-length subunit of DNA polymerase III is the ␣ subunit (129.9 kDa) ␶ product, , and therefore differs from Escherichia coli encoded by dnaE; it lacks a proofreading exonuclease (7). The dnaX that produces two proteins (␥ and ␶). Nonetheless, Ј Ј ⑀ ␶␦␦؅ proofreading 3 –5 -exonuclease activity is contained in the the A. aeolicus proteins form a complex. The dnaN ␣ ,␶␦␦؅ (27.5 kDa) subunit (dnaQ) that forms a 1:1 complex with (8 ␤ gene encoding the clamp was identified, and the ␣Ϫ⑀ ␤ 9). The pol III complex is found tightly associated to a third

complex is active in loading onto DNA. A. aeolicus http://www.jbc.org/ ␪ contains one dnaE homologue, encoding the ␣ subunit of subunit, called , to form the heterotrimeric E. coli DNA po- ␪ DNA polymerase III. Like E. coli, A. aeolicus ␣ and ␶ lymerase III core (10). The subunit (holE, 8.6 kDa) is not interact, although the interaction is not as tight as the essential for growth and is generally not conserved in bacteria .(؊␶ contact in E. coli. In addition, the A. aeolicus homo- (11␣ logue to dnaQ, encoding the ⑀ proofreading 3؅–5؅-exonu- The E. coli pol III ␣ subunit and pol III core subassembly act clease, interacts with ␣ but does not form a stable ␣⅐⑀ distributively on primed ssDNA and have only low activity; complex, suggesting a need for a brace or bridging pro- they are even further inhibited by the presence of SSB (7, 12). at Rockefeller University Library on August 2, 2015 tein to tightly couple the polymerase and exonuclease in However, after the ␤ clamp has been assembled onto a primed this system. Despite these differences to the E. coli sys- site, the efficiency of the pol III ␣ subunit is greatly stimulated, tem, the A. aeolicus proteins function to yield a robust and ␣⅐␤ extends the primer at a rate of ϳ300 nucleotides/s with replicase that retains significant activity at 90 °C. Simi- a processivity of 1–3 kb (9). The pol III ␣⑀ complex and pol III larities and differences between the A. aeolicus and E. core subassembly are even further stimulated by ␤ and extend coli pol III systems are discussed, as is application of DNA at a rate of about 1 kb/s with a processivity that exceeds thermostable pol III to biotechnology. the entire 7.2-kb M13mp18 ssDNA template (9). The E. coli clamp loader of pol III consists of five different subunits, ␥, ␦, ␦Ј, ␹, and ␺, but only three of them, ␥ (dnaX, 47.5 Chromosomal replicases of all cellular organisms studied kDa), ␦ (holA, 38.7 kDa), and ␦Ј(holB, 36.9 kDa), are essential thus far are composed of three components, the DNA polymer- for clamp loading activity in vitro (13). Homologues to E. coli ␹ ase, a ring-shaped DNA sliding clamp, and a clamp loader that (holC, 16.6 kDa) and ␺ (holD, 15.2 kDa) subunits can only be uses ATP to assemble the sliding clamp onto DNA (1–3). In identified in a few other organisms so far. The ␥ and ␦Ј subunits bacteria, the sliding clamp is a homodimer called ␤ (4). The are homologous to one another and are members of the AAAϩ ring-shaped ␤ dimer completely encircles DNA and slides along family of proteins (14–16). The ␦ subunit shows no homology to the duplex (5). The ␤ clamp also binds the DNA polymerase III, ␥ and ␦Ј, but the ␦⅐␤, ␦Ј and ␥ ␦␦Ј crystal structures show that thereby tethering it to DNA for high processivity (5). 3 ␦ has the same three domain structure and chain folding pat- This report on the Aquifex aeolicus pol III1 replicase is part ␥ ␦Ј of our continuing study of comparing and contrasting replicases tern as and (17–19). Crystal structure analysis reveals that ␥ ␦ ␦Ј from a variety of bacteria. Most knowledge of bacterial DNA the five subunits of the 3 1 1 complex are arranged as a circular pentamer (19). Mechanistic studies have outlined the overall mechanism of * This work was supported in part by National Institutes of Health the clamp loader and are consistent with the structural anal- Grants GM R01 38839 (to M. O. D.) and GM 45547 (to J. K.). The costs ysis. The ␥ subunit is the only subunit that interacts with ATP of publication of this article were defrayed in part by the payment of and therefore is the motor of the clamp loader (20). The ␦ page charges. This article must therefore be hereby marked “advertise- ␤ ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this subunit alone can open one interface of the dimer (21, 22). fact. The ␦ clamp opener is sequestered within ␥ complex via asso- ¶ Present address: Vertex Pharmaceuticals, 130 Waverly St., Cam- ␦Ј ␥ ciation to (21). ATP binding to 3 results in a conformational bridge, MA 02139. ␤ ␦ ␦Ј ʈ change, releasing the interactive site on from (21, 23). To whom correspondence should be addressed: The Rockefeller ⅐␥ ␦␦Ј ␤ University and Howard Hughes Medical Institute, 1230 York Ave., The ATP 3 species binds to , opens the ring, and binds New York, NY 10021, Tel.: 212-327-7255; Fax: 212-327-7253; E-mail: DNA (24, 25). Then hydrolysis of ATP brings ␦ back onto ␦Ј, [email protected]. severing connections to ␤, allowing ␤ to close around DNA (21, 1 The abbreviations used are: pol III, polymerase III; DTT, dithiothre- 26–28). itol; IPTG, isopropyl-1-thio-␤-D-galactopyranoside; TBS, Tris-buffered saline; BSA, bovine serum albumin; Elisa, enzyme-linked immunosor- In E. coli, the dnaX gene encoding ␥ also encodes the ␶ bent assay; SSB, single-strand DNA binding protein. subunit of DNA pol III holoenzyme (29–31). ␶ (71.1 kDa) is the

17334 This paper is available on line at http://www.jbc.org pol III Holoenzyme of Aquifex aeolicus 17335

full-length product of dnaX, whereas ␥ is shorter (47.5 kDa), CTCGGAAGTAAGGG-3Ј) contains a BamHI site (underlined). The being truncated by a translational frameshift. ␶ can fully re- PCR product was digested with NdeI and BamHI, purified, and ligated ␥ ␶ ␦␦Ј into the pET24 NdeI and BamHI sites to produce pETAadnaE. place in the clamp loader, and the 3 complex is active in ␭ ␶ The pETAadnaE plasmid was transformed into the BL21 ( DE3) clamp loading (13). The C-terminal sequences unique to are strain of E. coli. Cells were grown in 50 liters of LB containing 100 required for interaction with the pol III ␣ subunit (32) and also ␮ ϭ g/ml kanamycin, 5 mM MgSO4 at 37 °CtoA600 2.0, induced with 2 with the replicative DnaB helicase (33, 34). Therefore, within mM IPTG for 20 h at 20 °C, and then collected by centrifugation. Cells the holoenzyme, ␶ subunits must replace two (or all three) of were resuspended in 400 ml of 50 mM Tris-HCl (pH 7.5), 10% sucrose, the ␥ subunits in order to connect the two pol III core polym- 1 M NaCl, 30 mM spermidine, 5 mM DTT, and 2 mM EDTA. The following erases in the holoenzyme structure for simultaneous replica- procedures were performed at 4 °C. Cells were lysed by passing them twice through a French press (15,000 pounds/square inch) followed by tion of both leading and lagging strands (60). centrifugation at 13,000 rpm for 90 min at 4 °C. In this protein prepa- We have undertaken the study of other bacterial replication ration, as well as each of those that follow, the induced A. aeolicus systems in an effort to delineate those features of prokaryotic protein was easily discernible as a large band in an SDS-polyacrylamide replicases that are general to all bacteria. Study of the Gram- gel stained with Coomassie Blue. Hence, column fractions were assayed negative Thermus thermophilus dnaX gene showed that it pro- for the presence of the A. aeolicus protein by SDS-PAGE analysis, which duces both ␥ and ␶, like E. coli dnaX (35, 36). However, instead forms the basis for pooling column fractions. Ϫ The clarified cell lysate was heated to 65 °C for 30 min, and the of a 1 ribosomal frameshift, T. thermophilus employs a tran- precipitate was removed by centrifugation at 13,000 rpm in a GSA rotor scriptional slippage mechanism that results in both Ϫ1 and Ϫ2 for 1 h. The supernatant (1.4 g, 280 ml) was dialyzed against buffer A frameshifts (35, 37). We have also examined the pol III repli- (20 mM Tris-HCl (pH 7.5)), 10% glycerol, 0.5 mM EDTA, 5 mM DTT) case of a Gram-positive organism, Streptococcus pyogenes (38). overnight, and then diluted to 320 ml with buffer A to a conductivity Downloaded from This study showed that only one protein is produced from the S. equal to 100 mM NaCl. The dialysate was applied to a 150-ml fast flow pyogenes dnaX gene. This full-length ␶ protein (62.1 kDa) is Q-Sepharose column (Amersham Biosciences) equilibrated in buffer A, ␥ ␶ and eluted with a 1.5-liter linear gradient of 0–500 mM NaCl in buffer intermediate in length between E. coli (47.5 kDa) and (71.1 A. Eighty fractions were collected. Fractions 38–58 (1 g, 390 ml) were kDa) but retains the capacity to bind the DNA polymerase, like pooled, dialyzed versus buffer A overnight, and applied to a 250-ml ␶

E. coli . However, the strength of this interaction is weaker heparin-agarose column (Bio-Rad) equilibrated with buffer A. Protein http://www.jbc.org/ than in the E. coli system. Like other Gram-positive bacteria, was eluted with a 1-liter linear 0–500 mM NaCl gradient in buffer A. the S. pyogenes DNA polymerase, pol C (ϳ165 kDa), contains One hundred fractions were collected. Fractions 69–79 (320 mg in 200 Ј Ј ml) were pooled and dialyzed against buffer A containing 100 mM NaCl. an inherent 3 –5 -exonuclease activity instead of delegating ␣ Ϫ ⑀ The preparation was aliquoted and stored frozen at 80 °C. The this proofreading action to a separate subunit as observed in Coomassie Blue-stained SDS-polyacrylamide gel of the final ␣ prepa- the E. coli system (39). As in E. coli, the S. pyogenes, ␶, ␦, and ration is shown in Fig. 1. ␦Ј subunits are required to load ␤ onto DNA, and the clamp ⑀ Purification of Encoded by dnaQ—The A. aeolicus dnaQ was iden- at Rockefeller University Library on August 2, 2015 endows pol C with the same rapid speed and processivity as the tified in the genome sequence as a 202-residue protein of 17,132 Da entire E. coli DNA pol III holoenzyme (38). It is interesting to having significant homology to E. coli dnaQ (41). A. aeolicus dnaQ was amplified by PCR using the following oligonucleotide primers. The up- note that S. pyogenes also contains a second homologue to E. Ј ␣ stream 35-mer (5 -GTGTGTCATATGCGAGACAATCTCCTTGATGGC- coli , which we refer to as the DnaE polymerase (38). DnaE AG-3Ј) contains an NdeI site (underlined), and the downstream 44-mer polymerase is similar in size to E. coli ␣ (120 kDa) and like E. (5Ј-GTGTGGATCCTCAAAACTTACCCTTTTCCAGCCTTTTTAAGGA- coli ␣ it lacks 3Ј–5Ј-exonuclease activity (38). The processivity G-3Ј) contains a BamHI site (underlined). The PCR product was di- of S. pyogenes DnaE polymerase is stimulated by ␶␦␦Ј and ␤, gested with NdeI and BamHI, purified, and ligated into the pET24a but its intrinsic speed (60 nucleotides/s) is unaltered (38). vector to produce pETAadnaQ. This report examines the pol III replication machinery of the The pETAadnaQ plasmid was transformed into E. coli strain BL21(␭DE3). A single colony was used to inoculate 12 liters of LB media extreme thermophile, A. aeolicus. By using the known genome supplemented with 200 ␮g/ml ampicillin. Cells were grown at 37 °Cto sequence (41), we identify A. aeolicus replicase genes and pro- ϭ A600 0.5 at which point 0.5 mM IPTG was added. After a 3-h induction, duce and isolate recombinant ␣, ⑀, ␤, ␶, ␦Ј, ␦, subunits and SSB. cells were collected by centrifugation and resuspended in 50 mM Tris- We then compare and contrast the function and assembly of HCl (pH 7.5), 10% sucrose, 1 M NaCl, 30 mM spermidine, 5 mM DTT, 2 these replicase subunits from a thermophile to the Gram-neg- mM EDTA. Cells were lysed by two passages through a French press ative E. coli replicase and to our previous studies on the Gram- (15,000 pounds/square inch) followed by centrifugation at 13,000 rpm for 30 min at 4 °C. The resulting supernatant (1038 mg) was incubated positive S. pyogenes replicase. ina65°C waterbath for 30 min. The solution was clarified by centrifugation at 13,000 rpm for 30 min. The supernatant (426 mg) was EXPERIMENTAL PROCEDURES dialyzed against buffer A and loaded onto a 15-ml fast flow Q-Sepharose Materials—Radioactive nucleotides were from PerkinElmer Life Sci- column equilibrated with buffer A. The column was eluted with a ences; unlabeled nucleotides were from Amersham Biosciences and The 150-ml linear gradient of 50–500 mM NaCl in buffer A; 80 fractions Upjohn Co. DNA oligonucleotides were synthesized by Invitrogen. were collected. Peak fractions (fractions 36–44, 12.9 mg) were pooled, M13mp18 ssDNA was purified from phage that was isolated by two dialyzed against buffer A, and then loaded onto a 10-ml heparin-aga- successive bandings in cesium chloride gradients as described (42). rose column equilibrated with buffer A. The column was eluted with a M13mp18 ssDNA was primed with a DNA 30-mer (map position 6817– linear gradient of 0 mM to 1 M NaCl; 80 fractions were collected. Peak 6846) as described (9). The pET protein expression vectors and BL21 fractions (fractions 60–70) were pooled, aliquoted, and stored at (␭DE3) protein expression strain of E. coli were purchased from Nova- Ϫ80 °C. gen. DNA modification enzymes were from New England Biolabs. A. Identification of A. aeolicus holA and Purification of ␦—The A. aeoli- aeolicus genomic DNA and A. aeolicus cells were a gift of Dr. Robert cus holA gene was not identified previously by the genome sequencing Huber and Dr. Karl Stetter (Regensburg University, Germany). Protein project. We identified A. aeolicus holA by searching the A. aeolicus concentrations were determined using Protein Stain (Bio-Rad) and BSA genome with the amino acid sequence of the E. coli ␦ subunit (encoded as a standard. Polyclonal antisera was produced by rabbits injected by holA). Although the resulting match had too low a score to be with purified E. coli ␥ or ␣ (5). Antibodies directed to A. aeolicus ␶ were confident of its assignment as ␦, the studies of this report prove that the purified from antisera by transfer of E. coli ␶ from an SDS gel to gene truly encodes ␦ subunit of the replicase. The A. aeolicus holA gene nitrocellulose followed by incubation with antisera and elution of puri- was amplified by PCR using the following primers. The upstream 36- fied antibody from the nitrocellulose membrane. mer (5Ј-GTGTGTCATATGGAAACCACAATATTCCAGTTCCAG-3Ј) Purification of A. aeolicus ␣ Encoded by dnaE—The A. aeolicus dnaE contains an NdeI site (underlined); the downstream 39-mer (5Ј-GTGT- gene (41) was amplified from A. aeolicus genomic DNA by PCR using GTGGATCCTTATCCACCATGAGAAGTATTTTTCAC-3Ј) contains a the following primers. The upstream 37-mer (5Ј-GTGTGTCATATGAG- BamHI site (underlined). The PCR product was digested with NdeI and TAAGGATTTCGTCCACCTTCACC-3Ј) contains an NdeI site (un- BamHI, purified, and ligated into the pET24 NdeI and BamHI sites to derlined); the downstream 34-mer (5Ј-GTGTGTGGATCCGGGGACTA- produce pETAaholA. 17336 pol III Holoenzyme of Aquifex aeolicus

FIG.1.Protein subunits of A. aeolicus pol III holoenzyme system. A, subunit preparations were analyzed in a 15% SDS-polyacrylamide gel stained with Downloaded from Coomassie Blue. Proteins were prepared as described under “Experimental Proce- dures.” The positions of protein standards analyzed in the same gel are indicated to the left. B, the table gives the subunit gene name, mass predicted from the gene sequence for A. aeolicus pol III subunits, http://www.jbc.org/ and their percent identify to corresponding subunits in the E. coli pol III systems (aligned using ClustalX). The function of subunits, or combinations of subunits, are given in the column at the right. at Rockefeller University Library on August 2, 2015

The pETAaholA plasmid was transformed into E. coli strain BL21 derlined). The PCR product was digested with NdeI and BamHI, puri- (␭DE3). Cells were grown in 50 liters of LB media containing 100 ␮g/ml fied, and ligated into the pET24 NdeI and BamHI site to produce ϭ kanamycin. Cells were grown at 37 °CtoA600 2.0, then induced for pETAaholB. 20 h upon addition of 2 mM IPTG, and collected by centrifugation. Cells The pETAaholB plasmid was transformed into E. coli strain BL21 from 25 liters of culture were lysed as described above for purification (␭DE3). Cells were grown at 37 °C in 50 liters of media containing 100 of ␣. ␮ ϭ g/ml kanamycin to A600 2.0 and then induced for 3 h upon addition The cell lysate was heated to 65 °C for 30 min, and the precipitate of2mM IPTG. Cells were collected by centrifugation and were lysed was removed by centrifugation. The supernatant (650 mg, 240 ml) was using lysozyme by the heat lysis procedure. The cell lysate was heated dialyzed against buffer A, adjusted to a conductivity equal to 160 mM to 65 °C for 30 min, and precipitate was removed by centrifugation. The NaCl by addition of 40 ml of buffer A, and applied to a 220-ml heparin- supernatant (2.4 g, 400 ml) was dialyzed versus buffer A and then agarose column equilibrated in buffer A containing 100 mM NaCl. The applied to a 220-ml fast flow Q-Sepharose column equilibrated in buffer column was eluted with 1.0 liters linear gradient of 150–700 mM NaCl A. Protein was eluted with a 1-liter linear gradient of 0–500 mM NaCl in buffer A. One hundred and four fractions were collected. Fractions in buffer A; 80 fractions were collected. Fractions 23–30 were pooled 45–56 were pooled (250 mg, 210 ml), diluted with 230 ml buffer A to a and diluted 2-fold with buffer A to a conductivity equal to 100 mM NaCl conductivity equal to 230 mM NaCl, and then loaded onto a 100-ml fast and then loaded onto a 200-ml heparin-agarose column equilibrated in flow Q-Sepharose column equilibrated in buffer A containing 150 mM NaCl. The column was eluted with a 200-ml linear gradient of 150–750 buffer A. Protein was eluted with a 1-liter linear gradient of 0–1.0 M NaCl in buffer A; 84 fractions were collected. Fractions 46–66 were mM NaCl in buffer A; 73 fractions were collected. Fractions 16–38 were pooled (95 mg, 40 ml), aliquoted, and stored at Ϫ80 °C. pooled (1.3 g, 395 ml), dialyzed versus buffer A containing 100 mM NaCl, Ϫ Identification of A. aeolicus holB and Purification of ␦Ј—The A. then aliquoted, and stored frozen at 80 °C. aeolicus holB gene was identified by Blast search using the E. coli ␦Ј Purification of ␶ Encoded by dnaX—The A. aeolicus dnaX gene was sequence as a query. The A. aeolicus holB gene was amplified by PCR amplified by PCR from genomic DNA using the following primers. The using the following primers. The upstream 41-mer (5Ј-GTGTGT- upstream 41-mer (5Ј-GTGTGTCATATGAACTACGTTCCCTTCGCGA- CATATGGAAAAAGTTTTTTTTGGAAAAAACTCCAG-3Ј) contains an GAAAGTACAG-3Ј) contains an NdeI site (underlined); the downstream NdeI site (underlined); the downstream 35-mer (5Ј-GTGTGTGGATC- 36-mer (5Ј-GTGTGTGGATCCTTAAAACAGCCTCGTCCCGCTGGA-3Ј) CTTAATCCGCCTGAACGGCTAACG-3Ј) contains a BamHI site (un- contains a BamHI site (underlined). The PCR product was digested pol III Holoenzyme of Aquifex aeolicus 17337

with NdeI and BamHI, purified, and ligated into the pET24 NdeI and 60 fractions were collected. The ␦␦Ј complex elutes later than either ␦ or BamHI sites to produce pETAadnaX. ␦Ј. The ␦␦Ј complex was stored at Ϫ80 °C. The pETAadnaX plasmid was transformed into E. coli strain BL21 Constitution of ␶␦␦Ј Complex—The reaction mixture contained 400 (␭DE3). Cells were grown in 50 liters of LB containing 100 ␮g/ml ␮gof␦␦Ј and 1.2 mg of ␶ in a volume of 200 ␮l of buffer A containing 300 ϭ kanamycin at 37 °CtoA600 0.6 and then induced for 20 h at 20 °C mM NaCl. The protein mixture was incubated at 24 °C for 15 min and upon addition of IPTG to 2 mM. Cells were collected by centrifugation then injected onto an HR 10/30 Superose 12 column equilibrated with and lysed as described for purification of ␣. The clarified cell lysate was buffer A containing 300 mM NaCl. After the first 6.6 ml, fractions of 170 heated to 65 °C for 30 min, and the protein precipitate was removed by ␮l were collected. Fractions were analyzed in 10% SDS-polyacrylamide centrifugation. The supernatant (1.1 g in 340 ml) was treated with gels stained with Coomassie Blue. The controls included ␦␦Ј complex 0.228 g/ml ammonium sulfate followed by centrifugation. The ␶ subunit alone (400 ␮g) and ␶ alone (1.2 mg). remained in the pellet which was dissolved in buffer B (20 mM Hepes Quantitation of the Intracellular Concentration of A. aeolicus ␶—A. (pH 7.5), 0.5 mM EDTA, 2 mM DTT, 10% glycerol) and dialyzed versus aeolicus cells were a generous gift of Dr. Karl Stetter (Regensburg buffer B to a conductivity equal to 87 mM NaCl. The dialysate (1073 mg, University). A. aeolicus cells were resuspended in buffer A to ϳ2 ϫ 1011 570 ml) was applied to a 200-ml fast flow Q-Sepharose column equili- cells/ml by direct counting as follows. Serial dilutions of the cell sus- brated in buffer A. The column was eluted with a 1.5-liter linear pension (107,108,109, and 1010) were counted using a hemocytometer gradient of 0–500 mM NaCl in buffer A; 80 fractions were collected. (Hausser Scientific) under a high power microscope (Olympus IX70), Fractions 28–37 were pooled (289 mg, 138 ml), dialyzed against buffer and the results were averaged. A 50-␮l aliquot was then cracked in 100 A to a conductivity equal to 82 mM NaCl, and then loaded onto a 150-ml ␮l of a solution containing 0.3% SDS, 30 mM Tris base, 30 mM DTT, column of heparin-agarose equilibrated in buffer A. The column was 0.01% bromphenol blue, and 8% glycerol. Amounts equivalent to 2.33 ϫ eluted with a 900-ml linear gradient of 0–500 mM NaCl in buffer A; 32 108 and 9.32 ϫ 108 cells were analyzed in two lanes of a 12% SDS- fractions were collected. Fractions 15–18 (187 mg, 110 ml) were dia- polyacrylamide gel, followed by transfer to nitrocellulose. Western blot lyzed versus buffer A, then aliquoted, and stored at Ϫ80 °C. analysis was then performed using rabbit polyclonal antibody directed Downloaded from Purification of ␤ Encoded by dnaN—The A. aeolicus dnaN gene (41) against E. coli ␥ (which was purified against A. aeolicus ␶ as described was amplified from genomic DNA by PCR using the following primers. earlier). Known amounts of pure recombinant A. aeolicus ␶ were ana- The upstream 33-mer (5Ј-GTGTGTCATATGCGCGTTAAGGTGGACA- lyzed in the same gel for use as a standard curve to quantitate the ␶ in GGGAG-3Ј) contains an NdeI site (underlined); the downstream 35-mer A. aeolicus cells observed in the Western blot. The protein concentration (5Ј-TGTGTCTCGAGTCATGGCTACACCCTCATCGGCAT-3Ј) contains of A. aeolicus ␶ was calculated from its absorbance at 280 nm in 6 M

an XhoI site (underlined). The PCR product was digested with NdeI and guanidine hydrochloride using the molar extinction coefficient deter- http://www.jbc.org/ ⑀ ϭ ϫ ϩ BamHI, purified, and ligated into the pET24 NdeI and BamHI sites to mined from its 2 Trp, 14 Tyr, and 20 Phe content ( 280 5,690 2 Ϫ Ϫ produce pETAadnaN. 1,280 ϫ 14 ϩ 2 ϫ 20 ϭ 29,340 M 1 cm 1). The pETAadnaN plasmid was transformed into E. coli strain BL21 Assays of ␣, ␶␦␦, Ϯ␤—Titration of ␣ into replication reactions were (␭DE3). Cells were grown in 1 liter of LB containing 100 mg/ml kana- performed using circular M13mp18 ssDNA primed with a 700-fold ϭ mycin at 37 °CtoA600 1.0 and then induced for 6 h upon addition of molar excess of synthetic DNA 30-mer oligonucleotide. Reactions con- 2mM IPTG. Cells were collected (7 g) and lysed as described in the tained 138 fmol of primed M13mp18 ssDNA, 100 pmol of DNA 30-mer, ␣ ␮ ␶␦␦Ј ␮ ␤

purification of above. The cell lysate was heated to 65 °C for 30 min, 3.6 g of SSB (when present), 150 ng of , 0.4 gof (when present), at Rockefeller University Library on August 2, 2015 and the protein precipitate was removed by centrifugation. The super- and the indicated amount of ␣ in 50 ␮lof20mM Tris-HCl (pH 8.8), 10

natant (39 mg, 45 ml) was applied to a 10-ml DEAE-Sephacel column mM KCl, 10 mM (NH4)2SO4,2mM MgSO4, 0.1% Triton X-100, 4 mM ␮ (Amersham Biosciences) equilibrated in buffer A. The column was CaCl2, 0.5 mM ATP, and 60 M each dATP and dGTP. Reactions were eluted with a 100-ml linear gradient of 0–500 mM NaCl in buffer A; 75 mixed on ice, and then the mixture was brought to 65 °C for 2 min fractions were collected. Fractions 45–57 were pooled (18.7 mg), dia- before initiating synthesis upon addition of 2 ␮l of dCTP and lyzed versus buffer A, and applied to a 30-ml heparin-agarose column [␣-32P]dTTP (final concentrations, 60 and 40 ␮M, respectively). Aliquots equilibrated in buffer A. The column was eluted with a 300-ml linear were removed and quenched at the times indicated upon adding EDTA gradient of 0–500 mM NaCl in buffer A; 65 fractions were collected. and SDS to final concentrations of 20 mM and 0.5%, respectively. Fractions 27–33 were pooled (11 mg, 28 ml) and stored at Ϫ80 °C. Quenched reactions were then analyzed in a 0.8% alkaline-agarose gel. Purification of SSB Encoded by ssb—The A. aeolicus ssb gene (41) Size standards were also included in the same gel. Gels were dried was amplified from genomic DNA by PCR using the following primers. followed by analysis using a PhosphorImager. The upstream 47-mer (5Ј-GTGTGTCATATGCTCAATAAGGTTTTTAT- Replication time course reactions contained 70 ng (25 fmol) of AATAGGAAGACTTACGGG-3Ј) contains an NdeI site (underlined); the M13mp18 ssDNA, 100 pmol of 30-mer oligonucleotide, 2 ␮gofSSB downstream 39-mer (5Ј-GTGTGGATCCTTAAAAAGGTATTTCGTCCT- (when present), 100 ng of ␣, 200 ng of ␶␦␦Ј complex, and 40 ng of ␤ in 25 Ј ␮ CTTCATCGG-3 ) contains a BamHI site (underlined). The PCR product lof20mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4,10mM was digested with NdeI and BamHI, purified, and ligated into the MgSO4, 0.1% Triton X-100, 1 mM ATP, 4 mM CaCl2, 10% glycerol, and pET16b NdeI and BamHI sites to produce pETAassb. 60 ␮M each dGTP and dATP. Replication time courses were performed The pETAassb plasmid was transformed into E. coli strain BL21 at different temperatures as described above except that SSB was (␭DE3). Cells were grown in 6 liters of LB media containing 200 ␮g/ml omitted where indicated, and the mixture was brought to the indicated ϭ ␮ ampicillin. Cells were grown at 37 °CtoA600 0.6, then induced at temperature for 2 min before initiating synthesis by the addition of 2 l 15 °C overnight in the presence of 2 mM IPTG, and collected by centrif- of dCTP and [␣-32P]dTTP (final concentrations, 60 and 40 ␮M, respec- ugation. Cells were lysed as described above for purification of ␣ except tively). Aliquots were removed and quenched at the times indicated cells were resuspended in buffer C (20 mM Tris-HCl (pH 7.9), 500 mM upon adding EDTA and SDS to 20 mM and 0.5%, respectively. Quenched NaCl). reactions were then analyzed in 0.8% alkaline-agarose gels. Size stand- The cell lysate was heated to 65 °C for 30 min, and the resulting ards were included in the same gel. Gels were dried and exposed to film. precipitate was removed by centrifugation. The supernatant (1.4 g, 190 Detection of ␣ Interaction with ␶ and ⑀ by Elisa—The ␣Ϫ␶ interaction ml) was applied to a 25-ml chelating Sepharose column (Amersham was analyzed as follows: E. coli ␣ or A. aeolicus ␣ (2 ␮gin100␮l) were Biosciences) charged with 50 mM nickel sulfate and then equilibrated in placed in wells of a 96-well vinyl assay plate (Costar) and incubated for buffer C containing 5 mM imidazole. The column was eluted with a 12hat4°Cin20mM Tris-HCl (pH 7.5), 1 mM EDTA, 2 mM DTT, 5 mM

300-ml linear gradient of 5–100 mM imidazole in buffer C. Fractions of MgCl2, 100 mM NaCl, 20% glycerol. Solutions were removed, and wells 4 ml each were collected. Fractions 81–92 were pooled (ϳ240 mg in 48 were washed 4 times with 100 ␮l of TBS. Wells were then blocked with ml) and dialyzed overnight against 2 liters of buffer B containing 200 100 ␮l of TBS containing 5% non-fat milk for3hat23°C. Following the mM NaCl. The dialysate was diluted to a conductivity equal to 92 mM blocking step, wells were washed 4 times with TBS and then incubated NaCl using buffer A and then loaded onto an 8-ml MonoQ column with 4 ␮g of either E. coli ␶ or A. aeolicus ␶ for2hat23°C. Solutions equilibrated in buffer A containing 100 mM NaCl. The column was were then removed, and wells were washed as above and then incu- eluted with a 120-ml linear gradient of 100–500 mM imidazole in buffer bated overnight at 4 °C with 100 ␮l of 1:500 dilution of polyclonal rabbit A. Seventy four fractions were collected. Fractions 57–70 were pooled antibody raised against E. coli ␥ and purified against A. aeolicus ␶. (100 mg, 25 ml), aliquoted, and stored at Ϫ80 °C. Wells were washed three times with TBS (5 min each wash) and then Preparation of ␦␦Ј Complex—The ␦ (30 mg) and ␦Ј (30 mg) subunits incubated with anti-rabbit horseradish peroxidase-conjugated antibody were mixed in a volume of 23 ml of buffer A (final conductivity equiv- (Sigma) for 30 min and developed with an ECL detection kit (Amer- alent to 130 mM NaCl) and incubated at 24 °C for 10 min. The mixture sham Biosciences) as described by the manufacturer. was applied to an 8-ml MonoQ column equilibrated in buffer A. The Detection of ␣ and ⑀ interaction was performed as described above protein was eluted with an 80-ml gradient of 100 mM to 500 mM NaCl; with the following modifications. One hundred microliters of either E. 17338 pol III Holoenzyme of Aquifex aeolicus

coli ⑀ or A. aeolicus ⑀ (50 ␮g/ml) were incubated in wells for 12 h at 4 °C. each reaction were analyzed in a 15% denaturing (6 M urea) polyacryl- Following the block step and the washes with TBS, 100 ␮l of either E. amide gel. coli ␣ or A. aeolicus ␣ (100 ␮g/ml) were placed into the wells and Calf Thymus DNA Synthesis Assay—Assays contained 2.5 ␮gof incubated for2hat23°C. Solutions were then removed, and wells were activated calf thymus DNA and 0.2 of either E. coli ␣ subunit or A. washed with TBS as above and then incubated overnight at 4 °C with aeolicus ␣ in a final volume of 25 ␮lof20mM Tris-HCl (pH 7.5), 4% ␮ ␮ 100 l of 1:500 dilution of polyclonal rabbit antisera directed against E. glycerol, 0.5 mM EDTA, 5 mM DTT, 40 g/ml BSA, 8 mM MgCl2,1mM coli ␣. Wells were then washed with TBS (3 times for 5 min) and further ATP, 60 ␮M each dCTP, dGTP, dATP, and 20 ␮M [␣Ϫ32P]dTTP. Reac- incubated with a 1:10,000 dilution of anti-rabbit horseradish peroxi- tions were incubated at the indicated temperatures, and aliquots were dase-conjugated antibody (Sigma) for 30 min and then developed with removed at the times indicated. DNA synthesis was quantitated using an ECL detection kit (Amersham Biosciences) and exposed to film. DE81 paper as described (43). Detection of ␣-␶ Interaction by DNA Synthesis—DNA synthetic reac- Clamp Loading Assay—A C-terminal kinase site was introduced into tions contained 70 ng of M13mp18 ssDNA uniquely primed with a A. aeolicus ␤ by PCR using a primer with a sequence encoding a synthetic DNA 60-mer, 1 ␮gofE. coli SSB, 100 ng of polymerase 6-amino acid (RRASVP) recognition motif for cAMP-dependent protein subunit (either S. pyogenes polCorE. coli ␣), and when present 400 ng kinase (44). The gene encoding A. aeolicus ␤pk was placed into NdeI/ pk of ␶ (S. pyogenes ␶ or E. coli ␶), in 25 ␮lof20mM Tris-HCl (pH 7.5), 4% BamHI sites of pET16b, and the A. aeolicus ␤ was expressed and ␮ ␤pk glycerol, 0.1 mM EDTA, 5 mM DTT, 2 mM ATP, 8 mM MgCl2,40 g/ml purified. The was radiolabeled using cAMP-dependent protein ki- 32 pk BSA, and 60 ␮M each of dGTP and dCTP. Reactions were preincubated nase and [␥- P]ATP as described for E. coli ␤ (45). Clamp loading at 37 °C for 2 min, and then synthesis was initiated upon addition of 1.5 reactions contained 0.125 pmol of singly nicked M13mp18 plasmid DNA 32 ␮l of dATP and [␣-32P]TTP (final concentrations of 60 and 20 ␮M, (using gpII protein), 0.7 nmol (60 ng) of P-␤, 100 ng of ␶␦␦Јcomplex respectively). Reactions were allowed to proceed for 5 min prior to being (when present) and 2.4 ␮gofA. aeolicus SSB (when present) in 50 ␮lof quenched with an equal volume (25 ␮l) of 1% SDS, 40 ␮M EDTA. reaction buffer (20 mM Tris-HCl (pH 8.8, at 25 °C), 10 mM KCl, 10 mM Downloaded from One-half of the reaction was analyzed for total DNA synthesis using (NH4)2SO4,10mM MgSO4, 0.1% Triton X-100, and 1 mM ATP). Reac- DE81 filter paper as described (43). tions were incubated for 10 min at 70 °C and then applied to 5 ml Assays of A. aeolicus ␣ and ␶ utilized a different temperature and Bio-Gel A15m columns (Bio-Rad) pre-equilibrated with 20 mM Tris-HCl ␮ conditions as follows. Reactions contained 70 ng (25 fmol) of M13mp18 (pH 7.5), 8 mM MgCl2, 5% glycerol, 2 mM DTT, 40 g/ml BSA, and 50 mM ssDNA, 100 pmol of DNA 30-mer, 2 ␮gofA. aeolicus SSB, 50 ng of A. NaCl. Gel filtration was performed at room temperature (24 °C). Frac- aeolicus ␣, and 400 ng of A. aeolicus ␶ in 25 ␮lof20mM Tris-HCl (pH tions of 200 ␮l were collected, and 150 ␮l of each fraction was analyzed M M M by liquid scintillation counting. 8.8), 10 m KCl, 10 m (NH4)2SO4,10m MgSO4, 0.1% Triton X-100, http://www.jbc.org/ ␮ 1mM ATP, 4 mM CaCl2, 10% glycerol, 60 M each dGTP and dATP. The reaction mixture was incubated at 60 °C for 2 min before initiating RESULTS ␮ ␣ 32 synthesis by the addition of 1.5 l of dCTP and [ - P]TTP (final The A. aeolicus Clamp Loader—Studies in the E. coli system concentrations, 60 and 40 ␮M, respectively). Reactions were allowed to ␮ demonstrated the need for five proteins to form a processive proceed for 5 min before being quenched with an equal volume (25 l) ␣ ␶ ␥ ␦ ␦Ј ␤ of 1% SDS, 40 ␮M EDTA. One-half of the quenched reaction was ana- polymerase III: , (or ), , , and (9, 13, 46). A sixth protein, the ⑀3Ј–5Ј-exonuclease, further enhanced the speed

lyzed for total DNA synthesis using DE81 filter paper (43). at Rockefeller University Library on August 2, 2015 Gel Filtration Analysis of ␣, ␶, and ⑀—The mixtures contained 1 mg and processivity of the holoenzyme constituted from these five of ␣ and 200 ␮gof⑀ or 0.5 mg of ␶ in a volume of 200 ␮l of buffer A proteins (9). The report on the A. aeolicus genome sequence (41) containing 300 mM NaCl. Proteins were incubated at 24 °C for 15 min documented the presence of A. aeolicus dnaE (␣), dnaX (␶/␥), before injecting the mixture onto an HR 10/30 Superose 12 column dnaN (␤), and dnaQ (⑀), but holA and holB genes encoding ␦ equilibrated with buffer A containing 300 mM NaCl. In a further exper- ␦Ј ␦ iment, 4 mg ␣ (150 ␮M)and1mg␶ (92 ␮M) were mixed in a volume of and were not identified. As we have reported previously, is 200 ␮l of buffer A containing 300 mM NaCl and analyzed by gel filtra- a very poorly conserved subunit of the pol III replicase (38). We tion as described above. The controls included ␣ alone (1 mg, 37.5 ␮M), identified S. pyogenes holA by isolating a gene encoding a weak ␶ alone (0.5 mg, 46 ␮M), and ⑀ alone (200 ␮g) in 200 ␮l of buffer A homologue to E. coli ␦ and then showing that the putative ␦ containing 300 mM NaCl. formed an active clamp loader complex with other S. pyogenes ␣ ␤ ␶␦␦Ј ␣ ϩ ␶␦␦Ј Thermostability Assays—A. aeolicus , , , SSB, and subunits (38). In the experiments below, we follow a similar were tested for stability at different temperatures by incubating the ␦ ␦Ј proteins at the temperatures indicated and then testing them for activ- strategy to identify genes encoding both A. aeolicus and . ity in the M13mp18 replication assay. Heat treatment was performed in A search of the A. aeolicus genome for the A. aeolicus holA 0.4-ml tubes under mineral oil in 5 ␮lof20mM Tris-HCl (pH 7.5), 5 mM gene encoding ␦, using E. coli ␦ subunit as the query, produced DTT, 5 mM EDTA, and either (a) 0.352 ␮gof␣;(b) 0.2 ␮gof␤;(c) 0.125 very weak matches, the best having about 18% identity to E. ␮gof␶␦␦Ј;(d) 0.32 ␮g of SSB and 0.042 ␮g of primed M13mp18 ssDNA; coli ␦ (see Fig. 2A). Likewise, a search for the A. aeolicus holB ␮ ␣ ␮ ␶␦␦Ј or (e) 0.352 g and 0.125 gof . Heat treatment was for 2 min at gene encoding ␦Ј, using E. coli ␦Ј as query, produced a similarly either 70, 80, 85, or 90 °C in the presence of either (a) 0.1% Triton weak match (Fig. 2B). To determine whether the putative A. X-100; 0.05% Tween 20 and 0.01% Nonidet P-40; (b)4mM CaCl2;(c) 40% glycerol; (d) 0.01% Triton X-100, 0.05% Tween 20, 0.01% Nonidet aeolicus holA and holB genes truly encode the ␦ and ␦Ј subunits

P-40, 4 mM CaCl2;(e) 40% glycerol, 0.1% Triton X-100; (f) 40% glycerol, of the replicase, we expressed and purified the encoded proteins 0.05% Tween 20, 0.01% Nonidet P-40; (g) 40% glycerol, 4 mM CaCl2;(h) and then tested them for clamp loading activity with the A. 40% glycerol, 0.01% Triton X-100, 0.05% Tween 20, 0.01% Nonidet P-40, aeolicus dnaX product. The genes encoding the putative A. 4mM CaCl . After heating, reactions were shifted to ice, and 20 ␮lof 2 aeolicus ␦ and ␦Ј subunits were cloned into the T7-based pET11 replication assay buffer was added followed by incubation for 1.5 min at ␦ ␦Ј 70 °C; 15 ␮l was then spotted onto a DE81 filter and DNA synthesis was expression plasmid. Purification of A. aeolicus and were quantitated (43). The replication assay buffer contained the following: performed as described under “Experimental Procedures,” and

60 mM Tris-HCl (pH 9.1 at 25 °C),8mM MgCl2,10mM (NH4)2SO4,2mM a sample of each preparation is shown in the SDS-polyacryl- ATP, 60 ␮M each of dATP, dCTP, dGTP, and 20 ␮M [␣Ϫ32P]TTP (specific amide gel of Fig. 1A. activity 10,000 cpm/pmol), 2 ␮M DNA 30-mer, and 0.264 ␮g of primed To test ability of the putative A. aeolicus ␦ and ␦Ј subunits to ␤ ␣ ␶␦␦Ј M13mp18 ssDNA. To assay , 170 ng of and 100 ng of was added form a clamp loader complex with A. aeolicus ␶(␥), encoded by to the reaction. To assay ␶␦␦Ј, 0.9 ng of ␤ and 170 ng of ␣ were added to the reaction. To assay ␣ Ј2b ␶␦␦Ј,90ngof␤ was added to the reaction. dnaX, we needed to express and purify the product of the A. To assay ␣,90ngof␤ and 100 ng of ␶␦␦Ј was added to the reaction. To aeolicus dnaX gene. A. aeolicus dnaX lacks a ribosomal frame- assay SSB, 90 ng of E. coli ␤ and 100 ng of E. coli ␣␶␦␦Ј was added to the shift sequence, and it also lacks the six or more contiguous A reaction followed by incubation for 1.5 min at 37 °C. residues that are needed to produce ␥ in T. thermophilus by a Exonuclease Assay—Exonuclease assays contained 100 fmol of a transcriptional slippage mechanism (37). Consistent with the Ј ⑀ 5 -labeled 50-mer DNA oligonucleotide and 50 ng of either E. coli lack of sites for synthesis of the truncated ␥ subunit, expression subunit or A. aeolicus ⑀ in 20 ␮lof20mM Tris-HCl (pH 7.5), 4% glycerol, ␮ of the A. aeolicus dnaX gene in E. coli showed production of 0.1 mM EDTA, 5 mM DTT, 40 g/ml BSA, 8 mM MgCl2. Reactions were incubated at 37 °C for the indicated times and were quenched upon the only one protein that was the approximate size of the predicted addition of 20 ␮l of 95% formamide, 40 mM EDTA. Ten microliters of full-length product (54.3 kDa). As will be shown later in this pol III Holoenzyme of Aquifex aeolicus 17339

FIG.2.The sequences of A. aeolicus -and ␦؅ subunits. The amino acid se ␦ quences of A. aeolicus and E. coli ␦ (A) and Downloaded from ␦Ј (B) are aligned using ClustalX. The as- terisks below the sequence indicate amino acids that are identical between the two sequences. http://www.jbc.org/ at Rockefeller University Library on August 2, 2015

␥ ␦␦Ј ␶␦␦Ј ␶ ␦ report, examination of A. aeolicus cell extracts revealed the studies of 3 that the stoichiometry of A. aeolicus is 3 1 ␶ ␦Ј presence of only full-length subunit, a truncated version (i.e. 1 (235.8 kDa) (19, 47). Consistent with this, densitometric ␥) was not detected. The A. aeolicus ␶ subunit was purified as scans of SDS-polyacrylamide gels of Superose 12 column frac- described under “Experimental Procedures,” and a sample of tions of ␶␦␦Ј, constituted using excess ␦␦Ј, give a stoichiometry ␶ ␦ ␦Ј the protein preparation is shown in Fig. 1A. of 3.3 1.1 1.0. In the E. coli system, ␶ (or ␥) forms a heterotrimer with ␦ and Previous study of the E. coli and S. pyogenes pol III systems ␦Ј ␥ ␦␦Ј ␦ ␦Ј ␦␦Ј (13). Even though the crystal structure of E. coli 3 shows demonstrated that the and subunits form a 1:1 complex that ␥ contacts both ␦ and ␦Ј directly, mixtures of ␥ (or ␶) with (13, 38). Fig. 3D shows that A. aeolicus ␦ and ␦Ј also form a ␦␦Ј either ␦ or ␦Ј do not yield either heterodimer (i.e. ␶␦ or ␶␦Ј) complex. Analysis of a mixture of ␦ and ␦Ј demonstrates that complex (13). To examine the A. aeolicus ␶, ␦, and ␦Ј subunits the two subunits coelute in earlier fractions than the elution for protein interactions, combinations of these subunits were position of either ␦ or ␦Ј alone (compare D with A and B) and mixed and then analyzed by gel filtration on a Superose 12 thus form a ␦:␦Ј complex. Densitometric scans of the gel in Fig. ␦ ␦Ј column. The analysis in Fig. 3A demonstrates that a mixture of 3D yield a stoichiometry of 1.1 1.0 (a similar stoichiometry is ␶ ϩ ␦Ј does not yield a heterodimer complex, instead the two obtained from an analysis using a 2-fold molar excess of ␦Јover proteins elute at distinct positions. This result is similar to ␦ and scanning the fractions containing ␦␦Ј complex). observations in the E. coli system (13). Analysis of the ␶ ϩ ␦ Is the A. aeolicus ␶␦␦Ј complex active as a clamp loader? To mixture, in Fig. 3B, shows that the bulk of the ␶ and ␦ does not test this, a six-residue kinase recognition site was placed onto comigrate, although a slight amount of ␦ appears in the vicinity the C terminus of ␤, allowing it to be phosphorylated using of ␶ indicating a weak interaction between them. The elution [␥-32P]ATP and cAMP-dependent protein kinase. The 32P-␤ position of the free ␦ and ␦Ј subunits in Fig. 3, A and B, was then incubated with ␶␦␦Ј, ATP, and a circular plasmid compared with molecular mass standards, indicates that ␦ and having a single nick. After incubation at 70 °C for 10 min, the ␦Ј are each monomeric, similar to ␦ and ␦Ј of E. coli and S. reaction was applied to a 5-ml Bio-Gel A15m column. Bio-Gel pyogenes (13, 38). The ␶ subunit elutes as an oligomer, possibly A15m is a large pore resin that includes most proteins but a trimer or tetramer, like E. coli ␶ and S. pyogenes ␶ (38, 47). excludes the large circular DNA molecule. Therefore, if ␶␦␦Ј is A mix of all three subunits results in their comigration as a active in assembly of 32P-␤ onto DNA, some of the 32P-␤ should ␶␦␦Ј complex that elutes earlier than either ␶, ␦,or␦Ј subunits comigrate with the DNA in the excluded volume and resolve alone (Fig. 3C). We presume on the basis of E. coli structural from the free 32P-␤ in the included fractions. The results of this 17340 pol III Holoenzyme of Aquifex aeolicus Downloaded from http://www.jbc.org/ at Rockefeller University Library on August 2, 2015

FIG.3.Constitution of the A. aeolicus ␶␦␦؅ clamp loader. Combinations of the ␶, ␦, and ␦Ј subunits were incubated together and then analyzed for complex formation by gel filtration analysis on a Superose 12 column. Column fractions were analyzed in 10% SDS-polyacrylamide gels stained with Coomassie Blue. A, ␶ ϩ ␦Ј; B, ␶ ϩ ␦; C, ␶ ϩ ␦ ϩ ␦Ј; D, ␦ ϩ ␦Ј. Subunit positions in the gels are indicated to the right. The elution positions of protein standards are indicated at the bottom: 670 kDa, thyroglobulin; 440 kDa, horse spleen apoferritin; 158 kDa, bovine ␥ globulin; 44 kDa, chicken ovalbumin; and 17 kDa, horse myoglobin.

32P-␤ clamp loading experiment are shown in Fig. 4; about half duced. In Fig. 5A A. aeolicus whole cells were analyzed by of the 32P-␤ is observed to comigrate with DNA in the excluded Western using a rabbit polyclonal antibody raised against E. fractions (fractions 10–15). As a control, a similar experiment coli ␥ which cross-reacts with A. aeolicus ␶. The A. aeolicus ␶ was performed in the absence of ␶␦␦Ј complex. The result dem- subunit is clearly visible in the analysis. The limit of detection onstrates that essentially no 32P-␤ is assembled onto the DNA in this assay (e.g. due to background) is such that if a lower in the absence of ␶␦␦Ј. Hence, the A. aeolicus ␶␦␦Ј is capable of molecular weight ␥-like product is present then it is present at assembling A. aeolicus 32P-␤ clamps onto DNA. The tempera- less than 10% the intracellular level of ␶. We have performed ture resistance of the A. aeolicus ␶␦␦Ј complex and ␤ clamp will this Western several times and have also used even more cells, be demonstrated later in this report, along with the DNA with results similar to those in Fig. 5. The inability to detect ␥ polymerase. in A. aeolicus is consistent with the absence of recognizable DnaX Produces Only ␶ in A. aeolicus—The experiments of signals for transcriptional slippage or a translational frame- Fig. 5 address whether A. aeolicus cells produce a truncated shift in the A. aeolicus dnaX gene sequence. product (i.e. ␥) or whether only full-length protein (␶) is pro- To determine the average number of molecules of ␶ present in pol III Holoenzyme of Aquifex aeolicus 17341

⑀ (24.8% identity) (41). The A. aeolicus dnaQ gene was cloned into pET11 for expression and purification. A sample of the purified A. aeolicus ⑀ is shown in the SDS-polyacrylamide gel of Fig. 1. In Fig. 6A, A. aeolicus ␣Ϫ⑀ interaction was analyzed in an Elisa type assay in which ⑀ is adhered to wells of a 96-well plate, followed by blocking additional sites with nonspecific protein. Following this, A. aeolicus ␣ is added, and then unbound ␣ is washed away. Rabbit antibody directed against E. coli ␣ cross-reacts with A. aeolicus ␣ and was used to detect whether A. aeolicus ␣ had bound to ⑀. The results demonstrate a high signal of ␣ bound to the well that was treated with ⑀.No ␣ is detected if the well is not pretreated with A. aeolicus ⑀. Similar results were obtained using E. coli ␣ and ⑀ (also shown in Fig. 6A). To determine whether A. aeolicus ␣ and ⑀ form a stable complex, we analyzed a mixture of ␣ and ⑀ (37 ␮M ␣ and 44 ␮M ⑀) by gel filtration on an FPLC Superose 12 column;

however, the ␣ and ⑀ subunits do not comigrate (Fig. 6D). This Downloaded from result indicates that the ␣ and ⑀ subunits do not tightly interact under these conditions. This experiment has been repeated at a protein concentration of 150 ␮M ␣ and 90 ␮M ⑀, and in the presence of either ␶, ␶␦␦Ј,or␶␦␦Јϩ␤, all with negative results (not shown). A chromosomal replicase is generally expected to contain the http://www.jbc.org/ 3Ј–5Ј-exonuclease activity in tight association with the DNA polymerase activity. The fact that we only detect weak interaction rather than a tight gel filterable complex between A. aeolicus ␣ and ⑀ may be due to any of several factors. For .FIG.4.Demonstration of clamp loading by A. aeolicus ␶␦␦؅. The example, ␣ and ⑀ may interact tightly at high temperature at Rockefeller University Library on August 2, 2015 scheme at the top illustrates the assay. A C-terminal six-residue tag Another possibility is that a tight interaction between ␣ and ⑀ ␤ was cloned onto A. aeolicus that serves as an efficient substrate for may occur in the presence of primed DNA. It is also possible phosphorylation. A. aeolicus ␶␦␦Ј and 32P-␤ are incubated with a circular plasmid containing a single nick. Clamp loading by ␶␦␦Ј is detected that other proteins beyond those studied in this report are by analysis of the reaction on a resin that resolves 32P-␤ attached to the needed to act as a bridge between ␣ and ⑀. Yet another expla- 32 large DNA from the free P-␤ that remains off the DNA. Squares, nation may be that recombinant A. aeolicus ␣ or ⑀ is not prop- reaction lacking ␶␦␦Ј. Circles are the result of the reaction in which ␶␦␦Ј was present. The elution positions of 32P-␤ DNA and 32P-␤ free in erly folded (addressed below). solution are indicated above the plot. As one measure that the A. aeolicus ␣ and ⑀ subunits preparations are correctly folded, we examined them for catalytic activity. The A. aeolicus ␣ was active on gapped calf thymus an A. aeolicus cell, we directly counted the number of A. aeoli- DNA at 37 °C but became more active at 65 °C (Fig. 6B). By cus cells in the cell suspension using a microscope. This pro- comparison, E. coli ␣ was more active than A. aeolicus ␣ at vided us the number of cells that were analyzed in each lane of 37 °C but was essentially inactive at 65 °C. A. aeolicus ␣ re- ␶ the Western in Fig. 5A. The amount of detected in cells by tained significant activity even at 85 °C (about 40% relative to Western analysis was then quantitated by scanning the gel 65 °C). For exonuclease activity, a 5Ј 32P-end-labeled DNA 50- with a laser densitometer. The band intensity was converted to mer was used to determine whether the A. aeolicus ⑀ subunit nanograms of ␶ by comparison with known quantities of recom- was active. The results, in Fig. 6C, show an increase in mobility binant A. aeolicus ␶ that had been analyzed in the same gel as of the 5Ј 32P-DNA oligonucleotide upon incubation with A. the A. aeolicus cells. The result, in Fig. 5B, indicates that A. aeolicus ⑀, indicating the presence of 3Ј–5Ј-exonuclease activity. aeolicus has 110–150 molecules of ␶ as trimer (assuming three A similar experiment using E. coli ⑀ results in full degradation ␶ subunits per clamp loader complex). Thus, this quantity could of the DNA within 10 min. The greater activity of E. coli ⑀ may provide for 110–150 clamp loading assemblies per cell. Similar be due to use of 37 °C for the exonuclease assays, which is quantitative work in the E. coli system indicated the presence probably suboptimal for the A. aeolicus ⑀ subunit. The possi- of about twice as much ␥ (and ␶) than observed here for A. ␣ Ј aeolicus ␶ (61). However, the same study estimated about 150 bility that the A. aeolicus contains an intrinsic 3 –5-exonu- clamp loader complexes per E. coli cell due to limiting ␦Ј (61). clease activity was also tested in the exonuclease assay, but the The A. aeolicus DNA Polymerase—The A. aeolicus ␣ subunit result was negative (not shown). The presence of both polym- homologue has 40.7% identity to E. coli ␣. Cloning of the A. erase and exonuclease activities suggests that recombinant A. ␣ ⑀ aeolicus dnaE gene into pET11 provided a high level of expres- aeolicus and subunits are properly folded. Therefore, a lack sion from which 320 mg of ␣ were obtained from 50 liters of of interaction between them is probably not due to one of them induced cells. A sample of the protein preparation is shown in existing in a denatured state. Factors that may underlie the lane 1 of Fig. 1. inability of A. aeolicus ␣ and ⑀ to form a complex are explored Like E. coli ␣, the A. aeolicus ␣ homologue lacks a region of further under “Discussion.” homology to the E. coli ⑀ subunit exonuclease. Thus, it may be A. aeolicus ␣ and ␶ Interact—In the E. coli system, the ␶ presumed that A. aeolicus ␣ binds to another protein that subunit forms a tight contact with the ␣ subunit of the pol III supplies the proofreading exonuclease function, similar to in- core DNA polymerase, and this complex can be detected by gel teraction of E. coli ␣ to the ⑀ 3Ј–5Ј-exonuclease. The A. aeolicus filtration analysis at even low concentrations (100 nM) (46). Our genome contains a dnaQ gene encoding a homologue to E. coli studies in the S. pyogenes system demonstrate that the S. 17342 pol III Holoenzyme of Aquifex aeolicus Downloaded from http://www.jbc.org/

FIG.5.A. aeolicus contains ␶ but not ␥. A, A. aeolicus whole cells were analyzed by Western blot to determine whether a truncated (␥) product of dnaX is present or if only full-length product (␶) is observed. The position of A. aeolicus ␶ is noted on the left by comparison to the mobility of recombinant ␶ in an adjacent lane of the same gel (not shown). The position of size standards is shown to the right of the gel. B, the plot quantitates the amount of intracellular ␶ by comparison of the intensity of A. aeolicus ␶ in cells to the intensity of known amounts of pure recombinant A. aeolicus ␶ analyzed in the same gel. The table takes into consideration the number of A. aeolicus cells applied to lanes of the Western blot in A to arrive at the average number of molecules of ␶ (as trimer) per A. aeolicus cell. at Rockefeller University Library on August 2, 2015 pyogenes ␶ subunit interacts with S. pyogenes pol C; however, they do not form as tight of a complex as E. coli ␣⅐␶, which is the contact is weaker than in the E. coli system. stable to gel filtration even at concentrations below 0.1 ␮M. The In Fig. 7, we examine the A. aeolicus ␶ and ␣ subunits for concentrations of ␣ and ␶ used in the analysis of Fig. 7C were 37 interaction between them. In Fig. 7A, A. aeolicus ␣-␶ interac- and 46 ␮M, respectively. We have also analyzed A. aeolicus ␣ tion was analyzed in the Elisa type assay as described above for and ␶ at yet higher concentrations (150 and 92 ␮M, respectively) analysis of ␣-⑀. In this case, A. aeolicus ␣ is adhered to the but still observe no interaction between them (data not shown). wells, followed by blocking with nonspecific protein, and then A. aeolicus ␣ Functions with the ␤ Clamp—Does A. aeolicus adding A. aeolicus ␶. Unbound ␶ was washed away, and anti- pol III ␣ subunit function with A. aeolicus ␶␦␦Ј and ␤? This is body directed against E. coli ␶ (which cross-reacts with A. tested in the experiments of Fig. 8 in which a singly primed aeolicus ␶) was used to detect whether A. aeolicus ␶ had bound M13mp18 ssDNA circle (7.2 kb) is utilized as substrate. In the to ␣. The results demonstrate a high signal of ␶ bound to the E. coli system, this large ssDNA circular primed template must well treated with ␣.No␶ is detected if the well is not pretreated be coated with SSB in order for it to serve as an efficient with A. aeolicus ␣. Hence, A. aeolicus ␣ and ␶ directly interact. substrate for pol III holoenzyme. Without SSB, E. coli pol III Similar results were obtained using E. coli ␣ and ␶ (also shown holoenzyme is encumbered by DNA secondary structure which in Fig. 7A). SSB mostly eliminates by virtue of its tight interaction with the Next we designed another assay to examine A. aeolicus ␶ and ssDNA template, providing about a 10-fold stimulation in syn- ␣ for interaction, and this second method carries the advantage thesis relative to the absence of SSB. However, with SSB pres- of demonstrating that the interaction is functional. The assay ent, the E. coli holoenzyme extends the primer completely is based on the fact that E. coli ␣ has very low activity on a around the circular template within 11 s at 30 °C to form the singly primed M13mp18 ssDNA template coated with SSB, but RFII species. In the absence of ␤, the E. coli core polymerase addition of E. coli ␶ provides over 10-fold simulation. This does not extend the primer more than a few hundred nucleo- stimulation of E. coli ␣ is specific to E. coli ␶, as use of S. tides (12). However, when ␤ is first assembled onto the primed pyogenes ␶ does not stimulate the reaction (Fig. 7B). Likewise, site, core becomes rapid and processive in synthesis (5). S. pyogenes ␣ is stimulated by S. pyogenes ␶, but not by E. coli To develop this assay in the A. aeolicus system, we cloned the ␶ (Fig. 7B). In keeping with interaction between A. aeolicus ␣ A. aeolicus ssb gene into pET11 and expressed and purified the and ␶, A. aeolicus ␣ is stimulated by A. aeolicus ␶. We were SSB (a sample of the preparation is shown in lane 7 of Fig. 1). unable to examine whether E. coli ␶ or S. pyogenes ␶ stimulate With the A. aeolicus SSB, ␤ clamp and ␶␦␦Ј clamp loader in hand, A. aeolicus ␣ due to the 65 °C reaction temperature required by we examined their effect on the A. aeolicus pol III ␣ subunit in the A. aeolicus system for activity. extension of a primed site on the M13mp18 ssDNA genome. As a In Fig. 7C, A. aeolicus ␣ and ␶ were analyzed for ␣⅐␶ complex basis for comparison we first examined the behavior of ␣ on formation by gel filtration on an FPLC Superose 12 column. singly primed SSB-coated M13mp18 ssDNA in the absence of ␤ The top two panels show the elution profiles of A. aeolicus ␣ and subunit (Fig. 8A). In this experiment, product is labeled by poly- ␶ alone, and the bottom panel shows the analysis of a mixture merase-catalyzed incorporation of [␣-32P]deoxyribonucleoside of ␣ and ␶. The result shows that the A. aeolicus ␣⅐␶ complex triphosphate. Reactions were incubated at a temperature of cannot be isolated by gel filtration and therefore suggests that 65 °C, as the experiment of Fig. 6 demonstrates that the A. pol III Holoenzyme of Aquifex aeolicus 17343 Downloaded from http://www.jbc.org/ at Rockefeller University Library on August 2, 2015

FIG.6.Analysis of A. aeolicus ␣ and ⑀. A, Elisa dot blot assays of ␣Ϫ⑀ interaction. Wells were coated with either A. aeolicus ⑀ or no protein and then were blocked and treated with A. aeolicus ␣ or no additional protein prior to detection with antibody to E. coli ␣ and anti-rabbit antibody coupled to horseradish peroxidase. A similar experiment using E. coli ␣ and E. coli ⑀ is also shown. B, activity assays of A. aeolicus ␣ and E. coli ␣ (0.2 ␮g each) were compared at different temperatures using activated calf thymus DNA as a substrate. C, the activity of A. aeolicus ⑀ and E. coli ⑀ was compared at 37 °C using a 5Ј FIG.7.Analysis of A. aeolicus ␣ and ␶. A, Elisa type assays of ␣Ϫ␶ 32P-end-labeled DNA 50-mer. Reactions were analyzed on a 12% dena- interaction. Wells were coated with either A. aeolicus ␣ or no protein ␶ turing (6 M urea) polyacrylamide gel, followed by autoradiography. The and then were blocked and treated with A. aeolicus or no additional position of the 50-mer is indicated to the left of the gel. D, Superose 12 protein prior to detection with ␶-specific antibody and anti-rabbit anti- gel filtration analysis of a mixture of A. aeolicus ␣ and ⑀. Column body coupled to horseradish peroxidase. A similar experiment using E. fractions were analyzed in a 10% polyacrylamide gel stained with coli ␣ and E. coli ␶ is also shown. B, ␶ stimulates synthesis by ␣. The plot Coomassie Blue. Positions of ␣ and ⑀ are indicated to the right. to the left demonstrates that E. coli ␶ stimulates ␣, but S. pyogenes ␶ has no effect (1st three lanes). Species-specific stimulation, an indication of specific ␣Ϫ␶ interaction, is also observed by S. pyogenes polCbyS. aeolicus ␣ is more active at 65 than at 37 °C. The results dem- pyogenes ␶ but not E. coli ␶ (lanes 5–8). Controls of E. coli ␶ alone and S. onstrate that A. aeolicus pol III ␣ subunit by itself is capable of pyogenes ␶ alone are in lanes 4 and 7. The plot to the right shows that completely, or nearly completely, extending the single primer full A. aeolicus ␣ is stimulated by A. aeolicus ␶ (lanes 9–11). C, A. aeolicus ␣, ␶, and a mixture of ␣ ϩ ␶ were analyzed by gel filtration. Column circle provided it is supplied in a large amount and is given fractions were analyzed in a 10% SDS-polyacrylamide gel. The positions sufficient time (see the last time point at the highest ␣ concen- of ␣ and ␶ are indicated to the right of the gels. tration). The size of the product formed at any given time appears as a fairly tight band. Furthermore, the product length is depend- M13mp18 ssDNA no matter the time or concentration of polym- ent on the amount of ␣ added to the reaction. This pattern of erase used due to presence of insurmountable barriers to exten- product formation, and length dependence on ␣ concentration, sion (48). In fact, SSB inhibits chain extension by E. coli pol III indicates that synthesis is distributive. In other words, the ␣ core in the absence of ␤ (48). polymerase samples the entire population of primed templates, Next, the time course of DNA synthesis by A. aeolicus pol III synthesizing short stretches over and over on each template until ␣ subunit was examined in the presence of both ␶␦␦Ј and the ␤ all templates are finally completed. E. coli pol III core (and E. coli clamp (Fig. 8B). The results demonstrate formation of complete ␣ subunit) is also highly distributive in synthesis. However, E. RFII product within 2 min at all concentrations of ␣.Atthe coli core is unable to completely extend a single primer around highest amount of ␣ used, formation of the RFII product occurs 17344 pol III Holoenzyme of Aquifex aeolicus

F .8.Activity of the A. aeolicus pol Downloaded from III replicase. Singly primed M13mp18 ssDNA was extended using A. aeolicus ␣ and ␶␦␦Ј either without ␤ (A) or with ␤ (B). Reactions contained the indicated amounts of ␣. Time points are indicated at the bottom of each gel, and the positions of starting primed substrate http://www.jbc.org/ (ssDNA) and finished product (RF II) are indicated to the right. at Rockefeller University Library on August 2, 2015

within 1 min indicating a speed of at least 120 nucleotides per These next experiments were performed using subsaturating s. Indeed, at the two highest ␣ concentrations, the synthetic pol III ␣ subunit in order to assess which temperature is most time course is about the same indicating that the reaction is favorable, and therefore the rates of polymerization are less saturated with respect to ␣ at 2.64 ␮g and above. At ␣ subunit than the maximal rates observed in Fig. 8. The experiments concentrations lower than 0.88 ␮g, less RFII product is formed were also performed in either the presence of SSB (a)orits at the 1-min time point compared with the 2-min time point. absence (b). Proteins were preincubated with the DNA sub- Furthermore, the immature products formed at 15 and 30 s strate for 2 min with two dNTPs to allow time for protein become distinctly shorter as the ␣ subunit is titrated down- assembly onto DNA, and then synchronous synthesis was ini- ward. The dependence of chain length on ␣ concentration indi- tiated upon addition of the remaining two dNTPs. Timed ali- cates that ␣ does not remain attached to the ␤ clamp over the quots were quenched, and the products were analyzed in alka- synthesis of the entire 7.2-kb circle. In other words, processiv- line and native agarose gels. First we consider below the ity of the ␣ subunit with the ␤ clamp is less than 7 kb. Most reactions that were performed in the presence of SSB. likely, ␤ remains tightly bound to DNA continuously as ␣ At temperatures between 60 and 75 °C, the A. aeolicus sys- jumps from one ␤-containing template to the next during syn- tem yields significant levels of synthesis, and as the tempera- thesis. This behavior is similar to that of the E. coli system ture is elevated, the rate of synthesis increases (Fig. 9A). Very when ␣ subunit is used with ␤ and ␥ complex instead of core or little synthesis is observed at 55 °C, which is presumably too ␣⅐⑀ complex (9). In this case, the ␤ clamp confers a processivity low a temperature for the workings of this complex machinery of ϳ1–3 kb onto ␣, and the speed of the pol III ␣⅐␤ complex is from an extreme thermophile. The inactivity at low tempera- about 300 nucleotides per s at 37 °C (compared with about 1 ture may be ascribed, at least in part, to inactivity of the pol III kb/s when ␣⑀ complex is used at the same temperature). ␣ polymerase, as simple gap filling assays using ␣ alone showed The A. aeolicus pol III Replicase Is Thermostable—Next, we low activity at reduced temperature (see Fig. 6B). It remains examined the thermostability of the A. aeolicus pol III replica- possible that the clamp loading operation is also diminished at tion system and its rate of synthesis at different temperatures. low temperature. The A. aeolicus SSB, however, would appear pol III Holoenzyme of Aquifex aeolicus 17345 Downloaded from

FIG.9.The effect of SSB on the A. aeolicus pol III replicase. Reactions containing A. aeolicus ␣, ␶␦␦Ј, and ␤ were performed using singly primed M13mp18 ssDNA either in the presence (A) or absence (B) of SSB. Reactions were performed at either 55, 60, 65, 70, 75, or 80 °C, and aliquots were removed and quenched for analysis at the times indicated. Products were analyzed in alkaline-agarose gels followed by autoradiography. Lengths of size standards are indicated to the left of each gel. http://www.jbc.org/ to remain functional at low temperature as it stimulates the E. 2 min at elevated temperature in the absence of other compo- coli pol III system at 37 °C (data not shown). nents and then was shifted to ice before being assayed for Synthetic activity is greatly reduced at 80 °C indicating that activity. The thermostability of each component was tested one or more components of the A. aeolicus pol III replicase under several different buffer conditions containing either at Rockefeller University Library on August 2, 2015 denature at this temperature. DNA polymerase assays on 0.1% Triton X-100, 0.01% Nonidet P-40, 0.05% Tween 20, 4 mM gapped DNA at different temperatures also indicate that A. CaCl2, 40% glycerol, or combinations of these reagents. Heat- aeolicus pol III ␣ subunit loses activity at 80 °C (Fig. 6B) treated subunits were tested for activity by combining them suggesting that ␣ is the inactive component. However, it re- with the other (unheated) subunits in the primed M13mp18 mains possible that yet other A. aeolicus components also lose ssDNA synthesis assay. The assays contained ␣, ␶␦␦Ј, and ␤ activity at 80 °C and higher. The thermostability of these pro- (one of which was temperature-treated) and were conducted at teins will be examined below. It is also possible that the primed 70 °C for 1.5 min. Because this assay does not depend on the template may lose efficiency in supporting synthesis at these presence of SSB, we assayed the temperature stability of A. high temperatures. In these experiments, we utilize a DNA aeolicus SSB by substituting it for E. coli SSB in assays at ␣ ␶␦␦Ј ␤ 30-mer primer, the Tm of which is below 80 °C. However, we 37 °C using E. coli , , and subunit. have raised the primer concentration in the assays to increase The results in Fig. 10 show that A. aeolicus ␤ and SSB are by the occupancy of the primer on the ssDNA template at elevated far the most thermostable components (B and D). Seventy five temperature. To determine optimal primer concentration, we percent or more of their activity was retained under all condi- have titrated primer into reactions at different temperatures. tions tested, even at 90 °C. The ␶␦␦Ј clamp loader was also The experiments shown here utilize 2 ␮M primer; higher con- highly thermostable provided it was heat-treated in buffers centrations gave no greater synthesis at any temperature. We lacking Tween (C). The ␣ DNA polymerase was the most heat- have also titrated a 90-mer DNA oligonucleotide primer into sensitive of the A. aeolicus pol III replicase components (A). the assay at different temperatures but observed slightly lower Under most conditions, the ␣ subunit lost 25–50% activity upon activity than that obtained using the DNA 30-mer. incubation at 80 °C and lost 80–100% activity upon 90 °C treat- In the E. coli system, the primary role of SSB on synthesis is ment. However, polymerase activity was largely stabilized in thought to be the removal of ssDNA secondary structure. Tem- the presence of 4 mM CaCl2 and 40% glycerol, retaining over perature can also remove secondary structure in ssDNA. It 50% activity after incubation at 90 °C. As expected from the therefore seems reasonable to expect that SSB may not have as thermostability results of ␣ and ␶␦␦Ј, an equimolar mixture of great of a stimulatory effect on synthesis at high temperature ␣ and ␶␦␦Ј was generally thermolabile, but was most stabilized compared with a lower temperature. Ability of the A. aeolicus to heat treatment by 4 mM CaCl2 and 40% glycerol (E). replication system to function above 60 °C, where some second- DISCUSSION ary structure should melt without SSB, provides the opportu- Similarities to the E. coli pol III Replicase nity to assess the effect of SSB on synthesis at high temperature. The replication reactions in Fig. 9B are identical to those The results of this study confirm that bacteria that grow at in Fig. 9A, except that instead of adding SSB, only the SSB very high temperatures utilize the same overall strategy for buffer is added to the reactions. The comparison demonstrates processive replication as is observed in the mesophile, E. coli. that synthesis in the absence of SSB is about the same as in its Hence, A. aeolicus utilizes a pol III polymerase that is tethered presence, consistent with the idea that SSB stimulates polym- to DNA by a ␤ sliding clamp which, in turn, requires a clamp erase by removing secondary structure. loader complex for assembly onto DNA. Previous studies of The temperature stability of each replicase component was thermophilic DNA polymerases have focused mainly on homo- determined in the experiment of Fig. 10. Each individual sub- logues to DNA polymerase I (reviewed in Ref. 62). However, unit, or complex (as indicated in each panel), was incubated for recent studies (35, 36) demonstrated the presence of ␥ and ␶ 17346 pol III Holoenzyme of Aquifex aeolicus

reading frames in the A. aeolicus genome and the current study positively identifies these genes as holA and holB by expressing the proteins they encode and demonstrating their function in the clamp loading reaction. Of the A. aeolicus proteins studied in this report, these two subunits display the least percent identity to the corresponding E. coli subunit sequences (see Fig. 1B). Despite the rather wide sequence variations in A. aeolicus ␦ and ␦Ј compared with their E. coli counterparts, they are re- markably similar to the behavior of E. coli proteins in ability to form ␦␦Ј and ␶␦␦Ј complexes which remain associated during gel filtration analysis (13). Also like the E. coli system, the A. aeolicus ␤, once assembled onto DNA by ␶␦␦Ј, remains stably attached to DNA during gel filtration. Another similarity between these systems is that the A. aeolicus DNA polymerase III ␣ subunit, like E. coli ␣, lacks sequences that provide proofreading 3Ј–5Ј-exonuclease activity. The E. coli ␣ subunit is found tightly associated with the ⑀ 3Ј–5Ј-exonuclease which provides the proofreading function. Indeed, homologues to ⑀ are Downloaded from quite prevalent among bacteria. The dnaQ gene encoding A. aeolicus ⑀ was identified in the original A. aeolicus genome sequence report, and in this report we have cloned the gene and purified the corresponding protein. The A. aeolicus ⑀ homologue Ј Ј

has 3 –5 -exonuclease activity, and our studies demonstrate http://www.jbc.org/ that A. aeolicus ␣ and ⑀ interact. However, the association between the A. aeolicus ␣ and ⑀ subunits is not sufficiently strong to isolate an ␣⅐⑀ complex on a gel filtration column (as in the E. coli system). Inability to identify a tight A. aeolicus ␣⅐⑀ interaction will be elaborated upon below. The present study demonstrates that A. aeolicus pol III ␣ at Rockefeller University Library on August 2, 2015 subunit, when combined with ␤, is highly stimulated in synthesis of a primed M13mp18 ssDNA genome and extends the primer at approximately the same speed as E. coli ␣⅐␤. The current study also demonstrates that A. aeolicus ␣⅐␤ (plus ␶␦␦Ј) is not fully processive during primer extension around M13mp18, again like E. coli ␣⅐␤. Studies in the E. coli system demonstrate that the ⑀ proofreader is needed for optimal performance of the ␣ subunit (9). Whereas E. coli pol III core and FIG. 10. Thermostability of A. aeolicus pol III replicase compo- ␣⑀ complex function with ␤ to synthesize DNA at a speed of ␣ ␤ ␶␦␦Ј ␣ ␶␦␦Ј nents. Either (A), (B), (C), SSB (D), or a mixture of and about 1 kb/s (at 37 °C), with a processivity greater than the (E) were incubated in various buffers at 70, 80, 85, and 90 °C and then ␣ ␤ were assayed for activity on primed M13mp18 ssDNA with the other 7.2-kb M13mp18 genome, the subunit travels with at a components as described under “Experimental Procedures.” The buffers speed of about 300 nucleotides/s with a processivity of 1–3kb used are as follows: 0.1% Triton X-100 (filled diamonds); 0.05% Tween (9). Hence, ⑀ binding to ␣ would appear to result in a more 20 and 0.01% Nonidet P-40 (filled circles);4mM CaCl2 (filled triangles); active form of the polymerase. These comparisons suggest that 40% glycerol (inverted filled triangles); 0.01% Triton X-100, 0.05% A. aeolicus ␣ may also bind another protein(s) to form a pol III Tween 20, 0.01% Nonidet P-40, 4 mM CaCl2 (half-filled square); 40% glycerol, 0.1% Triton X-100 (open diamonds); 40% glycerol, 0.05% “core” that has similar speed and processivity with ␤ as E. coli

Tween 20, 0.01% Nonidet P-40 (open circles); 40% glycerol, 4 mM CaCl2 core. (open triangles); 40% glycerol, 0.01% Triton X-100, 0.05% Tween 20,

0.01% Nonidet P-40, 4 mM CaCl2 (half-filled diamonds). Differences to the E. coli pol III Replicase proteins in a thermophile which suggested they may use a Subunit Interactions—It is widely anticipated that the po- multicomponent replicase like E. coli pol III holoenzyme for lymerase and proofreading exonuclease activities will be chromosomal replication. Since those studies, the genomes of tightly associated in chromosomal replicases because of the several thermophilic organisms have been sequenced. These need for high fidelity to accurately duplicate an entire genome. genomes contain at least some of the genes encoding subunits Although A. aeolicus ␣ and ⑀ interact, the expectation that A. homologous to subunits of the E. coli pol III holoenzyme. This aeolicus ␣ and ⑀ would form a tight ␣⅐⑀ complex, as in the E. coli report confirms the presence of a working pol III machinery in system, was not met. Because replicases across the evolution- the hyperthermophile, A. aeolicus, by reconstituting the ma- ary spectrum contain or have tightly associated proofreading chinery from recombinant subunits. exonuclease activity, it seems likely that A. aeolicus ␣⅐⑀ forms a Studies in the E. coli pol III system showed that the ␥ (or ␶), tight complex in vivo. For example, the higher temperature at ␦, and ␦Ј subunits are required to load the ␤ sliding clamp onto which A. aeolicus grows may strengthen the interaction be- DNA (13). The A. aeolicus dnaX gene encoding ␶ was identified tween these two proteins. Alternatively, another protein may in the A. aeolicus genome report, but the holA and holB genes be required in A. aeolicus which serves as a brace, binding to encoding ␦ and ␦Ј were not (41). We noted in our earlier study both ␣ and ⑀, thereby stabilizing the ␣⅐⑀ contact (neither ␶ nor (38) of the S. pyogenes replicase that the ␦ subunit sequence ␶␦␦Ј served this function; data not shown). Finally, ␣ and ⑀ may was highly divergent from E. coli ␦. Sequence searches using associate tightly when present on a primed template. the E. coli ␦ and ␦Ј sequences yield weak matches to open Another difference to the E. coli system is the relatively low pol III Holoenzyme of Aquifex aeolicus 17347

affinity interaction between A. aeolicus ␣ and ␶. Although we A search of the A. aeolicus genome does not yield a significant show herein that A. aeolicus ␣ and ␶ directly interact, they do match to E. coli ␪, and in fact a search of the entire GenBankTM not form a complex that can be isolated by gel filtration as is yields only one significant match to ␪.2 readily demonstrated for E. coli ␣⅐␶ complex (32). In the E. coli In common with the genomic sequences of most other organ- system the C-terminal sequences of ␶ are required for binding isms, A. aeolicus shows no significant sequence matches to the to ␣ and the DnaB helicase (32–34, 40). In this regard, the A. E. coli ␹ and ␺ subunits of the clamp loader within DNA pol III aeolicus ␶ subunit is only 54.3 kDa, significantly less than E. holoenzyme. Biochemical studies in the E. coli system show coli ␶ (71.1 kDa) and only 7 kDa larger than E. coli ␥ (47.5 kDa), that these subunits are not required for clamp loading action and this discrepancy between the length of E. coli ␶ and A. (46, 54). What do these subunits do? The ␺ subunit binds to ␥ aeolicus ␶ is predominantly in the C-terminal region. For ex- and increases the stability of ␥␦Ј complex and the ␥␦␦Ј complex ample, the N-terminal 40 kDa of A. aeolicus ␶ contains the (54, 55). The ␹ subunit binds to ␺ in the ␥ complex clamp loader greatest extent of the homology to E. coli ␶ and corresponds to (54). The ␹ subunit also binds to SSB (56, 57). The ␹-to-SSB the sequences needed to bind ␦, ␦Ј, and ATP for motor protein contact is involved in displacement of primase from the RNA- function. However, it remains possible that A. aeolicus ␣-␶ primed site, thereby helping the polymerase and primase to interaction increases at the high temperatures where this ther- trade places on the RNA primer (51). This ␹-SSB interaction mophile lives. also aids clamp loading under conditions of elevated ionic SSB—The SSB generally stimulates its cognate DNA polym- strength, and stimulates polymerase elongation as well (56, erase. For example, T4 gp32 protein, E. coli SSB, and eukary- 57). A PsiBlast search of GenBankTM using E. coli ␹ and ␺ otic RPA all significantly enhance DNA synthesis by their sequences as queries shows a somewhat broader distribution of Downloaded from respective DNA polymerase holoenzyme. The basis for this homologues to these two proteins among different bacterial enhancement by SSB is generally believed to be the elimination species, although the list is still confined to only a few organ- of DNA secondary structure “road blocks” to polymerase trans- isms.3 It remains quite possible that yet other organisms may location. Theoretically, SSB should slow polymerase chain ex- contain homologues to these two subunits, but sequence

tension, because some of the energy of nucleotide polymeriza- changes, combined with their small size, may preclude their http://www.jbc.org/ tion must be expended to displace tightly bound SSB from identification by sequence comparison. This scenario is under- template ssDNA during its conversion to duplex. If SSB dis- scored by the difficulty of recognizing the essential ␦ subunit placement is rate-limiting, or partially rate-limiting, its dis- that is present apparently in all bacteria but difficult to iden- placement will slow the intrinsic rate of DNA synthesis. If this tify by sequence searches. is the case, the observed SSB stimulation of synthesis is prob- Lack of ␥ Subunit—Examination of dnaX genes in Gen- ably due to elimination of kinetic barriers (i.e. DNA hairpins) BankTM shows that the dnaX gene of some bacteria contains a at Rockefeller University Library on August 2, 2015 that exert much more effect on polymerase speed than the frameshift sequence but several others do not. A. aeolicus dnaX barrier of SSB displacement. Thus, the net effect of substitut- contains neither the ribosomal translational frameshift se- ing the SSB displacement barrier for the DNA secondary struc- quence nor the transcriptional slippage sequence. Consistent ture barrier is a stimulation of synthetic rate. with this, we demonstrate in this report that only the full- At high temperatures many DNA secondary structures may length protein, designated ␶, is detected by Western analysis of spontaneously melt. Therefore, for organisms that grow at high A. aeolicus whole cell extracts. Production of only the full- temperature, the DNA melting role of SSB may be less signif- length product of dnaX is by no means unique as we have icant to polymerase progression. Indeed, the results of this shown previously (38) that only ␶ is produced from dnaX in the report demonstrate that at elevated temperature, where most Gram-positive organism, S. pyogenes (as recombinant protein DNA secondary structure is probably eliminated, SSB has no in E. coli). significant effect on the efficiency of synthesis. Of course, it is The genes encoding clamp loader subunits in the T4 phage, possible that SSB may stimulate the hypothetical A. aeolicus yeast, human, and archaeal systems are not known to produce core polymerase (in the presence of ␤, ␶␦␦Ј). It seems likely that a truncated product. Hence, it may be more appropriate to ask SSB is needed for other important roles and thus is retained in why E. coli, and some other bacteria, go through the trouble of thermophiles even if there is no significant need to melt DNA producing a truncated product from dnaX. The evolution of hairpins. For example, SSB coating of ssDNA on the lagging both translational and transcriptional mechanisms to produce strand may be important to protect DNA against digestion by ␥ (E. coli and T. thermophilus, respectively) would suggest that nucleases. Also, E. coli SSB helps in ordering the handoff of an the truncated product plays an important role in the organisms RNA primer from primase to the DNA polymerase (51). This that produce it. However, genetic studies show that the dnaX primase-to-polymerase switch has been conserved in evolution gene in E. coli can be mutated such that ␥ is not produced, with and involves specific protein-protein contacts with SSB. SSB is no growth defects (58). This dnaX mutant strain should make also required in some types of DNA repair and recombination only the ␶ complex clamp loader, in which the three ␥ subunits (52). are replaced by three ␶ subunits. This should also have the effect of dedicating the clamp loader to the replication fork ␶ Similarities to Other Replicases because the C-terminal domains of will connect the clamp loader to core polymerases and the replicative DnaB helicase. Absence of ␹, ␺, and ␪ Subunits—Besides replicase subunits The fact that some bacteria have evolved frameshifting strat- that play catalytic roles in clamp loading and polymerization, egies to produce ␥ suggests that there is some role(s) for a ␥ the E. coli DNA pol III holoenzyme contains three small sub- complex clamp loader. For example, ␤ clamps must not only be units that are not absolutely required for these processes. The smallest of these, ␪ (8.6 kDa), associates directly with ⑀ subunit in the heterotrimeric pol III core polymerase (53). Absence of ␪ 2 The PsiBlast search of GenBankTM identifies a homologue to E. coli shows no defect in polymerase action or function of polymerase ␪ in Pasteurella multocida. 3 TM with the clamp loader and clamp in vitro (9, 53). Consistent The PsiBlast search of GenBank identifies homologues to both E. ␹ ␺ with this, the holE gene encoding ␪ can be deleted without coli and in Hemophilus influenzae, Pasteurella multocida, and Vibrio cholerae. In addition, ␹ homologues were present in Pseudomo- noticeable consequence to E. coli growth and viability (11). nas aeruginosa, Xylella fastidiosa, Neisseria meningitides, and Cau- Moreover, homologues to ␪ are not widespread among bacteria. lobacter crescentus. 17348 pol III Holoenzyme of Aquifex aeolicus loaded onto DNA but also need to be unloaded from DNA so 11. Slater, S. C., Lifsics, M. R., O’Donnell, M., and Maurer, R. (1994) J. Bacteriol. ␥ 176, 815–821 they can be reused (45). The complex has been shown to be 12. Fay, P. J., Johanson, K. O., McHenry, C. S., and Bambara, R. A. (1982) J. Biol. capable of removing ␤ clamps from DNA (21). Thus, a clamp Chem. 257, 5692–5699 loader that is physically separate from the replication fork may 13. Onrust, R., and O’Donnell, M. (1993) J. Biol. Chem. 268, 11766–11772 14. Dong, Z., Onrust, R., Skangalis, M., and O’Donnell, M. (1993) J. Biol. Chem. be important to clamp recycling. Furthermore, recent studies 268, 11758–11765 (59) reveal that ␤ interacts with all the E. coli DNA polym- 15. O’Donnell, M., Onrust, R., Dean, F. B., Chen, M., and Hurwitz, J. (1993) Nucleic Acids Res. 21, 1–3 erases and also with other proteins such as MutS and DNA 16. Neuwald, A. F., Aravind, L., Spouge, J. L., and Koonin, E. V. (1999) Genome ligase. Therefore, there may be circumstances in which there is Res. 9, 27–43 an advantage to having a clamp loader that is not dedicated to 17. Guenther, B., Onrust, R., Sali, A., O’Donnell, M., and Kuriyan, J. (1997) Cell 91, 335–345 the replication fork via strong attachment to ␣ and DnaB 18. Jeruzalmi, D., Yurieva, O., Zhao, Y., Young, M., Stewart, J., Hingorani, M., helicase. In this regard, we would like to make the correlation O’Donnell, M., and Kuriyan, J. (2001) Cell 106, 417–428 ␶ ␥ 19. Jeruzalmi, D., O’Donnell, M., and Kuriyan, J. (2001) Cell 106, 429–441 that , produced by organisms that do not make (i.e. A. 20. Onrust, R., Stukenberg, P. T., and O’Donnell, M. (1991) J. Biol. Chem. 266, aeolicus and S. pyogenes), does not form as tight a contact with 21681–21686 ␣ as observed (between ␣ and ␶)intheE. coli system. Hence, it 21. Turner, J., Hingorani, M. M., Kelman, Z., and O’Donnell, M. (1999) EMBO J. ␶ 18, 771–783 is conceivable that the resulting complex may not be as 22. Stewart, J., Hingorani, M. M., Kelman, Z., and O’Donnell, M. (2001) J. Biol. committed to action at a replication fork and thus would be Chem. 276, 19182–19189 available to act at other sites, either to recycle ␤ from DNA or 23. Naktinis, V., Onrust, R., Fang, L., and O’Donnell, M. (1995) J. Biol. Chem. 270, 13358–13365 to load ␤ onto DNA for use in different processes, such as repair 24. Hingorani, M. M., and O’Donnell, M. (1998) J. Biol. Chem. 273, 24550–24563 and recombination. 25. Bertram, J. G., Bloom, L. B., Turner, J., O’Donnell, M., Beechem, J. M., and Downloaded from Goodman, M. F. (1998) J. Biol. Chem. 273, 24564–24574 Thermophilic pol III and Its Use in Technology—Current 26. Bertram, J. G., Bloom, L. B., Hingorani, M. M., Beechem, J. M., O’Donnell, M., DNA amplification techniques make use of relatively distribu- and Goodman, M. F. (2000) J. Biol. Chem. 275, 28413–28420 tive single subunit DNA polymerases whose physiological role 27. Hingorani, M. M., Bloom, L. B., Goodman, M. F., and O’Donnell, M. 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erase I, than a chromosomal replicase. It seems quite likely http://www.jbc.org/ 29. Flower, A. M., and McHenry, C. S. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, that a rapid pol III-type enzyme endowed with a sliding clamp 3713–3717 for much higher speed, processivity, and fidelity would provide 30. Blinkowa, A. L., and Walker, J. R. (1990) Nucleic Acids Res. 18, 1725–1729 advantages over polymerases of a more distributive type. For 31. Tsuchihashi, Z., and Kornberg, A. (1989) J. Biol. Chem. 264, 17790–17795 32. Studwell-Vaughan, P. S., and O’Donnell, M. (1991) J. Biol. Chem. 266, example, a rapid and processive thermophilic DNA replicase 19833–19841 may yield larger products in “long chain PCR” applications and 33. Kim, S., Dallmann, H. G., McHenry, C. S., and Marians, K. J. (1996) Cell 84, could perhaps deliver more reliable performance in these pro- 643–650 34. Yuzhakov, A., Turner, J., and O’Donnell, M. 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Irina Bruck, Alexander Yuzhakov, Olga

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