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).