5074–5082 Nucleic Acids Research, 2008, Vol. 36, No. 15 Published online 28 July 2008 doi:10.1093/nar/gkn489 The proofreading exonuclease subunit e of Escherichia coli DNA polymerase III is tethered to the polymerase subunit a via a flexible linker Kiyoshi Ozawa1, Slobodan Jergic1,2, Ah Young Park1, Nicholas E. Dixon1,2 and Gottfried Otting1,* 1Research School of Chemistry, Australian National University, Canberra ACT 0200 and 2School of Chemistry, University of Wollongong, NSW 2522, Australia Received June 5, 2008; Revised July 14, 2008; Accepted July 16, 2008 ABSTRACT exonuclease activity (4) and the y-subunit has no identified enzymatic activity (5). The a:e:y core complex is active Escherichia coli DNA polymerase III holoenzyme is alone as a proofreading DNA polymerase, and copurifica- composed of 10 different subunits linked by nonco- tion of these three subunits demonstrates their tight phys- valent interactions. The polymerase activity resides ical association (6,7). Direct interactions between e and a in the a-subunit. The e-subunit, which contains the (8) and e and y (5) have been demonstrated using purified proofreading exonuclease site within its N-terminal subunits, but no interaction has been detected between 185 residues, binds to a via a segment of 57 addi- a and y. tional C-terminal residues, and also to h, whose The y-subunit is not essential, as a ÁholE mutant is function is less well defined. The present study normally viable (9) and y has only a modest stimulatory shows that h greatly enhances the solubility of e effect on the exonuclease activity of e on a mispaired during cell-free synthesis. In addition, synthesis of primer terminus (5). Genetic studies with the temperature- e in the presence of h and a resulted in a soluble sensitive dnaQ49 mutant allele indicated that y stabilizes the structure of e (10), and this effect may also be achieved ternary complex that could readily be purified and by the y homolog HOT (homolog of theta) encoded by analyzed by NMR spectroscopy. Cell-free synthesis bacteriophage P1 (11). Remarkably, the phage genome of e from PCR-amplified DNA coupled with site- 15 (94 kb) relies on E. coli Pol III for its replication but, directed mutagenesis and selective N-labeling with the exception of the Ban (DnaB analog) DNA helicase provided site-specific assignments of NMR reso- and SSB (single-stranded DNA-binding protein), does not nances of e that were confirmed by lanthanide- encode any other replication proteins (12). induced pseudocontact shifts. The data show that Two crystal structures of a have been determined: of a the proofreading domain of is connected to a via a C-terminally truncated version of E. coli a (13), and of flexible linker peptide comprising over 20 residues. full-length Thermus aquaticus a (14). Structures are also This distinguishes the a : e complex from other known of the N-terminal globular domain of e (e186), proofreading polymerases, which have a more both alone (15) and in complex with HOT (16,17). The rigid multidomain structure. structure of the e186:y complex determined by nuclear magnetic resonance (NMR) spectroscopy (18,19) is in agreement with the e186:HOT structure. No structure has been determined of full-length e but residues following e186 within its C-terminal region (in the following referred INTRODUCTION to as C-terminal segment (CTS) of e or eCTS; Figure 1) The DNA polymerase III (Pol III) holoenzyme is the are known to be responsible for binding of a (20,21). This major chromosomal replicase in Escherichia coli (1). This 57-residue segment contains a Q-linker sequence proposed enzyme complex is composed of 10 different polypeptide to provide a flexible tether between domains (22), followed subunits. The catalytic core contains one each of the a by the C-terminal 40-residue segment that has been shown (130 kDa), e (27 kDa) and y (8.8 kDa) subunits encoded to interact tightly with a when fused to maltose-binding by the dnaE, dnaQ and holE genes, respectively. The protein (21). a-subunit contains the 50–30 DNA polymerase active site The present study was carried out to obtain structural (2,3), the e-subunit is responsible for 30–50 proofreading information about the a:e:y complex. By studying the e:y, *To whom correspondence should be addressed. Tel: +61 2 61256507; Fax: +61 2 61250750; Email: [email protected] ß 2008 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Research, 2008, Vol. 36, No. 15 5075 1 MSTAITRQIV LDTETTGMNQ IGAHYEGHKI IEIGAVEVVN RRLTGNNFHV 15 NH4Cl (Cambridge Isotope Laboratories, Andover, 51 YLKPDRLVDP EAFGVHGIAD EFLLDKPTFA EVADEFMDYI RGAELVIHNA 101 AFDIGFMDYE FSLLKRDIPK TNTFCKVTDS LAVARKMFPG KRNSLDALCA MA, USA) as described (27). T7 RNA polymerase (28) 151 RYEIDNSKRT LHGALLDAQI LAEVYLAMTG GQTSMAFAME GETQQQQGEA and e186 (25) were as described. Concentrations of samples 201 TIQRIVRQAS KLRVVFATDE EIAAHEARLD LVQKKGGSCL WRA of soluble pure proteins (a, e186 and y) were determined spectrophotometrically at 280 nm, using calculated values Figure 1. Amino acid sequence of full-length e. Residues 7–180 have a defined conformation in the crystal structure of e186 (15). The 57 of e280 of 95440, 7680 and 8250/M/cm, respectively (29). residues of the eCTS are highlighted in bold. Secondary structure pre- diction suggests an a-helical segment in the eCTS with high propensity; Cell-free synthesis of the e-subunit the corresponding residues are underlined. Alanine and threonine resi- dues in the CTS of e are highlighted in red and yellow, respectively. S30 cell extracts from either E. coli strain Rosetta::DE3/ pRARE (from Novagen, Gibbtown, NJ, USA) or BL21Star::DE3 (from Invitrogen, Carlsbad, CA, USA) a:e and a:e:y complexes and probing potential weak inter- were prepared by the procedure of Pratt (24,30,31), fol- actions between a and e186 by a novel two-pronged NMR lowed by concentration with polyethylene glycol 8000 as assignment strategy, we show that residues in the eCTS described by Kigawa et al. (31,32), and were used inter- are flexible and that those in the Q-linker region remain so changeably. Cell-free protein synthesis was carried out for even when assembled in the Pol III core complex. This 6–7 h either using an autoinduction system that uses plas- provides the first experimental evidence that e is indeed mid pKO1166 to direct production of T7 RNA polymer- tethered to a by a flexible peptide linker, suggesting a ase in S30 extracts (33) at 378C, or a standard coupled mechanism for transition between polymerization and transcription/translation system with purified T7 RNA proofreading modes in Pol III that is fundamentally dif- polymerase at 30 or 378C as described previously ferent from those in other polymerases whose structures in (28,31). The plasmid template pSH1017 (25) was used at both modes are known. In addition, experiments to pro- a concentration of 16 mg/ml for production of e. All reac- duce e by cell-free protein synthesis shed further light on tion mixtures containing expressed proteins were clarified the function of y. by centrifugation (30 000g, 1 h) at 48C. Cell-free synthesis of selectively 15N-labeled e in complex MATERIALS AND METHODS with h Materials Samples of the e:y complex containing 15N-Ala or 15N-Thr L-[15N]Alanine and L-[15N]threonine were obtained from labeled e were prepared by synthesis of e in the cell-free Spectra Stable Isotopes. Synthetic oligonucleotides were system in the presence of separately purified unlabeled y purchased from GeneWorks (Hindmarsh, SA, Australia); (0.5 mg/ml), with 0.6 ml reaction mixtures at 308C for 7 h. sequences of oligonucleotides used are listed in Table S1. Following centrifugation, the supernatant was dialyzed against 2 l of NMR buffer (20 mM Tris–HCl, pH 6.9, Protein purification 1 mM EDTA, 1 mM dithiothreitol) and concentrated to For large-scale isolation of a, E. coli cells (BL21::DE3 a final volume of about 0.5 ml using Millipore Ultra-4 recA) harboring the plasmid pND517 encoding the dnaE centrifugal filters (molecular weight cutoff 10 000). D2O gene under control of a tac promoter (23) were grown was added to a final concentration of 10% (v/v) prior to aerobically at 308C and in 20 l of Z medium of pH 7.3 NMR measurements. (24) supplemented with 100 mg/l ampicillin in a fermenter Cell-free synthesis and purification of the a:e:h complexes with pH control (BIOSTAT C, B. Braun Biotech with selectively 15N-labeled e International, Melsungen, Germany). Overexpression of 15 a was induced by addition of 0.5 mM isopropyl-b,D-thioga- Samples of the a:e:y complex containing N-Ala or 15 lactoside at A595 = 1.5 and the induced culture was grown to N-Thr labeled e were prepared by cell-free synthesis of an A595 of 10 (4 h), yielding about 250 g of cells. The e in the presence of separately purified unlabeled y a-subunit was purified from 54 g of cells using a modified (0.5 mg/ml) and a (5.0 mg/ml). Reaction mixtures (0.9 ml) version of the procedure of Wijffels et al. (23), with two steps were incubated at 308C for 7 h. Following centrifugation, of chromatography on columns of DEAE-Toyopearl 650 M the supernatant was dialyzed against 2 l of buffer A (2.5 Â 45 cm) and phosphocellulose (5.5 Â 22.5 cm).