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A Separate Editing Exonuclease for DNA Replication

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A Separate Editing Exonuclease for DNA Replication Proc. Nati. Acad. Sci. USA Vol. 81, pp. 7747-7751, December 1984 Biochemistry A separate editing exonuclease for DNA replication: The £ subunit of Escherichia coli DNA polymerase III holoenzyme (protein overproduction/fidelity of DNA replication/dnaQ gene) RICHARD H. SCHEUERMANN AND HARRISON ECHOLS Department of Molecular Biology, University of California, Berkeley, CA 94720 Communicated by I. Robert Lehman, August 20, 1984 ABSTRACT DNA polymerase Ill (polIII) holoenzyme of of the multisubunit polIII holoenzyme. To provide an excess Escherichia coli has 3' -* 5' exonuclease ("editing") activity in of E and facilitate its purification, we used an overproducing addition to its polymerase activity, a property shared by other strain that expresses the dnaQ gene under the control of a prokaryotic DNA polymerases. The polymerization activity is strong promoter, PL of phage X, and an efficient ribosome- carried by the large a subunit, the product of the dnaE gene. binding region. We have purified the E subunit of polIII to Mutations affecting the fidelity of DNA replication in vivo and homogeneity. We have found that E carries a 3'-* 5' exonu- the activity of 3' -* 5' exonuclease assayed in vitro are found in clease activity with characteristics closely similar to those of the dnaQ gene, which specifies the £ subunit. To determine purified polIII core enzyme. Thus, the editing and polymer- whether e carries the 3' -* 5' exonuclease activity, we have ization activities ofpolIII holoenzyme reside on distinct sub- used an overproduction protocol to purify £ separately from units. the other subunits of polIII holoenzyme. We find that £ has 3' -- 5' exonuclease activity indistinguishable from that of polIII MATERIALS AND METHODS core, the subassembly of polilI holoenzyme consisting of the a, Bacterial Strains and Plasmids. The E. coli strains used are e, and 0 subunits. We conclude that the editing and polymer- MC1000 lac deletion (12) and C600 (13). The plasmids used ization activities of polIll holoenzyme reside on distinct sub- are pRK248-cIts2-tet (14), pMC1403-lac'Z-amp (15), pRC23- units, in contrast to DNA polymerase I of E. coli and DNA XPL (16), pNS121-dnaQ and pNS221-XPL-dnaQ (8), and polymerase of phage T4. This functional separation may pro- pMD1, a pBR322 derivative containing the 1.6-kilobase-pair vide for regulation of exonucleolytic editing independently of (kbp) EcoRI dnaQ fragment. polymerization, allowing cellular control of replication fideli- Materials. M9 minimal medium and LB broth were the ty. standard recipes (17). The minimal medium was supplement- ed with 0.2% glucose, thiamine (20 mg/liter), and amino ac- Duplication of the Escherichia coli genome is an extremely ids (20 mg/liter). Where needed, ampicillin (100 mg/liter) accurate process: error frequencies are typically -10-9- and tetracycline (15 mg/liter) were added. Minimal plates for 10-10 per base replicated (1). This high fidelity is thought to the screening of Lac' colonies were spread with 5-bromo-4- occur through a multistage process: (i) selection of the com- chloro-3-indolyl-,3-D-galactoside (Sigma) (20 mg/ml in N,N- plementary base in the initial 5'-* 3' incorporation, (ii) exo- dimethylformamide) prior to use. Antibiotics and ADP were nucleolytic 3' -* 5' editing of a noncomplementary base at from Sigma. EcoRI linkers were from New England Biolabs. the growing point, and (iii) postreplicative mismatch repair. [3H]dCTP (58 Ci/mmol; 1 Ci = 37 GBq), [3H]dTTP (108 The summation of these steps could achieve the accuracy Ci/mmol), and [y-32P]ATP (>5000 Ci/mmol) were from observed (2-4). DNA polymerase III (polIII) holoenzyme is Amersham. (dT)18, poly(dA), and all other nucleotides were the primary enzyme involved in chain elongation in E. coli from P-L Biochemicals. Guanidine HCl was from Whittaker and is, therefore, likely to be the major determinant of fideli- (Delaware Water Gap, PA). DEAE-Sephacel was from Phar- ty (2, 3). To study fidelity mechanisms in DNA replication macia. Gel matrix Blue A was from Amicon. and explore the possibility that fidelity might be regulated, Enzymes. E. coli polIII core fraction VI (6) was generously we have been seeking to assess the contribution of polIII provided by C. McHenry. Calf thymus terminal deoxynu- subunits to base selection and editing. cleotidyl transferase was from P-L Biochemicals. T4 polynu- The polIII holoenzyme has at least seven subunits: a, E, 6, cleotide kinase was from Boehringer Mannheim. All restric- r, y, 8, and f3 (2, 5). The smallest subassembly of polIII holo- tion enzymes, T4 DNA ligase, BAL-31, E. coli DNA poly- enzyme prepared in native form is polIII core, which con- merase I (polI), and poll Klenow fragment were from New tains the a, E, and 0 subunits (6); a is the dnaE gene product England Biolabs. (7), and e is the dnaQ gene product (8). polIII core carries Preparation of Plasmid DNA and Transformation. Plasmid both the polymerase and the 3' -* 5' exonuclease activities DNA isolation and bacterial transformation were carried out ofpolIII holoenzyme (6). Enzyme assays with subunits sepa- as described (8). rated by NaDodSO4/PAGE have indicated that the large (a) Plasmid Constructions. To obtain overproduction of the E subunit has the polymerase activity and might also carry the protein, we first prepared a convenient derivative of the 3' -*5' exonuclease activity (9). However, an important role cloning vector pRC23 (16); this was done by replacing the for e in 3' -* 5' exonuclease (and replication fidelity) has 760-base-pair (bp) Pst I/EcoRI fragment of pMC1403 with been inferred from the observation that mutator mutations in the 1-kbp Pst I/EcoRI fragment of pRC23. The resultant the dnaQ gene render polIII holoenzyme defective in the plasmid pNS3 carries the PL promoter of phage X, a consen- editing exonuclease (10, 11). sus Shine-Dalgarno sequence for efficient translation, and To determine the role of E in exonucleolytic editing by pol- EcoRI, BamHI, and Sma I sites preceding a truncated lacZ III, we wanted to study E separately from the other subunits gene. Thus, insertion of a properly aligned dnaQ gene frag- ment within the polylinker sequence generates a ,3-galacto- The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: pollIl, DNA polymerase III; polI, DNA polymerase in accordance with 18 U.S.C. §1734 solely to indicate this fact. I; bp, base pair(s); kbp, kilobase pair(s). 7747 Downloaded by guest on September 29, 2021 7748 Biochemistry: Scheuermann and Echols Proc. Natl. Acad Sci. USA 81 (1984) sidase fusion protein containing the NH2 terminus of E pro- cated for 2 min to reduce the viscosity. After centrifugation tein subject to the efficient transcription and translation sig- for 30 min at 20,000 rpm in a Spinco type 30 rotor, the pellet nals of the plasmid vector. Once efficient expression of the was resuspended and washed twice with 100 ml of buffer S fusion gene was achieved, as judged by 03-galactosidase as- containing 1.0 M NaCl, washed once with 100 ml of buffer S, says, the intact dnaQ gene was reconstructed by inserting and resuspended with 25 ml of buffer S. This washed pellet the 3' end of the gene at the fusion site. fraction contained >95% of the e present in the harvested Plasmid DNA containing the dnaQ gene, pNS121 (8), was cells and only small amounts of other cellular proteins. How- digested to completion with EcoRI and Acc I and subjected ever, E remained in an insoluble form. to agarose gel electrophoresis, and the 1069-bp Acc IlEcoRI The E protein in the washed pellet fraction was converted fragment was isolated (18). From sequence analysis, this Acc to soluble form by a denaturation-renaturation protocol I site lies ==60 bp upstream from the ATG translational start with guanidine HCl. A portion (1.0 ml) was diluted 1:10 with signal of the dnaQ gene (19). The purified Acc I/EcoRI frag- buffer S, and an equal volume of 6 M guanidine HCl in buffer ment was treated with BAL-31 nuclease, after which the S was added. After the solution cleared, it was warmed to ends were filled in with polI Klenow fragment, and EcoRI room temperature and slowly diluted to 1 M guanidine HCl linkers were added by T4 DNA ligase (20). After treatment by the addition of buffer S. The resultant solution was dia- with EcoRI and removal of excess linkers, the fragments lyzed twice against buffer S containing 0.5 M NaCl and then were cleaved with EcoRI and BamHI and inserted into the against buffer S containing 50 mM NaCl. After this proce- EcoRI and BamHI sites of pNS3. The desired plasmids were dure, -40% of the e in the washed pellet was soluble and selected as Lac+AmprTetr transformants (at 300C) of the lac remained so during subsequent manipulations. This soluble deletion strain MC1000 carrying pRK248-cIts-tet. To ensure fraction was assayed for the presence of 3' -+ 5' exonuclease that the resultant plasmids carried the 5' end of the dnaQ activity for mispaired bases by the copolymer assay de- gene, plasmid DNA was checked for the presence of an scribed above; potent exonuclease activity was found HgiAI site that overlaps the translational start codon of (53,000 units/mg of protein). dnaQ (19). Strains that met this criterion were then exam- The soluble E fraction was diluted with buffer S to a con- ined for inducible expression of the fusion protein after a ductivity equivalent to 50 mM NaCl and applied to a DEAE- shift to 42°C to derepress the PL promoter. For the highest Sephacel column.
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