Proc. NatL Acad. Sci. USA Vol. 80, pp. 2189-2192, April 1983 Biochemistry Mutator strains of Escherichia coli, mutD and dnaQ, with defective exonucleolytic editing by DNA polymerase III holoenzyme- (DNA replication/fidelity of replication/mutagenesis/proofreading) HARRISON ECHOLStt, CHI Lutf, AND PETER M. J. BURGERSt§ tDeartament of Molecular Biology, University of California, Berkeley, California 94720; and tDepartment of Biochemistry, Stanford University School of Medicine, StOrd. California 94305 Communicated by I. Robert Lehman, January 17, 1983 ABSTRACT The closely linked mutD and dnaQ mutations clease. We infer that the mutD (dnaQ) gene product controls confer a vastly increased mutation rate on Escherichia coli and the editing capacity of pol III. thus might define a gene with a central role in the fidelity of DNA replication. To look for the biochemical function of the mutD gene MATERIALS AND METHODS product, we have measured the 3' -* 5' exonucleolytic editing ac- tivity of polymerase m holoenzyme from mutD5 and dnaQ49 mu- Materials. Unlabeled deoxynucleoside triphosphates and the tants. The editing activities of the mutant enzymes are defective polymers (dA)1,5oo and (dT)17 were obtained from P-L Bio- compared to wild type, as judged by two assays: (i) decreased ex- chemicals. [3H]dTTP and [3H]dTMP were purchased from New cision of a terminal mispaired base from a copolymer substrate England Nuclear and Schwarz/Mann, respectively. [a-32P]dTTP and (i) turnover of dTTP to dTMP during replication with a phage was obtained from Amersham. Polyethylenimine (PEI)-cellu- G4 DNA template. Thus, the mutD (dnaQ). gene product is likely lose plates were from Machery-Nagel and DEAE-paper (DE81) to control the editing (proofreading) capacity of polymerase HI was from Whatman. DEAE-Sephacel was purchased from holoenzyme. Pharmacia and phosphocellulose (P11) was from Whatman. Phage G4 DNA was purified as described (10). (dT)17-([3H]dC)1.6, pre- Mutation rates are typically extremely low, about 10-9-10`1 pared by extension of (dT)17 with [3H]dCTP by terminal trans- per base replicated (1). This fidelity factor for duplication of the ferase (11), was annealed with (dA)1500 (in a 10:1 molar ratio of genome is astoundingly high, given that the correct and in- adenine to thymine) at 37°C for 15 min in 20 mM Tris-HCl, pH correct substrates are not strikingly dissimilar (e.g., a G'C pair 7.5/1 mM EDTA/100 mM NaCl. Holoenzyme (fraction V; 7 vs. a G-T). The vast reduction of error frequency compared to x 105 units/mg), pol II*, the subassembly ofholoenzyme lacking that expected from base-pairing energetics is thought to involve the /3 subunit (4 x 105 units/mg), and ,B subunit (5 x 106 units/ three stages of base selection: (i) the original incorporation of mg) were prepared as described (12, 13). Primase (1 X 106 units/ the complementary nucleotide; (ii) exonucleolytic proofreading mg) was prepared as described (10) from an over-producing strain ofthe newly added nucleotide; and (iii) postreplicative scanning (14). Single-strand binding protein (7 x 104 units/mg) (15) was for mismatched base pairs (2-4). the gift of D. Soltis. The DNA polymerases ofEscherichia coli (polymerases I, II, DNA Replication. DNA replication with a phage G4 DNA and III) and phage T4 all have a 3' -* 5' exonuclease activity template was used to monitor holoenzyme or pol III* activity (2). For phage T4, there is direct evidence for the biological during purification steps. The standard reaction mixture (25 1I) importance of this activity in the fidelity of DNA replication. contained: 20 mM Tris-HCl (pH 8.1), 8 mM dithiothreitol, bo- Phage mutant in the gene for DNA polymerase (gene 43) may vine serum albumin at 80 jig/ml, 4% glycerol, 8 mM MgCl2, exhibit either increased or decreased mutation rates compared 2 mM ATP, 100 mM (each) CTP, GTP, and UTP, 48 ,uM (each) to wild type (5, 6). For several mutants, the enzyme specified dATP, dGTP, and dCTP, 18 AM [3H]- or [a-32P]dTTP, 220 pmol by the mutator phage has decreased 3' -- 5' exonuclease ac- as nucleotide of G4 DNA, 20 ng of primase, 600 ng of single- tivity, whereas the polymerase encoded by the antimutator phage strand binding protein, and holoenzyme or pol III* with 4 ng has increased activity (7). of ,B subunit. After 10 min at 30°C, the reaction was stopped We have undertaken a combined genetic and biochemical by the addition of sodium pyrophosphate to 10 mM, and the analysis of the role of polymerase III (pol III) holoenzyme in incorporation of labeled dTMP into DNA was determined by mutation rate for several reasons: (i) the central involvement of precipitation with 10% trichloroacetic acid, followed by wash- this enzyme in the replication of the E. coli genome; (ii) the ing with 1 M HC1 and then, 95% ethanol (16). possible participation of additional components of the multi- Turnover Assay. To assay for turnover of dTTP into dTMP subunit enzyme besides the polypeptide responsible for base by the 3' -- 5' exonuclease activity of the polymerase, the G4 incorporation; (iii) the possibility that mutation rate might be reaction was carried out as above, except that [32P]dTTP was controlled at the enzyme level in response to environmental added at 9 ,M. The assay mixture contained 5-10 units ofwild- signals (as in the SOS response). type or mutant pol III* (see Enzyme Purification); the reaction In the work reported here, we have studied the pol III en- was allowed to proceed for 4 or 5 min. Under these conditions zyme from two mutator strains of E. coli, mutD5 (8) and dnaQ49 the incorporation of dTMP into DNA was linear for at least 8 (9); the closely linked mutations of these strains confer a large min. Free dTMP and DNA were fractionated by PEI-cellulose increase in mutation rate and have given prior indications for thin-layer chromatography ofa 5-,u aliquot (17-19), after which involvement in DNA replication. We have found that the pol III from both mutator strains is defective in 3' -> 5' exonu- Abbreviations: kDa, kilodaltons; PEI, polyethylenimine; pol III, poly- merase III; pol III*, the subassembly of pol III holoenzyme lacking the The publication costs of this article were defrayed in part by page charge P3 subunit. payment. This article must therefore be hereby marked "advertisement" 9Present address: Dept. of Biological Chemistry, Washington Univ. in accordance with 18 U. S. C. §1734 solely to indicate this fact. School of Medicine, 660 S. Euclid, Box 8094, St. Louis, Missouri 63110. 2189 Downloaded by guest on September 25, 2021 2190 Biochemistry: Echols et al. Proc. Natl. Acad. Sci. USA 80 (1983) the PEI-cellulose plate was sliced and radioactivity was deter- ative ease of preparation of partially purified pol III* free from mined by scintillation counting. For free dTMP, the chroma- polymerases I and II (2) (see Materials and Methods). The dnaQ tography solvent was 1 M formic acid/0.4 M LiCl; the dTMP enzyme shows a less effective exonuclease activity than wild type was located precisely by the use of marker [3H]dTMP in the at 300C (Fig 1); at 450C wild-type activity increases but dnaQ does same sample. For dTMP in DNA, the chromatography solvent not, indicating that exonuclease is thermolabile in the case of was 1 M formic acid/i M LiCl. dnaQ. Exonuclease Assay. For measurement of exonuclease with the To be sure that the pol III from dnaQ49 cells can act on the (dT)17-([3H]dC)1.6/(dA)1,50 substrate, the standard reaction synthetic substrate, we have also measured exonuclease activity mixture (125 ,1) contained: 20mM Tris'HC1 (pH 8.1), 8 mM di- under conditions in which replication can occur (Fig. 2). The dnaQ thiothreitol, bovine serum albumin at 80 tkg/ml, 4% glycerol, 8 enzyme is defective in exonuclease compared to wild-type en- mM MgCl2, 1,650 pmol as nucleotide of DNA, and pol III* as zyme, even for identical rates of DNA replication. To achieve indicated in the figure legends. For measurement of exonucle- equal rates of replication, a 2-fold excess of dnaQ enzyme over ase and DNA synthesis bypol III*, 100 ,.M [32P]dTTP and 3 ,g wild-type enzyme is required (based on the standard G4 DNA ofsingle-strand bindingprotein were added; for holoenzyme as- assay). Thus, replication from the proofreading template may be says, 2 mM ATP and 5 ng of (3 subunit were also added. During inhibited by slow removal of the mispaired base. The dnaQ49 incubation at 300C, 15-1Ld aliquots of the reaction mixture were mutation confers a slight thermolability on DNA replication in spotted on DEAE-cellulose paper (2 x 2cm squares). The squares vivo (9). We have not found a consistent thermolability for DNA were washed three times for 10 min in 500 ml of 0.3 M ammo- replication in vitro; some preparations ofpartially purified holo- nium formate (pH 7.8), rinsed with 95% ethanol, and dried, and enzyme from dnaQ49 exhibit a slight temperature sensitivity, but the radioactivity was determined by scintillation counting. others do not. Enzyme Purification. DNA pol III holoenzyme consists of a From the data of Figs. 1 and 2, we conclude that a defective "core" polymerase of a, e, and 0 subunits [140, 25, and 10 kilo- 3'-- 5' exonuclease activity can explain the mutator phenotype daltons (kDa), respectively] and accessory subunits (3, of 40 kDa, of the dnaQ49 strain. y, of 52 kDa, 8, of 32 kDa, and T, of 83 kDa (20). Phosphocellu- The mutD Enzyme: Drastic Exonuclease Deficiency.