Proc. NatL Acad. Sci. USA Vol. 80, pp. 2189-2192, April 1983 Biochemistry

Mutator strains of , mutD and dnaQ, with defective exonucleolytic editing by DNA 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). (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 ofEscherichia coli (polymerases I, II, DNA Replication. DNA replication with a phage G4 DNA and III) and phage T4 all have a 3' -* 5' 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. The lose chromatography partitions holoenzyme into the (8 subunit mutD5 and dnaQ49 mutations are closely linked, as judged by and an assembly containing all the other subunits, termed polIII* transduction frequencies with respect to an outside marker (refs. (20). For exonuclease assays, we have carried out a partial puri- 8, 9, 23; unpublished data). Because of the close linkage and fication ofpol III* that takes advantage ofchromatography on both similar (although not identical) mutator phenotypes, mutD5 and positively and negativelycharged matrices, DEAE-Sephacel, and dnaQ49 are likely to be different mutations in the same gene. phosphocellulose. For assay ofpol III* with G4 DNA, 3subunit Thus, we might expect to find an exonuclease deficiency for the was added to reconstitute pol III holoenzyme. Wild-type en- pol III from mutD5 bacteria if the lowered exonuclease that we zyme was prepared from strain JMI (21); dnaQ enzyme, from KH1116 dnaQ49 (9) (from M. Sekiguchi) and RS2, a transduc- tant of JM1 with the dnaQ49 mutation; and mutD enzyme, from LE30mutD5 (8) (from L. Enquist). Fraction II was prepared from 10-20 g of lysed cells by ammonium sulfate precipitation and backwashing, as described by McHenry and Kornberg (22). Fraction II was desalted on 5 ml of Sephadex G-25 and applied to DEAE-Sephacel in buffer D (50 mM TrisHCI, pH 7.5/1 mM EDTA/5 mM dithiothreitol/10% dextrose/10% glycerol) with 30mM NaCl. The column was washed with 60 and 150mM NaCl in buffer D and III was eluted buffer D with pol holoenzyme by E 300 mM NaCl (fraction III). Fraction III was concentrated by C. precipitation with ammonium sulfate, and then was desalted on 0' Sephadex G-25 and applied to a phosphocellulose column in CP buffer P (buffer D with 15% glycerol instead ofdextrose) with 20 O mM NaCl. The column was eluted with a NaClgradient (70-200 E mM) in buffer P; pol III* was eluted at about 110 mM NaCl 0 (fraction IV). Fraction IV was stored at -70°C in the elution buffer a- with 10% dextrose. The specific activities (pmol/min per mg of 0 234 protein) .of the fraction IV enzymes were: wild type, 53,000; mutD, 49,300; and dnaQ, 42,200. Under the same assay condi- tions, pol III* prepared from holoenzyme purified by the com- plete protocol of McHenry and Kornberg (22) had an activity of 180,000 pmol/min per mg ofprotein. RESULTS The dnaQ Enzyme: Exonuclease Deficiencyand Thermola- bility. The dnaQ49 mutation confers a drastic temperature-de- 0 2 ~~~~4 6 pendent increase in mutagenesis. Because the mutagenic effect Time, minutes might derive from a less active 3' -- 5' exonuclease, we have -compared the exonuclease activity of wild-type andidnaQ en- FIG. 1. Exonuclease defect of dnaQ pol III*. Wild-type or dnaQ pol zymes. For this purpose, we have measured the release of mis- m* was added to the synthetic copolymer (dT)17-([3H]dC)1.6/(dA)1,500; dCMP residues from the 3' end of a 17-base dT the 3'-- 5' exonuclease was measured by the remaining [3H]dCMP in paired oligonu- polymer form. The data presented are for equal units (12 units) of wild- cleotide under conditions in which the oligo(dT)-DNA is base- type and dnaQ enzyme based on the standard G4 replication assay. o, paired to long-chain poly(dA) (7, 11). For the exonuclease as- Wild-type enzyme at 300C;e, dnaQ at 300C; A, wild-type enzyme at 450C; says, we have used the pol HII*form ofpol III because ofthe rel- A, dnaQ at 450. Downloaded by guest on September 25, 2021 Biochemistry: Echols et al. Proc. Natl. Acad. Sci. USA 80 (1983) 2191

4 0 0~~~~~~~~~~ -d FIG. 2. EouesdftodaplmudroE 0 E

E' 6 a 0 0~~~~~~~~~~~~~~~~~~~~~ 0. d. W C.P - 0. C e 0 o

0 a

E 0 2-~~~~~~~~~~~~~~~~~~a c) a. ~~~~~~~~~~~~~~~~~~~~a- I tLa. ~0-2 CY V- I

2 4 6 Time, minutes O.

FIG. 2. Exonuclease defect of dnaQ pal under replication con- Time, minutes ditions. Wild-type or dnaQ enzyme was added to the synthetic copoly- mer, as in Fig. 1, but replication by pol occurred in the presence of FIG. 4. Exonuclease defect of mutD holoenzyme under replication [32P]dTTP (99 cpm/pmol) and single strand binding protein. A 2-fold conditions. The nuclease and replication assays were carried out as in excess of dnaQ enzyme over wild-type enzyme was added, based on the Fig. 2, except that pol Ill holoenzyme (pol II* and 1 subunit) was used standard G4 replication assay. o, Wild-type exonuclease; *, dnaQ exo- in the presence of ATP. A 2-fold excess of mutD enzyme over wild-type nuclease; A, wild-type DNA replication; A, dnaQ DNA replication. enzyme was added, basedon the standard G4 replication assay. o, Wild- type exonuclease; e, mutD exonuclease; A, wild-type DNA replication; have found for the dnaQ enzyme is responsible for the mutator *, mutD DNA replication. phenotype of dnaQ49 cells. The mutD enzyme is indeed highly defective in 3' -*5' exonuclease under a variety of assay condi- sented in Fig. 4. The pol III* form of the mutD enzyme is also tions. Comparative exonuclease activities for pol III* are shown defective in exonuclease activity under replication conditions (data in Fig. 3. Exonuclease activities under replication conditions for not shown). reconstituted holoenzyme (pol III* and ,B subunit) are pre- Editing Efficiency of dnaQ and mutD Enzymes During Replication with a G4 DNA Template. All of the exonuclease 10 assays presented so far have utilized the synthetic copolymer. To assay 3' 5' exonucleolytic activity under replication con- ditions with a natural DNA template, we have studied the con- version of dTTP to dTMP during replication ofphage G4 DNA. This "turnover" assay measures the production of dTMP by re- lease of incorporated dTMP from DNA by the 3' -- 5' exonu- 63 clease (18, 19, 24). For this assay, [32P]dTMP was separated from dTTP and DNA by PEI-cellulose chromatography; the ratio of

3 - dTMP released to dTMP incorporated into DNA measures the editing capacity of the polymerase (18, 19, 24). We have used E reconstituted holoenzyme (pol III* and J8 subunit) for these ex- periments. The turnover assays also indicate a defect in 3' -+ 5' exonuclease for the mutD and dnaQ enzymes (Table 1). Be-

a. Table 1. Exonuclease defect of mutD and dnaQ enzymes by E turnover assay dTMP Editing efficiency, dTMP in released, dTMP released/ Enzyme DNA, pmol pmol total dTMPt I .6_\ Wild type 22 1.3 0.059 ± 0.009 mutD 17 0.35 0.012 ± 0.009 dnaQ 20 0.63 0.028 ± 0.003 The replication reaction was carried out with a G4 DNA template. The dTMP numbers shown for a representative experiment have been I I expressed in terms of the total 25-,ul assay. 0 2 4 6 t Numbers are the mean ± SEM of five experiments for the wild-type Time, minutes and mutD enzymes and the mean ± SEM of three experiments for dnaQ. The background of dTMP in the original dTTP substrate was FIG. 3. Exonuclease defect of mutD pol m*. The assay was carried subtracted on the basis of an incubation with all of the components out at 30°C by the same protocol as in Fig. 1. o, Wild-type enzyme; *, of the replication reaction except pol II*, followed by PEI-cellulose mutD. chromatography; the background was about 0.3 pmol. Downloaded by guest on September 25, 2021 2192 Biochemistq: Echols et al. Proc. Natl. Acad. Sci. USA 80 (1983)

cause both mutD and dnaQ enzymes exhibit a decreased ca- view, we are intrigued by our preliminary indication for a pro- pacity for excision of a 3' nucleotide, we consider it highly likely tein that might control the editing capacity of pol III. that the enhanced mutation rates of dnaQ49, and mutD5 bac- Note Added in Proof. Since this paper was submitted, in collaboration teria result from a defective editing function of pol III. with R. Scheuermann we have concluded that the dnaQ gene product is the E subunit ofpol III holoenzyme, as judged by comigration in two- DISCUSSION dimensional gel electrophoresis. We have also learned that Bhatnagar et. al. have independently found that pol III from a mutD5 mutant is The Role of the mutD Gene in Editing. The mutD gene was defective in 3' -> 5' exonuclease (33). We note also that an editing de- discovered and has been extensively studied by Cox and co- fect for mutD5 polymerase was predicted earlier on the basis of the workers, who have suggested an involvement with DNA rep- frequency of transition and transversion mutations for wild-type and mutD5 strains lication (8, 25). In addition to mutD5, other closely linked mu- (34). tations have been isolated with a similar mutator phenotype (9, We thank Lynn Enquist and Mutsuo Sekiguchi for bacterial strains 23, 26). In the following discussion, we will make the likely but and we thank Arthur Kornberg and Robert Lehman for laboratory fa- unproved assumption that these are all mutations in the same cilities and much valuable advice. This work was supported in part by gene. The participation of the mutD protein in the replication grants from the American Cancer Society (ACS MV-131 to H.E.) and complex has been indicated by a variety of observations, ofwhich from the National Institutes of Health and the National Science Foun- H.E. was a Fellow at Stan- two are particularly pertinent. First, the dnaQ49 mutation im- dation (to Arthur Kornberg). Guggenheim ford University. P.M.J.B. was a Senior Fellow (S6-81) of the American pairs DNA replication in addition to its mutator phenotype (9). Cancer Society, California Division. Second, the dnaQ gene product appears to interact at least functionally with the a subunit of pol III because many sup- 1. Drake, J. W. (1969) Nature (London) 332, 1132. pressors of dnaE temperature-sensitive mutations (of Salmo- 2. Kornberg, A. (1980) DNA Replication (Freeman, San Francisco). are in Maurer, 3. Loeb, L. A. & Kunkel, T. A. (1982) Annu. Rev. Biochem. 51, 429- nella) dnaQ (R. personal communication). 458. Our biochemical experiments indicate that the decrease in 4. Echols, H. (1982) Biochimie 64, 571-575. replication fidelity found for mutations in the mutD gene de- 5. Speyer, J. F., Karam, J. D. & Lenny, A. B. (1966) Cold Spring rives from a defective 3' -+ 5' exonuclease component of pol Harbor Symp. Quant. Biol. 32, 345-350. III. The mutD5 case is particularly striking; the pol III is in- 6. Drake, J. W., Allen, E. F., Forsberg, S. A., Preparata, R. M. & distinguishable from wild type in temperature and salt sensi- Greening, E. D. (1969) Nature (London) 221, 1128-1131. tivity for G4 replication and in capacity to replicate 4X174 DNA 7. Muzyczka, N., Poland, R. L. & Bessman, M. J. (1972)J. Biol. Chem. 247, 7116-7122. (unpublished data). Thus, the mutD protein is likely to carry or 8. Degnen, G. E. & Cox, E. C. (1974)J. Bacteriol. 117, 477-487. control the 3' -* 5' exonuclease activity of the enzyme and af- 9. Horiuchi, T., Maki, H. & Sekiguchi, M. (1978) Mol. Gen. Genet. fect DNA replication only by the interaction with other pro- 163, 277-283. teins. Assays with subunits separated by acrylamide gel elec- 10. Rowen, L. & Kornberg, A. (1978) J. Biol. Chem. 253, 758-764. trophoresis have indicated that the a subunit of pol III has the 11. Brutlag, D. & Kornberg, A. (1972)J. Biol. Chem. 247, 241-248. 12. K. 0. & C. S. Biol. Chem. catalytic site for polymerization and also may have the 3' -> 5' Johanson, McHenry, (1980) J. 255, could not be 10984-10990. exonuclease (27). Although the gel experiments 13. Burgers, P. M. J. & Kornberg, A. (1983)J. Biol Chem., in press. definitive because of a difficulty of quantitative assay, the most 14. Wold, M. & McMacken, R. (1982) Proc. Natl. Acad. Sci. USA 79, direct interpretation of the available data is that the mutD pro- 4907-4911. tein has a control function for the activity of the 3' -* 5' exonu- 15. Weiner, J. H., Bertsch, L. L. & Kornberg, A. (1975)J. Biol. Chem. clease. By radiochemical identification of the product of the 250, 1972-1980. cloned dnaQ gene, the dnaQ protein has been assigned a mo- 16. Eisenberg, S. & Kornberg, A. (1979)J. Biol. Chem. 254, 5328-5332. 17. Randerath, K. & Randerath, E. (1967) Methods Enzymol. 12, 323- lecular mass of 25 kDa; this indicates that dnaQ might be the 347. e'subunit of pol III holoenzyme (28). A clarification of the con- 18. Hershfield, M. S. & Nossal, N. G. (1972)J. Biol. Chem. 247, 3393- tribution of the subunits of pol III to 3' -* 5' exonuclease will 3404. require experiments with separated subunits. 19. Fersht, A. R., Knill-Jones, J. W. & Tsui, W.-C. (1982) J. Mol. Biol. The Contribution of Exonucleolytic Editing to Fidelity of 156, 37-51. Replication. If the high mutation rates-for mutD5 and dnaQ49 20. McHenry, C. & Kornberg, A. (1981) in The Enzymes, ed. Boyer, P. D. (Academic, New York), Vol. 14, pp. 39-50. bacteria derive solely from a defective 3' -- 5' exonuclease, the 21. Mount, D. W. (1977) Proc. Natl. Acad. Sci. USA 74, 300-304. contribution of editing to the fidelity of replication by E. coli 22. McHenry, C. & Komberg, A. (1977)J. Biol. Chern. 252,6478-6484. is very large, in the 10 range. This fidelity contribution is con- 23. Cox, E. C. & Homer, D. L. (1982) Genetics 100, 7-18. siderably higher than that estimated from in vitro studies with 24. Clayton, L. K., Goodman, M. F., Branscomb, E. W. & Galas, D. pol III and polymerase I, which led to a 10-102 number (19). J. (1979)J. Biol. Chem. 254, 1902-1912. Our estimate might be high if base selection is also affected by 25. Fowler, R. G., Degnen, G. E. & Cox, E. C. (1974) Mol. Gen. Ge- net. 133, 179-191. mutD5 and dnaQ49; alternatively, the lower calculated fidelity 26. Horiuchi, T., Maki, H. & Sekiguchi, M. (1981) Mol. Gen. Genet. factor might suffer from the difficulties of in vitro analysis. 181, 24-28. Quantitative discrepancies aside, both analyses point to the im- 27. Spanos, A., Sedgwick, S. J., Yarranton, G. T., Hubscher, U. & portance of editing for the fidelity of DNA replication by E. Banks, G. R. (1981) Nucleic Acids Res. 9, 1825-1839. coli. Based on the combined data with pol III and T4 poly- 28. Horiuchi, T., Maki, H., Maruyama, M. & Sekiguchi, M. (1981) Proc. Natl. Sci. USA 3770-3774. merase, the importance of the 3' -* 5' nuclease for the fidelity Acad. 78, 29. Devoret, R., Blanco, M., George, J. & Radman, M. (1975) in of replication by prokaryotes seems to be well established. Molecular Mechanisms for Repair of DNA, ed. Hanawalt, P. & An interesting feature of replication fidelity is the increase Setlow, R. B. (Plenum, New York), pp. 155-171. in mutation rate associated with the SOS response to DNA 30. Ichikawa-Ryo, H. & Kondo, S. (1975)J. Mol. Biol. 97, 77-92. damage, an event which occurs to some extent even in the ab- 31. Witkin, E. M. & Wermundsen, I. E. (1979) Cold Spring Harbor sence of a DNA lesion ("untargeted mutagenesis") (4, 29-31). Symp. Quant. Biol. 43, 881-886. 32. A possible explanation for this phenomenon is a general control Echols, H. (1981) Cell 25, 1-2. 33. Bhatnagar, S. K., DiFrancesco, R. A., Brown, A. L. & Bessman, mechanism for replication fidelity, which provides for a higher M. J. (1983) Fed. Proc. Fed. Am. Soc. Exp. Biol., in press. mutation rate as a source of genetic variation for a population 34. Topal, M. D. & Fresco, J. R. (1976) Nature (London) 263, 285- under severe environmental stress (4, 32). From this point of 289. Downloaded by guest on September 25, 2021