JOURNAL OF BACTERIOLOGY, Mar. 1992, p. 1974-1982

Vol. 174, No. 6

0021-9193/92/061974-09$02.00/0 Copyright X) 1992, American Society for Microbiology

An Escherichia coli dnaE Mutation with Suppressor Activity toward Mutator mutD5 ROEL M. SCHAAPER* AND ROBIN CORNACCHIO Laboratory ofMolecular Genetics, National Institute of Environmental Health Sciences, P.O. Box 12233, 111 T. W. Alexander Drive, Research Triangle Park; North Carolina 27709 Received 18 September 1991/Accepted 16 January 1992

The Escherichwa coli mutator mutDS is a conditional mutator whose strength is moderate when the strain is growing in minimal medium but very strong when it is growing in rich medium. The primary defect of this strain resides in the dnaQ gene, which encodes the e (exonucleolytic proofreading) subunit of the DNA polymerase HI holoenzyme. In one of our mutD5 strains we discovered a mutation that suppressed the mutability of mutD5. Interestingly, the level of suppression was strong in minimal medium but weak in rich medium. The mutation was localized to the dnaE gene, which encodes the a (polymerase) subunit of the DNA polymerase III holoenzyme. This mutation, termed dnaE910, also conferred improved growth of the mutD5 strain and caused increased temperature sensitivity in both wild-type and dnaQ49 backgrounds. The reduction in mutator strength by dnaE916 was also observed when this allele was placed in a mutL, a mutT, or a dnaQ49 background. The results suggest that dnaE910 encodes an antimutator DNA polymerase whose effect might be mediated by improved insertion fidelity or by increased proofreading via its effect on the exonuclease activity.

ficity of mutagenesis in mutD strains. In MM, a majority of mutations are transversion base substitutions, while in rich medium they are mostly transitions (25); a predominance of transition mutations is characteristic of strains defective in mismatch repair (29). The hypothesis was confirmed by directly measuring mismatch repair by transfection of heteroduplex M13 DNAs in mutD5 cells (25, 26). When grown in rich medium, mutD cells could not repair heteroduplex molecules, but when grown in MM, they could. Similar results were obtained for transfections with bacteriophage lambda heteroduplexes (7). Thus, the main difference between the mutability of mutD strains in rich medium and that of mutD strains in MM appears to be the presence or absence of mismatch repair, this difference quantitatively accounting for the 100- to 1,000-fold difference in mutability between the two media. The precise reason why mutD strains are deficient in mismatch repair in rich medium is not entirely clear. One possibility is that the dnaQ-encoded 3' exonuclease is directly required for mismatch repair. This hypothesis, if correct, would require further delineation of why this requirement would be different in different media. A second hypothesis, supported by several lines of evidence, is that the deficiency of mismatch repair is transient and caused by an overwhelming of the capacity of the mismatch-repair system by the high level of DNA replication errors in mutD cells (30). This saturation model assumes that one or more components of the mutHLS mismatch-repair system is in relatively short supply or perhaps consumed during the repair reaction. It is supported by the observation that mismatch repair can be restored in rich medium by stopping or slowing down DNA replication, giving the cell an opportunity to replenish the supply of mismatch-repair proteins. Likewise, introducing a multicopy plasmid carrying the mutL+ or mutH+ gene restored mismatch repair to cells growing in rich medium and substantially reduced the mutation rate (30). The saturation model, while attractive, does not explain why saturation occurs in rich medium but not in MM. The

Among the mutators of the bacterium Escherichia coli, mutator mutD5 (5, 8) takes a special place for two reasons. First, it is an extraordinarily strong mutator, mutation rates being elevated up to 105-fold above the wild-type level. Second, its mutation rate is conditional upon the medium in which it grows. The very high mutation rates are obtained with rich medium (such as Luria-Bertani [LB] broth), but only moderate mutation rates (102-fold elevation) are obtained with a minimal medium (MM). The mutD5 mutation resides in the dnaQ gene (20, 35). This gene encodes the e subunit of the DNA polymerase III holoenzyme (9, 31, 32), which is responsible for replicating the bacterial chromosome (21). The £ subunit provides the 3' exonucleolytic proofreading (editing) activity (31), while the ao subunit encoded by the dnaE gene provides the polymerase activity (37). The a and e subunits bind tightly together (with the 0 subunit) in the holoenzyme core (21). The a and e subunits are the main determinants of fidelity, a because of polymerase insertion fidelity and £ because of exonucleolytic removal of incorrectly inserted nucleotides. Together, they provide a fidelity of -10' error per base pair replicated (12, 28). Following polymerization, the overall fidelity of DNA replication is increased to about 10-10 by the methylationinstructed DNA mismatch repair system, encoded by the mutH, mutL, and mutS genes (4, 23, 24). This system is capable of recognizing and correcting mismatches resulting from replication errors, distinguishing the correct half from the incorrect half of the mismatch by way of the transient undermethylation of adenine (at 5'-GATC-3' sites) in the newly synthesized strand. The absence of exonucleolytic proofreading in a mutD5 strain (10) can, however, provide only a partial explanation for the strong mutator effect of this strain. Experiments from our laboratory (25, 26) and that of others (7) have indicated that the very high levels of mutability in rich medium result from an additional deficiency in mutHLS-encoded DNA mismatch repair. This was initially suggested by the speci*

Corresponding author. 1974

dnaE SUPPRESSOR OF MUTATOR mutD5

VOL. 174, 1992

1975

TABLE 1. Bacterial strains and plasmids Strain

Genotype [derivation]a

Source or reference

Bacteria CD4 ES1293 KA796 KH1214 NR9073 NR9082 NR9455 NR9458 NR9515 NR9516 NR9670 NR9675 NR9681 NR9686 NR9694 NR9695 NR9728 NR9729 NR9803 NR9812 NR9813 SG13082

HfrC metD88 proA3 A(lacI-Y)6 tsx-76 X- reLAI mai436(Xr) metBI mutL::TnS ara thi Aprolac mutD5 zaf-13::TnlO ara thi mutDS zaf-13::TnlO dnaE910 Aprolac F' prolacl28-27 KA796, but mutTI azi KA796, but mutDS zaf-13::TnlO dnaE910 [Pl(NR9073) x KA796] KA796, but mutD5 zaf-13::TnlO [Pl(NR9073) x KA796] CD4, but mutDS zaf-13::TnlO metD+ dnaE910 [Pl(NR9455) x CD4] CD4, but mutDS zaf-13::TnlO [Pl(NR9455) x CD4] NR9082, but leu::TnlO [Pl(SG13082) x NR9082] CD4, but metD+ dnaE910 (Pl(NR9455) x CD4] CD4, but metD+ [Pl(NR9455) x CD4] NR9675, but mutL::TnS [Pl(ES1293) x NR9675] CD4, but mutL::TnS [Pl(ES1293) x CD4] KD1088, but dnaQ49 zae-502::TnJO CD4, but muTI leu::TnJO [Pl(NR9670) x CD4] NR9675, but mutTI leu::TnlO [Pl(NR9670) x NR9675] CD4, but dnaQ49 zae-502::TnlO pro' [Pl(NR9695) x CD4] NR9675, but dnaQ49 zae-502::TnlO pro' dnaE+ [Pl(NR9695) x NR9675] NR9675, but dnaQ49 zae-502::TnlO pro' dnaE910 [Pl(NR9695) x NR9675] suiA366 his leu::TnlO ion-100

B. Bachmann E. Siegel (33) 28 E. Cox (6) This work 28 This work This work This work This work This work This work This work This work This work R. Fowler This work This work This work This work This work S. Gottesman

E. coli dnaQ+(mh+) inserted into EcoRI site of pBR322 E. coli dnaE+ inserted into EcoRI site of pBR325

BRLb H. Maki (20) H. Maki

Plasmids pBR322 pMM5

pMK9

Derivation indicates the donor and recipient of P1 transduction used to construct the strain. b BRL, Bethesda Research Laboratories.

a

most straightforward explanation is that saturation is a function of the growth rate of the bacterium. However, this is less likely, on the basis of the observation of Degnen and Cox (8) that at identical generation times in the two media, rich-medium cells still mutate several orders of magnitude more frequently than their MM counterparts. It remains possible, however, that the different media differentially affect the expression of key repair and replication proteins, so that saturation is achieved in one medium and not in the

other. Alternatively, specific components of rich media may affect the mutational state of the cell. One component that appears to act in this manner is thymidine, which, when added to MM, greatly enhances mutability (although not to the rich-medium level). An effect of one other, as yet unidentified component of broth has been suggested previously (11). The mechanisms by which such components could act are unclear, although the discovery of a dTTP binding site on the PolII E subunit (3), together with the requirement that thymidine be phosphorylated before it can exert its effect (11), has suggested that some regulatory aspect of the proofreading activity may be involved. This paper concerns the discovery of an apparently adventitious mutation in one of our laboratory mutD5 stocks. This mutation was characterized by its rather dramatic effect on the mutability of mutD5 in MM, its effect in rich medium being small. Because this mutation might shed further light on the mechanisms underlying the mutD mutator effect, its nature was investigated. Here we demonstrate that it resides in the dnaE gene and exerts a strongly antimutagenic effect in MM, presumably by reducing DNA replication errors. Possible reasons why this effect appears limited to MM will be discussed.

MATERIALS AND METHODS Strains and media. The E. coli strains used in this study and their derivations are listed in Table 1. All P1 transductions involving mutDS strains were done on MM to ensure maximum genetic stability. MM consisted of lx VogelBonner salts (36), 0.4% glucose, 5 jig of thiamine per ml, and 50 ,ug of amino acids per ml as required. LB broth contained 10 g of Bacto Tryptone (Difco) per liter, 5 g of yeast extract (Difco) per liter, and 10 g of NaCl per liter. Salt-free broth was the same, except that the NaCl was omitted. The following antibiotics were added at the following concentrations: rifampin (RIF), 100 ,ug/ml; nalidixic acid (NAL), 40 ,ug/ml; streptomycin, 200 ,ug/ml; ampicillin (AMP), 50 ug/lml; tetracycline (TET), 15 ,ug/ml; kanamycin (KAN), 25 ,ug/ml. Solid media contained 1.5% agar. Strain constructions. P1 transductions were performed by using PlvirA according to the method of Miller (22). The mutD5 allele was cotransduced with the zaf-13::TnlO insertion, selecting for tetracycline resistance on MM-TET, followed by screening of the transductants for mutator phenotype by toothpicking single colonies into 1 ml of LB-TET and spotting 3 ,ul of the saturated cultures on an LB-RIF or an LB-NAL plate. mutD strains typically produce 50 or more mutants per spot, while wild-type strains produce none. Class I- and class II-type transductants (see Results) were distinguished by first restreaking transductants for single colonies on MM and then toothpicking one or two colonies of each into 1 ml of MM. After growth overnight (37°C), 0.1 ml of the cultures was spread on MM-NAL plates. No significant differences between class I and class II in final cell count are observed. Class I transductants produce about 20 to 40 nalidixic-acid-resistant mutants per

1976

SCHAAPER AND CORNACCHIO

plate, while class II transductants produce about 400 to 800 nalidixic-acid-resistant mutants per plate. mutL::TnS was introduced on the basis of its kanamycin resistance; dnaQ49 was introduced by cotransduction with zae-502::TnlO (tetracycline resistance); and mutTI was introduced by cotransduction with leu::TnlO (tetracycline resistance). The presence of the mutator function was determined via spot tests as described above. Strains NR9675 and NR9681 (Table 1) were constructed by P1 transduction selecting for metD+ and then tested further as described in the text. Transformations. Transformation with plasmids pBR322, pMM5, and pMK9 was performed by electroporation with a Bio-Rad Gene Pulser apparatus and protocol provided by the manufacturer and with MM-AMP plates to select the transformants. Mutagenesis measurements. All mutD and dnaQ49 strains were maintained on MM plates. To measure mutation frequencies, 6 to 15 colonies were toothpicked into 1 ml of liquid medium, either LB or MM, and grown overnight with agitation. The temperature was 37°C unless otherwise indicated. To counter the effects of possible secondary mutations in the mutator strains that might affect mutation rates, colonies were usually taken from two to four independent isolates of each strain. Aliquots of appropriate dilutions of the saturated cultures were then plated to determine both the total cell count and the frequency of rifampin-, nalidixicacid-, and/or streptomycin-resistant mutants. If prior growth was in LB medium, all plates were of the LB type. If prior growth was in MM, MM plates were used. Measurements of temperature sensitivities. For each isolate to be tested, a saturated culture was diluted 104-fold in 0.2% NaCl to yield -105 cells per ml. About 100 cells in 1 ,ul were then spotted on LB or salt-free LB plates. The plates were incubated overnight at various temperatures and scored the next morning for growth. RESULTS Two distinct classes of mutator mutDS strains derived from mutD5 donor strain NR9073. In the course of experiments investigating the mechanisms responsible for the mutD mutator effect, we isolated on several occasions new mutD5 isolates by P1 transduction with one particular strain, NR9073 (mutD5 zaf-13::TnlO), as a donor strain. Selection was for the tetracycline resistance conferred by the zaf-13::TnlO insertion (5) and was followed by screening for a mutator phenotype. Because of the close linkage between the dnaQ gene (where mutDS resides) and the zaf-13 locus, more than 90% of the transductants are also mutator (5). However, a closer examination of several such new isolates suggested that they did not represent a homogeneous group. Judging from spot tests, all isolates had similar high levels of mutability in rich medium, but in MM their levels of mutability, while lowered in all cases, appeared to fall into two distinct classes. These were called class I (lower level of mutability) and class II (higher level of mutability). The phenomenon was further investigated by more accurately determining their mutation frequencies. Representative results are shown in Table 2. It is concluded that indeed two classes of mutD mutators which differ specifically in their mutability in MM are obtained. The effect is true for several mutational markers, including Rif' and Nalr (Table 2), as well as valine resistance or forward lacI mutagenesis (data not shown). On the basis of several experiments, the magnitude of the class I-class II difference for Nalr in MM is 20- to

J. BACTERIOL.

TABLE 2. Differential mutability of two classes of mutator mutD5 isolates No. of mutants per 106 cellsb

Rich medium

Classa

Expt

Rifr

1

119 53

I II

2

44 21

NDC ND

I II

MM

Nal

72 66

41 31

2.4 11.3

33±10 48

Nalr

Rif

0.05 ± 0.03 1.5 0.3

1.1 4.0

±

0.11±0.09 5.4 ± 2.6

ND ND

21

a Strains used were NR9455 (class I) and NR9458 (class II). Mutants resistant against rifampin (Rif) or nalidixic acid (Nalr). Frequencies (± standard deviation) are averages of six independent cultures. c ND, not determined. b

50-fold, whereas this difference between the two classes in rich medium is generally twofold or less. To further examine the origin of the two classes of mutators, a larger-scale experiment in which some 80 mutators derived from the above transductions were measured for their mutability toward Nalr when growing in MM was performed. The results are presented in Fig. 1. A strikingly bimodal distribution was obtained, confirming that this transduction indeed yielded two classes of mutators. About 40% of the transductants appeared to be class I and about 60% appeared to be class II. Over several experiments, the class I mutators were 40 to 55%. We hypothesized that the mutD5 donor strain NR9073 carried, in addition to mutDS, a second (linked) mutation that modifies the mutability of mutDS in MM and that, upon transductional transfer, either remains linked or is lost, thus giving rise to the two classes.

a

U 0 0 0

10

E z

0 C W-

46

1 comq .e at 8 a 8 1 6 9 9 9 v-

v-

cm

v-

v-

v-

40

cm

4 8 CO

§

8

Nal mutants per culture FIG. 1. Distribution of mutation frequencies among 80 mutD5 isolates obtained after transductional transfer of the mutDS allele from donor NR9073 into wild-type strain KA796. One purified colony of each mutator isolate was grown to saturation in MM and 0.1 ml of the culture was plated on MM containing nalidixic acid to determine the number of Nair mutants.

VOL. 174, 1992

dnaE SUPPRESSOR OF MUTATOR mutD5

1977

FIG. 2. Colony sizes of class I (A) and class II (B) mutDS mutators. The parent strain CD4 (C) is shown for comparison. Strains were grown on LB, but similar differences are seen with MM.

Two possibilities for this second mutation were envisaged: an antimutator mutation, reducing the mutability of mutD5 (retained in the class I transductants), or a mutator mutation, enhancing the mutability of mutD5 (retained in the class II

transductants). Different growth rates of class I and class II isolates. After the distribution among the two classes had been established, a strong correlation between the class of each mutator and its growth rate on plates, as judged by colony size, was noted. Class I mutator colonies grew almost as well as the parental wild-type strain, whereas class II mutators lagged considerably behind, forming smaller colonies which were, in general, somewhat heterogeneous in size (see Fig. 2). The difference in colony size is readily observed at early times (ca. 10 h for the plates shown in Fig. 2) but less noticeable on plates that are incubated for longer times. This observation may account for the fact that a small-colony phenotype has not been normally attributed to mutD5 strains. The difference in colony size was observed on both LB and MM plates and in both the CD4 and KA796 backgrounds (Table 1), although it was more pronounced for CD4, presumably because CD4 is a slightly more slowly growing strain. A measurement of doubling times in liquid medium (LB, 37°C) yielded 33.3 (+1.1), 33.8 (±0.6), and 37.9 (±1.9) min for CD4 and its classes I and II mutD5 derivatives, respectively. Class I mutDS derivatives carry an antimutator mutation. To test whether the second mutation was of the mutator or antimutator phenotype, another set of P1 transductions was performed, now with a class I and a class II isolate as the

mutDS donor. For each transduction, 96 transductants were purified and the mutators among them were tested for their mutability to Nalr in MM. The results are presented in Fig. 3. The class I donor produces the bimodal distribution observed before (class I and class II), but the class II donor produces predominantly the more frequently mutating class (class II). We conclude that the class I mutator carries the second mutation, which must be considered an antimutator mutation. Mapping the antimutator mutation to the dnaE gene. Approximate mapping of the mutation responsible for the class I phenotype was undertaken by P1 transductions with a class I mutator as a donor and strain CD4 as a recipient. Strain CD4 carries the metD mutation, the metD gene being located 0.5 min to the left of the dnaQ gene on the E. coli map (see Fig. 4). CD4 can therefore be used to determine the location of the antimutator mutation relative to metD and dnaQ. As the transductional data of Fig. 4 indicate, the mutation resides to the left of metD in the approximate region of dnaE. Because dnaE encodes the DNA polymerase subunit of PollI holoenzyme (37), it is a prime candidate for hosting the antimutator mutation. To test whether the mutation resides in dnaE, we performed a complementation experiment using a plasmid that carries the dnaE+ gene. The results (Table 3) show that whereas the class I and class II isolates differ significantly in their mutability without any plasmid or in the presence of pBR322, this difference disappears in the presence of plasmids pMK9(dnaE+) and pMM5(dnaQ+). The dnaQ+ plas-

1978

SCHAAPER AND CORNACCHIO

J. BACTERIOL.

(zae502::.Tn 10)

7

40

dnaE 4.4

30

metDj I 4.7

a

zaf-13::Tn 10

dnaO I

5.2

4.9

2

0

(84)

20

(31) (50)

E z

10

cm

0o

t Nts

0o

0

0

0

_

V_O 0

0

0

0

r

Nal mutants per culture FIG. 3. Distribution of mutation frequencies among mutators resulting from transductions with a class I or a class II mutD5 mutator as donor. Strains NR9455 and NR9458 were the respective class I and class II donors, and CD4 was the recipient. Ninety-five and 92 transductants were tested as described in the legend to Fig. 1.

mid cancels the mutD mutator activity because of the recessive nature of mutDS when faced with a high-copy mutD+ plasmid (6). Interestingly, the dnaE+ plasmid specifically increases the mutation frequency of the class I mutator to the level of the class II mutator. We cannot exclude the possibility that this represents a case of extragenic suppression. However, the simplest explanation for this and the mapping experiment is that the antimutator effect results from a recessive mutation in the dnaE gene. The mutation was designated dnaE910 (2). Properties of dnaE910 in the absence of mutD5. To gain further insights into the properties of dnaE910, we investigated whether it was possible to separate it from mutDS. This was done by transducing strain CD4 with P1 phage derived from a dnaE910 mutDS strain (class I), selecting metD+ colonies, and testing for their mutator phenotypes. We expected that a significant proportion of the metD+ nonmutators would carry dnaE910 (Fig. 4). To test whether this was the case, a total of 11 such metD+ nonmutators were then made zaf-13::TnlO mutDS with P1 phage from a class II mutator. The progeny of these transductions was then tested for the presence of class I and class II mutators, the appearance of class I indicating the presence of dnaE910 in the metD+ nonmutator strain. Seven out of the 11 metD+ nonmutator strains indeed produced both class I and class II mutators (the remaining 5 yielding only class II mutators). It was concluded that the seven strains contained dnaE910 uncoupled from mutDS. To assess whether dnaE910 might also contribute an antimutator effect in strains other than those carrying mutD5, we measured mutation frequencies in dnaE910 mutL and dnaE910 mutT strains (Table 4). In the mutL background, 2.8- and 3.8-fold antimutagenic effects were observed for Rif and Nalr, respectively. The effects were

FIG. 4. Location of the antimutator mutation (X) determined by P1 transduction. The positions of the various genes are in minutes (1). The positions of the zae and zaf transposon insertions are from this work (zaj) or unpublished data (zae). The P1 donor was NR9515 (containing zaf-13::TnlO, mutD5, and the antimutator mutation, X), and the recipient was CD4 (metD). The selection was for tetracycline resistance (+-) or for metD+ (). Ninety-six transductants were examined in each case. The beginning of the arrow (@) indicates the selected marker, the head of the arrow indicates the cotransduced marker, and the number in parentheses indicates the cotransduction frequency (%). X was placed to the left of the metD gene because 30 of the 31 class I mutators were metD', whereas only 1 of 12 metD+ mutators was class I.

similar whether the strains were grown in LB or MM. Introduction of plasmid pMK9(dnaE+) again nullified the antimutagenic effect (results not shown), indicating that the effect resulted from the mutant dnaE gene. Since in mutL strains (defective in DNA mismatch repair) mutations may be ascribed to DNA replication errors, the simplest interpretation is that the mutant DNA polymerase subunit in dnaE910 strains causes a reduced level of DNA replication errors, accounting for the antimutator effect in this strain. In the mutT background, a 3.8-fold reduction in the mutation frequency toward Rif was observed. The mutT mutator is characterized by a specific increase in A. T-&C G transversions, which arise as a consequence of a high frequency of A. G mispairings by DNA polymerase III holoenzyme (1, 28). Thus, dnaE910 also probably affects the frequency with which the polymerase commits this particular error. Effect of dnaE910 on thermolability of and mutagenesis in a dnaQ49 strain. The relatively moderate antimutagenic effect of dnaE910 in the mutL background, in contrast to its large antimutagenic effect in the mutDS background (at least in MM), prompted us to explore the effect of dnaE910 on a dnaQ49 strain. This strain contains a temperature-sensitive dnaQ gene product and exhibits a temperature-sensitive mutator activity (13, 20). Only a weak mutator effect is observed below 30°C, but a very strong mutator effect, which is similar to the mutDS effect in rich medium, is

TABLE 3. Complementation of two classes of mutD5 mutator by various plasmidsa No. of Nalr mutants per 10 cells with plasmid

Class

I II

None

pBR322

pMK9(dnaE+)

pMM5(dnaQ+)

0.32 9.6

0.28 2.1

7.0 7.7

0.02 0.02

a Strains used were NR9515 (class I) and NR9516 (class II), grown in and plated on MM for total cell count and plated on MM-NAL for mutant counts.

TABLE 5. Temperature sensitivities of dnaQ49 and dnaE910 strainsa

TABLE 4. Effect of dnaE910 on mutation frequency in mutL and mutT backgrounds

Sensitivity at":

No. of mutants per 106 cellsa Strain (genotype)

1979

dnaE SUPPRESSOR OF MUTATOR mutD5

VOL. 174, 1992

Rifr

Nalr

NR9694 (mutL) NR9686 (mutL dnaE910)

7.0 + 1.9 2.5 ± 0.7 (2.8)

2.9 ± 0.9 0.79 ± 0.20 (3.8)

NR9728 (mutT) NR9729 (mutT dnaE910)

1.4 ± 0.8 0.34 + 0.18 (3.8)

NDb ND

Frequencies (± standard deviation) are averages of 12 cultures for each strain grown in LB and plated on LB plates. The fold effect of dnaE910 is

44'Cc

370C

350C

Genotype

LB Salt-free LB LB Salt-free LB LB Salt-free LB +

+

+

+

+

+ dnaQ49 + dnaE910 dnaQ49 dnaE910 +

+ +

+ +

+ +

+ +

-

-

-

-

wt

+

-

a

given in parentheses. The experiment with the mutL and mutL dnaE910 strains was also performed in minimal media, and identical results were obtained (data not shown). b ND, not determined.

observed above 35°C (13, 16). No significant increases in mutability occur upon further temperature increases. At 44.5°C, the strain becomes inviable (in salt-free broth). Takano et al. (35) proposed that the dnaQ49 protein is temperature sensitive in its binding to the dnaE gene product. Increasing the temperature leads to an increasing population of DNA polymerase III holoenzyme molecules without £, causing a concomitant increase in the mutation rate. At 37°C, most polymerase molecules will already have lost their e subunit, and further increases in the mutation rate will be small. At 44.5°C in salt-free broth, the cell ceases to grow, presumably because of the intrinsic thermolability of the polymerase subunit in the absence of E (17). In view of this model, we thought that the dnaQ49 strain might provide a useful system to investigate whether dnaE910 might exert its effect through a changed interaction between the dnaE and dnaQ gene products. A dnaE910 dnaQ49 strain was constructed by transducing dnaQ49 linked to the transposon zae-502::TnlO (about 60% linkage; Fig. 4) into strain NR9675 (dnaE910) at 30°C. We noted that this cross yielded, in addition to nonmutator cells (dnaQ+), two types of mutators. One type, which apparently had become dnaE+ during the transduction (dnaE+ dnaQ49), was highly mutable at 37°C and formed normalsized colonies at this temperature. A second type, which apparently had retained the dnaE910 mutation (dnaE910 dnaQ49), grew extremely poorly at 37°C but moderately well at 35°C, at which temperature it showed significant mutator activity. Table 5 presents a comparison of the temperature sensitivities of these and related strains on both LB and salt-free LB media, the latter medium enhancing the temperature sensitivity of many dna(Ts) strains (16, 17). Note that the dnaE910 strain itself is temperature sensitive at 44°C on salt-free medium. Table 6 presents the mutability of the two types of dnaQ49 mutators at 30 and 35°C. It is clear that dnaE910 again exerts an antimutagenic effect. At 30°C, the reduction in mutagenesis is 5.8-, 6.7-, and 12-fold for Rif, Nalr, and Strr, respectively. At 35°C, mutagenesis levels are significantly enhanced, as is expected for dnaQ49 strains. However, at this level the antimutagenic effect of dnaE910 is slight or absent; the reduction factors at this temperature are 1.5-, 1.5- and 0.65-fold for the three markers. This differential effect of dnaE910 at the two levels of mutagenesis is reminiscent of its differential effect on the mutD5 mutator in rich medium versus MM (Table 2) and may reflect the same mechanism.

Sensitivities were determined by spot tests as described in Materials and Methods. Strains used were CD4, NR9812, NR9675, and NR9813 (Table 1). b +, growth; -, no growth. C (44 + 1)°C. a

DISCUSSION In this paper we describe a mutation in the E. coli dnaE gene, dnaE910, that acts as a partial suppressor of the mutD5 mutator. dnaE910 presumably arose adventitiously in one of the mutD5 strains maintained in our laboratory, because it was present neither in the parent mutD5 strain KH1214 (6) nor in any of the other strains whose mutational properties we have described in some detail (25, 26). dnaE910 produces three main effects. It (i) suppresses the mutD5 small-colony phenotype, (ii) causes temperature sensitivity, and (iii) produces a distinct antimutator phenotype. This antimutator effect is very strong for mutD5 in MM but only weak in rich medium, and it can also be observed in mutL, mutT, and dnaQ49 backgrounds. The DNA sequence alteration(s) in dnaE910 is not yet known, but in the simplest case one mutation would be responsible for all three phenotypes, and a satisfactory model for its mechanism of action would have to account for all three phenotypes. At this time, we are not yet able to produce such a comprehensive model, mostly because of lack of insight into the precise mechanism of the mutD medium effect. Below, we will discuss each of the three phenotypic characteristics separately. Suppression of colony-size phenotype. Insight into the mechanism by which dnaE910 suppresses the small-colony phenotype requires, first of all, an understanding of why mutD5 cells produce smaller colonies. Slow growth may result from the accumulation of deleterious or lethal mutations in this strong mutator, but this seems unlikely, because the phenomenon is observed in both MM and rich medium, for which mutation rates are vastly different. Also, other strong mutators (such as mutL) mutate as strongly as mutD in MM and do not produce smaller colonies. Thus, alternatively, the small-colony phenotype may result from the lack of exonuclease but for reasons other than the role of the exonuclease in fidelity. Such an additional role for the exonuclease could reside in one or more aspects of DNA replication, repair, or recombination. In Salmonella typhimurium, a deletion of the dnaQ gene which resulted in exceptionally poor growth has been described, but normal viability could be restored by suppressor mutations in dnaE (15). Because the deletion strain containing the suppressor retained a high level of mutability, it was concluded that the dnaQ gene fulfilled an essential function different from its role in fidelity. Despite this analogy, the mechanisms responsible for suppression in S. typhimurium and in our system need not be identical, because in the former case the £ subunit is completely absent (the major function of the suppressor possibly being the stabilization of at in the absence of £), while in the

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TABLE 6. Effect of dnaE910 on mutator activity of dnaQ49 at two temperaturesa Mutant frequency per 106 cellsb Genotype

300C Rifr

dnaQ49 dnaQ49 dnaE910

5.3 ± 1.6 0.91 ± 0.45

Nalr

2.9 1.0 0.44 ± 0.21 +

350C

Strr

1.2 + 0.5 0.10 ± 0.05

Rifr

Nalr

Strr

241 ± 60 157 ± 59

88 ± 36 59 ± 49

60 ± 13 92 ± 20

a The strains used were NR9812 and NR9813 (Table 1). bEach value is the average for 12 cultures grown in LB broth at the indicated temperature.

case of mutD5 the £ subunit is presumed present and functionally binding (35), as deduced from the dominant character of mutDS (5, 8). Assuming that the small-colony phenotype of mutDS results from the lack of exonuclease, suppression of the phenotype might occur by a partial restoration of the exonuclease activity by dnaE910. Such a possibility would, at the same time, provide a ready explanation for the dnaE910 antimutator phenotype. That a mutation in the polymerase might affect the activity of the exonuclease is not unreasonable, considering the tight interaction between the a and £ subunits (21) and the reciprocal stimulation of their activities (18, 34). Furthermore, both kinetic (14) and genetic (19) studies suggest that the polymerase plays an important role in determining the exonuclease activity, at least for fidelity purposes. Alternatively, suppression of the small-colony phenotype might proceed by altered protein-protein interactions between the a subunit (or a-£ complex) and other relevant proteins. Temperature sensitivity. dnaE910 leads to increased temperature sensitivity, as first observed when it was combined with the temperature-sensitive dnaQ49 allele. Because the temperature sensitivity and the temperature-dependent mutator activity of the dnaQ49 strain result from temperaturesensitive binding of £ (35), our experiments were aimed at measuring a possibly changed interaction between the a and £ subunits. Improved a-£ binding was expected to result in increased thermostability and reduced mutagenesis, while decreased binding would result in reduced stability and increased mutagenesis. Neither result was observed, with the dnaE910 dnaQ49 strain displaying reduced stability and reduced mutagenesis. The simplest interpretation of this result is that a-£ binding is not drastically affected but that, instead, the thermolability of the dnaE910-encoded a subunit is increased. The wild-type a subunit is inherently unstable, particularly in the absence of the c unit, and this is thought to be responsible for the temperature sensitivity of a series of dnaQ alleles (including dnaQ49) at 44.5°C in saltfree media (17). We assume that the permissive temperature of the dnaE910 a subunit has been lowered from 44.5 to 35°C or less in salt-free medium. At 35°C, when most dnaQ49encoded E subunits will have abandoned the polymerase, the dnaE910-encoded a subunits will be exposed and inactivated. Consistent with this increased thermolability is the greater temperature sensitivity of the dnaE910 dnaQ+ strain. In this case, it is possible that at 44°C even wild-type c subunit is no longer sufficient to stabilize the a subunit. Alternatively, the effect might reflect somewhat reduced a-c binding. Antimutator activity. The antimutator effect of dnaE910 may be most easily interpreted on the basis of an altered DNA polymerase complex producing fewer errors of DNA replication. Such improved fidelity could originate at the

level of base selection or at the level of proofreading. In the former case, the dnaE910-encoded a subunit would be more accurate in selecting correct over incorrect bases. In the latter, the dnaE910-encoded a subunit would stimulate the activity of the exonuclease. The latter possibility is most easily reconciled with the suppression of the small-colony phenotype (see above). It is also possible that both mechanisms operate simultaneously, since a more accurate DNA polymerase might be expected to act more slowly, providing increased opportunity for exonucleolytic proofreading to take place. The antimutator activity of dnaE910 against mutDS is characterized by its large effect in MM and small effect in rich medium. We can envision three possible explanations. First, the effect may reflect the specificity of the antimutator. Previous studies from our laboratory on the precise nature of mutD5-induced mutations have shown that in MM these were predominately transversions, but in rich medium they were predominately transitions (25). Thus, it is possible that the antimutator effect is strong for the transversions but weak for transitions. Studies on the specificity of the dnaE910 effect may provide an answer to this question. The second possibility concerns the precise mechanism by which the medium-induced switch occurs. If it were to occur by the action of certain effector molecules that interact with the c subunit, as has been suggested previously (3, 11), then the dnaE910-caused antimutator effect might proceed in the absence but not in the presence of such an effector. The third possibility is that the different dnaE910 antimutator activities in the two media reflect a difference in mismatch repair. The large difference in mutability of mutD5 strains in the two media has been attributed to the presence (MM) or absence (rich medium) of mismatch repair (7, 25, 26), the absence resulting from saturation of the system by an excess of DNA replication errors (30). However, one need not think of saturation as an all-or-none phenomenon. Instead, one may think of the two conditions as points on a scale of saturabilities, MM representing a low level of saturation and rich medium representing a high level of saturation. It is then instructive to consider how a given reduction in replication errors will affect cells at either end of the spectrum. Simple calculations can be done, assuming that, once enough errors are being made to initiate saturation, the repair activity is proportional to the number of remaining repair complexes. It then follows that at the low end of saturation, small changes in the number of repair complexes can cause large changes in mutation rates. On the high end of saturation, such small changes will not cause any observable effects, with even large changes causing only modest effects. At the low end, for example, the loss of only 5% of the repair complexes could reduce the efficiency of mismatch repair from, say, 99.9% (only 1 out of 1,000 errors escaping repair) to 95% (50 out of 1,000 errors escaping

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VOL. 174, 1992

repair), causing a 50-fold effect increase in the mutation rate. At the high end, the loss of 50% of the repair complexes could reduce the efficiency of mismatch repair from, say, 75% (one out of four errors escaping) to 25% (three out of four errors escaping), causing a threefold increase in the mutation rate. Thus, the dnaE910-mediated antimutator effect may be relatively small (three- to fourfold) at the level of DNA replication errors, but its consequences for the observed mutation rate may be significantly enhanced in MM by the parallel recovery of mismatch repair, while no such enhancement occurs in rich medium. Among these three hypotheses, we slightly favor the last one, because it also provides a relatively simple explanation for the antimutator effects observed in mutL, mutT, and dnaQ49 backgrounds. The effects in mutL and mutT strains are modest (three- to fourfold) and are independent of the medium. Because in these strains mismatch repair is either fully absent (mutL) (4, 23, 24) or fully present (mutT) (27), one expects to measure in these strains only the primary antimutator effect, which should be independent of the medium. Antimutagenesis in the dnaE910 dnaQ49 strain may be similarly explained. At 30°C, the mutator strength of dnaQ49 is only moderate and mismatch repair may be only marginally affected. Small differences in the mutation rate are thus likely to have large consequences. However, at 35°C the mutation rate is high (presumably by breakdown of the mismatch-repair system), and small differences in the extent of saturation will not largely affect the mutation rate. This is precisely what is observed. Summary. Taken together, the data of this paper indicate that a mutant derivative of the dnaE gene, dnaE910, produces an altered a subunit of the DNA polymerase III holoenzyme. This altered subunit (i) overcomes the growth defect conferred by mutDS, possibly by restoring some level of exonuclease activity or by improving on protein-protein interactions impaired by mutDS, (ii) has increased thermolability, and (iii) has an antimutator activity. The latter may result from more accurate base selection during polymerization or from better error correction through an effect on the proofreading subunit. The further study of this DNA polymerase allele, including the cloning of the gene and the determination of its DNA sequence changes, may be helpful in elucidating the mechanisms that ensure the accurate replication of the E. coli chromosome, as well as the mechanisms that underlie the peculiar nature of the mutD mutator. ACKNOWLEDGMENTS We thank J. Drake, I. Fijalkowska, R. Fowler, T. Kunkel, A. Oller, and M. Sander for their critical reading of the manuscript and helpful suggestions. R. Fowler, H. Maki, and E. Siegel are acknowledged for providing strains and plasmids. REFERENCES

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