11

Mutation Research, 52 ( 1 9 7 8 ) 1 1 - - 2 4 © Elsevier/North-Holland Biomedical Press

B R O M O U R A C I L MUTAGENESIS AND MISMATCH R E P A I R IN M U T A T O R STRAINS OF Escherichia coli

BJ(JRN RYDBERG *

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Conn. 06520 (U.S.A.) (Received 6 December 1977) ( R e v i s i o n r e c e i v e d 29 M a r c h 1 9 7 8 ) (Accepted 3 May 1978)

Summary A screening procedure based on the formation of papillae on individual bacterial colonies was used to isolate mutants of Escherichia coli with high mutation rates in the presence of bromouracil. Most of th~ mutants obtained had high spontaneous mutation rates and mapped close to the previously known mutators mutT, mutS, mutR, uvrE and mutL. Except for mutants of m u t T type, these mutators also showed high mutability by bromouracil. Transfection experiments were performed with heteroduplex lambda DNA to test for mismatch repair. The results suggest a reduced efficiency of repair of mismatched bases in mutators mutS, mutR, uvrE and mutL, whereas mutants mapping as m u t T appear normal. The results support a connection between spontaneous and bromouracil-induced mutability and repair of mismatched bases in DNA.

Introduction A low spontaneous mutation frequency is probably maintained by a complex set of cellular functions: In Escherichia coli, more than 10 genetic loci are known that control spontaneous mutation rates (review by Cox [6]). The nature of the gene products is largely unknown, except for a few cases where DNA polymerases are involved [4,9,25]. The "proof-reading" capability of bacterial DNA polymerases [3,13] has been emphasized in this connection [24]. * Present Institute: R e s e a r c h Institute of National Defence, Sweden. Present address: Swedish University of AgJcicultural Sciences, D e p a r t m e n t of Pathology) S-750 07 Uppsala (Sweden). By acceptance of this article, the publisher a n d / o r r e c i p i e n t a c k n o w l e d g e s the U.S. G o v e r n m e n t ' s right to retain a non-exclusive, royalty-free llcence in and to any copyright covering this paper. A b b r e v i a t i o n : BrU, 5-bromouracil.

12 Nevers and Spatz [17] recently demonstrated, in a transfection assay with heteroduplex lambda DNA, that the E. coli mutator uvrE- (as well as the nonmutator uvrD-) appeared defective in repair of mismatched bases in DNA. Such repair {review: [18]), is thought to occur by excision, i.e. by recognition of mismatches by (an) endonuclease(s) followed by excision and resynthesis of one of the strands. The repair tracts, at least under some conditions, appear to be as long as 2000--3000 nucleotides [26,27]. There is no indication that the genes uvrA, uvrB, uvrC and poIA, involved in excision of pyrimidine dimers and other lesions, are involved in excision of mismatched bases [17]. A connection between spontaneous mutability and mismatch repair has also been suggested in pneumococcus [23]. Mutagenesis by the base-analog bromouracil probably occurs by a mispairing mechanism [8,12,20,28]. There is indirect evidence that the mismatched bases introduced by bromouracil are repaired in such a way that the original genotype is restored. This process appears to be defective in uvrE- and uvrD- strains of E. coli [20]. In the present investigation, mutator strains mutS-, mutR-, mutL-, as well as uvrE-, are shown to have enhanced bromouracil mutagenesis; and transfection experiments with heteroduplex DNA suggest a defective repair of mismatched bases in these strains. Materials and methods

Cells and phages The Escherichia coli K12 strains and phages used are listed in Table 1. Phage ~cI857 with temperature-sensitive repressor produces turbid plaques at 30°C. Phage Xc4221, which produces clear plaques at 30°C, was obtained by bromouracil mutagenesis of Xci857. (See [12] for procedure.) Phage Xc4222, which also produces clear plaques at 30°C, was isolated after growth of Xci857 in m u t a t o r strain M5 for one lyric cycle {whereby the frequency of clear plaque formers increased by a factor of 10). Strain M5 probably has the marker mutT (see below). Growth media and plates Lambda buffer contained 6 mNI Tris (pH 7.2), 10 mM MgSO4 and 0.005% gelatin. SSC was 0.15 M NaC1 and 0.015 M sodium citrate (pH 7.5}. M9 buffer contained 19 mM NH4C1, 42 mM Na2HPO4, 22 mM KH2PO4, 1 mM MgSO4, 0.1 mM CaC12 and thiamine (B1) at 0.1/ag/ml. K medium was made up by adding 5 ml 20% glucose and 10 ml 10% casamino acids (decolorized and vitamin-free) per 100 ml M9 buffer. Thymine at 0.1 mM was added when required. Luria broth contained tryptone (10 g/l), yeast extract (Bacto) (5 g/l), NaC1 (0.5 g/l) and NaOH (0.12 g/l). Luria plates contained Luria broth with the addition of agar to 1.85%. Lambda (X) plates contained tryptone (5 g/l), peptone (8 g/l), NaC1 (1 g/l) and agar (15 g/l). Medium 56 agar contained: KH2PO4 (2.64 g/l), Na2HPO4 {4.34 g/l), (NH4)2SO4, (1 g/l), MgSO4 • 7H20 (100 mg/1), Ca(NO3)2 (5 mg/1), FeSO4 • 7H20 (0.25 mg/1) and agar (1.5%). SEMhis- plates were made from medium 56 agar with the addition of glucose

13 TABLE 1 BACTERIAL STRAINS AND PHAGES Bacterial strain AB2497

F-

Relevant genetic markers

Source

thr-1, leu-6, proA2, his-4, argE3, lacY1, galK2, ara-14, xyl-5, mtl-1, thi-1, tsx-33, strA31, sup-37, t h y A - ,

P. H o w a r d - F l a n d e r s

drm-

M1, M 2 - - M 9 6 KMBL1854 KMBL1851 KMBL1853 ES455 ES568 AB1931 PC0273 AT2427 KL16

As A B 2 4 9 7 . also m u t a t o r s multiple auxotroph As K M B L 1 8 5 4 , also uvrDl01 As K M B L 1 8 5 4 , also uvrE502 routS3 mutL13

metE46 purA46 cysC43

Hfr

early m a r k e r s e n t e r e d are:

This w o r k A. RSrsch A. Rbrsch A. R 6 r s c h B. B a c h m a n n B. B a c h m a n n B. B a c h m a n n B. B a c h m a n n B. B a c h m a n n E. Bardwell

t h y A +, m u t R +, r o u t s + W3110 W 3 1 1 0 (k ci857)

wild t y p e , h.~. l y s o g e n o f W 3 1 1 0

J. Stein J. Stein

Phage strain

Genetic markers

Source

k ci857 k c4221 k c4222 Plvir

c1857 (ts) c1857 (ts), c 4 2 2 1 c1857 (ts), c 4 2 2 2

J. Stein This w o r k This w o r k E. B a r d w e l l

(2 g/l), DL-threonine (83 pg/ml), DL-leucine (83 pg/ml),'L-proline (167 pg/ml), L-arginine (147 pg/ml), thiamine (0.17 pg/ml, thymine (12.6 pg/ml) and Luria broth. (1%). SEMthr- plates had the same composition except that threonine was omitted and DL-histidine (87 pg/ml) was added. Z plates for growth of Plvir contained Luria broth and 0.1% glucose, 2.5 mM CaCl~ and 0.8% agar. Soft agar for plating Plvir was 0.6% agar, 0.8% NaC1 and 2.5 mM CaC12. Selective plates for mapping experiments were medium 56 agar supplemented with the proper amino acids and vitamins as described previously [20]. Adenine at 25 pg/ml was added when necessary. Streptomycin at 200 gg/ml was included for counter-selection of males after conjugation. G plates for isolation and screening of mutator strains (in AB2497 background) contained 25.5 g Antibiotic Medium 2 (Bacto) per 1 0 0 0 ml water, with the addition of galactose to 1%, thymine to 0.075 mM and bromouracil to 0.025 mM. Drug-resistant mutants were tested on Luria plates containing either streptomycin (150 gg/ml), rifampicin (100 pg/ml), or ampicillin (10 pg/ml). Soft agar for phage ~ transfection experiments had the same composition as had X plates, except that the agar was reduced to 0.7%. Luria broth was supplemented with 0.1 mM thymine when strains requiring thymine were grown to high cell densities (overnight growth).

Mapping Conjugation was carried out by mixing equal numbers of exponentially growing male and female cells in Luria broth at an OD6s0 (absorbance at

14 650 nm) of 0.2. Incubation in a roller drum was continued for 15 min, after which the cultures were vortexed vigorously, diluted and plated on selective plates with streptomycin for counter-selection. For experiments involving Plvir phage, cells were grown in Luria broth to an OD6s0 of 0.5, with CaC12 present at a concentration of 2.5 mM for the last 10 min of incubation. After the addition of phage, the mixture was held at 37°C for 20--25 min for adsorption. Titers were obtained by ,olating strain W3110 to which phage had been adsorbed. Lysates of Plvir were obtained by plating 0.3-ml samples of the donor strain to which 107 phages had been adsorbed, and harvesting the phage from the soft agar layer after 8 or 18 h of incubation at 37°C (by using essentially the procedures described by Miller [ 16 ]). The phage lysates so obtained contained 1011 phage per ml for wild-type strain W3110 and 108--109 phage per ml for strain AB2497 and derivatives. Transduction was carried out by adsorbing Plvir to the recipient strain at an m.o.i. (multiplicity of infection) of 0.1 or lower, centrifuging the cells, washing, and resuspending in M9 buffer before plating on appropriate selective plates. If the yield of recombinants was too low with this procedure, the m.o.i. was increased to 0.5--1. In this case, an equal volume of SSC was added at the end of the adsorption period to prevent readsorption of phage. The cells were then centrifuged, taken up in a small volume of SSC, and plated on selective plates. When strains M1--M96 were the recipients in conjugation or transduction (selection for leu +, proA + or thyA+), the frequency of mutators and non-mutators among recombinants was tested by picking up colonies, streaking them out on selective plates of the same kind for partial purification, and then replicaplating these onto G plates. After 4 days incubation, papillae revealed m u t a t o r activity. The Plvir (W3110) lysate used in these experiments was tested for sterility. When strains AB1931, PC0273 or AT2427 were recipients in transduction by Plvir previously grown on strains M1--M96 (selection of m e t e +, purA + or cysC ÷ respectively), purified recombinants were grown overnight in 1 ml Luria broth and aliquots were plated on two different drug-containing plates. Mutator activity was identified by a high level (10--100 times the control) of both types of drug-resistant mutants. When UV-sensitive strains were the donors, both m u t a t o r activity (one drug-resistance marker) and UV sensitivity were tested. The selective plates used in these experiments did not support growth of the donor strains (M1--M96).

Preparation o f phage D N A Phage Xci857 was obtained by thermo-induction of W3110 (Xci857). Cells were grown in Luria broth at 29°C to an OD6s0 of 0.4, brought to 43°C for 20 min, and then kept at 37°C with vigorous aeration until lysis after about 1 h. Phage Xc4221 and 7~c4222 were obtained by infecting exponentially growing cultures of strain W3110 at OD6s0 of 0.4 in Luria broth with phage at an m.o.i, of 3. Lysis occurred after growth for 1 h at 37°C with vigorous aeration. Chloroform was added t o the lysates, which were centrifuged (5 k, 15 min) to remove most cellular debris. Titers at this stage were at 1--3 × 101° phages

15 per ml. The phages were concentrated with high speed centrifugation (25 k, 2 h, type 30 rotor). Free DNA was degraded with DNAase treatment (DNAase I at 40 pg/ml, 30 min, 37°C). The phages were purified by banding (30 k, 1 h, SW50.1 rotor) in a step gradient of CsC1 (in 0.1 M Tris, 0.04 M MgSO4, pH 8.0) followed by equilibrium banding (30 k, 15--20 h, SW50.1 rotor) in CsC1 at 1.50 g/ml (same buffer). The phage was then dialyzed twice against 0.025 M Tris, 0.01 M MgSO4 (pH 8.0), to remove CsC1. The phage DNA was extracted with phenol; 1 ml purified phage at 1012 per ml in 0.025 M Tris, 0.01 M MgSO4 (pH 8.0) was mixed with 1 ml redistilled phenol saturated with the same buffer. The tube was agitated by hand for 1 min, after which the phases were separated by centrifugation (5 k, 10 min). The upper aqueous phase was recovered, phenol added, and the procedure repeated. The DNA was dialyzed 3 times against 0.01 M Tris, 0.001 M EDTA (pH 8.3). For transfection, this DNA was diluted in TEN buffer (0.02 M Tris, 0.001 M EDTA, 0.02 M NaC1, pH 7.8 [17]). Strand separation was accomplished by heating 10--20 pg DNA/ml in 0.01 M Tris, 0.001 M EDTA (pH 8.3), to 92--93°C for 2.5 min, followed by chilling on ice [27]. Reannealing was accomplished by adding NaC1 to a final concentration of 0.2 M and holding the sample at 60°C for 10 min. The sample was then diluted 10-fold with 0.02 M Tris, 0.001 M EDTA (pH 7.8) to reconstitute the TEN buffer. Reannealed DNA regained 5--10% of the initial biological activity.

Transfection Transfection was carried out essentially according to the procedure of Mandel and Higa [15]. The cells were grown in Luria broth in a roller drum at 37°C to an OD6s0 of 0.5--0.7 and cooled on ice. After centrifugation, the cells were resuspended in half the initial volume of ice-cold 0.05 M CaC12, allowed to stand at 0°C for 20 min, centrifuged again and resuspended in 1/20 initial volume of ice-cold 0.05 M CaC12 {corresponding to an OD6s0 of 10). One part precooled DNA in TEN buffer was mixed with two parts cell suspension and the mixture allowed to stand at 0°C for 20 min followed by 15 min at 37°C. Aliquots were mixed with AB2497 as indicator strain and spread in 2.5 ml soft agar onto ~ plates, which were incubated at 30°C. With this indicator strain, plaques arising from mixed bursts could be distinguished as " m o t t l e d " plaques under good plating conditions [12]. The efficiency of transfection for most bacterial strains was about l 0 s plaque-forming units per pg of native DNA, but considerably lower for some strains.

Assay for mismatch repair of heteroduplex DNA The principle for the assay of mismatch repair was as follows. A mixture of (usually) 1 part ~c4221 DNA (or ~c4222 DNA) and 9 parts ~cI857 DNA was denatured and reannealed according to the procedure given above. If both DNA preparations had the same proportion of intact DNA single strands, random assortment of the strands would give rise to 1% molecules of type c:c', 9% of t y p e c:t', 9% of type t:c' and 81% of type t:t', where c:c' and t:t' denote the complementary strands in the respective phages, and c:t' and t:c' denote two

16 different mismatched configurations. After transfection, the frequency of clear plaques was counted and the frequency of mottled plaques was roughly estimated. (Mottled plaques and turbid plaques could not always be distinguished with certainty.) In the absence of any mismatch repair, one would theoretically expect to get 1% clear plaques (c:c') and 18% mottled plaques (c:t' and t:c') for the above example. If the mismatched bases are efficiently repaired before replication to yield the t or c genotype with equal probability one would expect to get 10% clear plaques and no mottled plaques.

Other techniques Uptake of bromouracil was estimated by the use of radioactively labeled DNA precursors [21]. Burst sizes after transfection were estimated by first titering total plaqueforming units (complexes) directly after DNA adsorption, diluting to 2 × 107 cells/ml in K medium with Mg2÷ reduced to 10-4M [12], and titering free phage at various times of incubation by plating chloroformed samples. Ultraviolet irradiation was performed with a low pressure mercury germicidal lamp (15 W) radiating predominantly at 254 nm. The dose rate was 1.0 J/m 2 per sec. Results

Isolation of mutants When a few hundred cells of strain AB2497, which has the marker galK2, were plated on rich agar (Difco Antibiotic Medium No. 2) with excess galactose and incubated for 3--4 days, papillae (i.e. microcolonies within previously formed colonies) developed at a frequency of about 1 papilla per 20 colonies. Presumably these were slow-growing gal ÷ revertants. With 0.075 mM thymine and 0.025 mM bromouracil present (G-plates), the frequency was still low (about 1 papilla per 10 colonies). With increasing concentrations of bromouracil in the plates, however, the frequency of papillae rose sharply as a result of mutagenesis to reach a maximum of 10--100 papillae per colony at high concentrations. The basis for the screening of mutators was to find colonies with many papillae on plates with a low level of bromouracil. Cultures of strain AB2497 in exponential growth were diluted in M9 buffer, mutagenized by exposure to UV radiation at 100 J/m 2 and plated directly on Luria plates to give a few hundred survivors per plate (survival was about 1%). The Luria plates were incubated at 37°C for one day. (In one experimental series, mutagenized cells were grown overnight in broth before being plated on Luria plates.) To locate mutants with m u t a t o r properties, the plates were replica-plated with velvet onto G-plates, which were incubated at 37°C for 3--5 days. Of 130 000 colonies inspected on the G-plates, 121 papillated colonies (4 or more papillae per colony) were observed; 96 of the corresponding colonies were picked from the Luria plates, purified and confirmed to give papillation on G-plates (strains M1-M96). Each strain probably arose from an independent mutation.

17 10 of these mutants did not grow well on minimal plates and could not be properly characterized. These were excluded from further characterization. Also, some weak mutators were excluded because of difficulties in distinguishing m u t a t o r and non-mutator properties in mapping experiments. Some of these strains appeared as mutators only in the presence of bromouracil.

Mutator activity A b o u t 107 cells from stationary phase (overnight)cultures of strains M1-M96 were spread on SEMthr- and SEMhis- plates, which contained either 0.1 mM thymine or 0.09 mM thymine + 0.01 mM bromouracil. These semienriched plates edlowed a limited growth before exhaustion of threonine or histidine respectively [20], after which only the revertants could continue to grow and yield visible colonies after two days of incubation. Results are shown in tables 2 and 3. Some mutants (listed as Class I) were characterized by efficient spontaneous reversion of both the thr-1 (amber} and his-4 (ochre) markers, with no significant further increase by bromouracil. The other mutants (Classes II--V) were also spontaneous mutators but showed enhanced mutagenesis by bromouracil as well, with the thr-1 marker reverting much more efficiently than the his-4 marker. Mapping studies (to be described below) make it likely that Class I mutants are alleles of mutT, whereas the other mutants are alleles of uvrE, mutR, routS and mutL. The m u t a t o r m u t T is known to give almost exclusively AT-* CG transversions, whereas the other four listed give transitions and frameshifts [6]. It has been shown previously that bromouracil is very inefficient in reverting the his-4 marker in wild-type AB2497 [20]. Uptake of bromouracil relative to thymine was measured for the strains listed in Table 3. Except for strain M82, which had a low uptake of bromouracil, the values were normal for drm- strains, with a bromouracil/thymine ratio in newly synthesized DNA of about half that in the medium [21]. U V sensitivity About 500 cells from dilutions of overnight cultures were spread on duplicate Luria plates, one of which was irradiated with 20 J/m 2. Tables 2 and 3 show that mutants listed as Class II were UV-sensitive, while the other classes had normal survival. Mapping experiments Mapping experiments were done as described in Materials and methods. The loci used in these experiments are shown in the map in Fig. 1. Cotransduction frequencies (not corrected for spontaneous reversion of the selected marker; see below) are shown in Tables 2 and 3. The m u t a t o r activity of the strains listed Class I cotransduced with leu. The cotransduction frequencies obtained (14--50%) are low estimates because the leu-6 marker in the recipient m u t a t o r strains was efficiently reverted by the m u t a t o r activity, resulting in the same order of magnitude of leu ÷ revertants as recombinants on the selective plates. The m u t a t o r m u t T has previously been shown to cotransduce with leu at a frequency of around 50% [ 10]. Two of the UV-sensitive mutants (Class II) were tested for cotransduction with metE, strain AB1931 being used as recipient. Of 13 metE* colonies tested

OF MUTATOR

STRAINS

(AVERAGE

VALUES)

--

Wild-type AB2497

--

29% 27% 70% 31% 90%

with with with with with

leu a m e t E a,b thyA cysC b purA

Cotransduction (%)

36

40 (0.5 38 39 39 c

Survival % UV at 20 J/m 2

8

2440 650 620 580 480

thy

SEMthr-

18

2730 1810 1500 1430 1350 14

4570 31 29 32 38

thy

12

4090 128 104 98 115

BrU (10%)

SEMhis- plates

of colonies per plate)

BrU (10%)

plates

Mutability (number

100

102 54 61 53 51

(65--135) d (51--59) (28--75) (36--85) (36--69)

Heteroduplex +/c4221 DNA transfection assay. Fraction clears, % of control (range)

L o w e s t i m a t e , see t e x t . Based on tests on two mutants only. A v e r a g e s u r v i v a l a t 4 J / m 2 w a s 7%. T w o m u t a n t s i n C l a s s I ( M 1 4 a n d M 9 5 ) h a d s i g n i f i c a n t l y l o w n u m b e r s o f c l e a r p l a q u e s . I f t h e s e are e x c l u d e d , t h e n e w a v e r a g e v a l u e w i l l b e 1 1 4 , r a n g e 9 5 - - 1 3 5 . I n o t h e r r e s p e c t s , M 1 4 a n d M 9 5 c o u l d n o t b e d i s t i n g u i s h e d f r o m t h e o t h e r m u t a n t s i n C l a s s I.

9 3 13 30 8

I (mutT?) II ( u v r E ? ) III ( m u t R ? ) IV (mutS?) V (mutL?)

a b c d

Number of mutants included

Class

M u t a n t s w e r e d i v i d e d i n t o 5 classes, b a s e d o n m a p p i n g s t u d i e s . W i t h i n e a c h class, t h e 8 0 % o f t h e m u t a n t s w i t h t h e h i g h e s t m u t a t i o n r a t e s (as m e a s u r e d o n S E M t h r plates w i t h 10% B r U ) were u s e d to c a l c u l a t e average. In t h i s w a y , ' l e a k y ' m u t a n t s were e x c l u d e d a n d each class was r a t h e r u n i f o r m w i t h regard t o p r o p e r t i e s .

PROPERTIES

TABLE 2

3

M5 M19

M41 M88

M10 M33

M1 M35

M36 M82

I (mutT?)

II (uvrE?)

III (mutR?)

IV (routS?)

V (mutL?)

12/13 13/14

6/20 5/16

20/29 24/33

37 40 39 45 34 42

(thyA) (thyA)

(cysC) (cysC)

(PurA) (PurA)

1855 2060 1738 2034 1340 1376 1560 1307a

637 636 1054 963 439 388 396 714

2710 3888

BrU (10%)

thy

2980 3698

plates

SEMthr-

Mutability (number average two plates)

less BrU into DNA than did the other strains.