Bromouracil mutagenesis: mispairing or misrepair?

Mutation Research, 25 (I974) 407-409

,~) Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

407

Bromouracil mutagenesis: mispairing or misrepair?* PIETRZYKOWSKA1 has postulated that BU mutagenesis is due to misrepair of potentially excisable D N A damage resulting from B U incorporation, and t h a t BU and UV share a c o m m o n mechanism of m u t a t i o n induction. This hypothesis was based upon experiments showing t h a t BU mutability of lambda bacteriophage is amplified b y uvr mutations and diminished by recA or lex mutations in the host strain of Escherichia coli. Mutations in the uvrA, uvrB or uvrC loci eliminate excision repair, and greatly increase UV mutability in E. coli; mutations in the recA or lexA (exrA) loci eliminate UV mutability entirely, probably b y inactivating an error-prone type of D N A repair 4. The responses of BU mutability of lambda bacteriophage to host mutations resulting in these repair deficiencies suggested that BU, like UV, induces mutations via error-prone repair activity, rather than via mispairing in D N A replication. We found PIETRZYKOWSKA'S results surprising, not only because they contradicted the classical interpretation of BU mutagenesis, but also because one of us (E. M. W.) had observed, in unpublished experiments, t h a t recA and lexA derivatives of E. coli B/r were no less mutable b y BU and other base analogues than wild t y p e strains. We have re-examined the BU mutability of repair-deficient strains carrying uvrA, lexA and/or recA mutations as compared to that of a strain carrying the wild type alleles at all three of these loci. Our results indicate that, at least in E. coli DNA, BU mutagenesis is indifferent to the presence or absence oI the products of these three genes. TABLE

I

DERIVATIVES OF Escherichia coli B / r USED IN THIS STUDY a

Strain

Parent strain

Method of derivation Genotype

U V Mutability (see ref. 4)

WP2s

WP2

trp uvrA

hypermutable

WPI

WP2 s

trp

WP5o

WP2 s

normal (wild type) nonmutable

WPloo

"WP2s

isolated as Hcrmutant by R. HILL PI transduction (this laboratory) P1 transduction (this laboratory) Conjugation b (this laboratory)

trp uvrA lexA trp uvrA recA

nonmutable

a Thymine-requiring derivatives, obtained by the trimethoprim method S, were used. b The donor was an Hfr strain of KI2 which transfers recA as an early marker; mating was interrupted after 7 min. Thymine-requiring derivatives of the four strains described in Table I, obtained b y the trimethoprim m e t h o d s, were used in the experiments to be described. Log phase cultures of all strains were grown in minimal m e d i u m " E ''3, supplemented with 0.4% glucose, 0.4% Difco Casamino Acids, 2o/~g/ml t r y p t o p h a n e and 5 o # g / m l thymine, on a shaker at 37 °, to a titer of about 2. lO 8 cells per ml. The bacteria were centrifuged, washed in unsupplemented minimal medium, centrifuged again, and * This work supported by research grant AI-io778 from the National Institute of Allergy and Infectious Diseases of the U.S. Public Health Service. Abbreviations: BU, 5-bromouracil.

408

SHORT (~OM.MtNI('A-I I~)N~

resuspended at the same titer in minimal medium supplemented as described abow,, except that thymine was omitted. These suspensions were incubated on a shaker for one hour to exhaust thymine reserves and facilitate subsequent BU uptake. As-avs made just before and just after the hour of thymine starvation showed that this treat ment resulted in neither thymineless death nor thymineless mutagenesis, i.,'., neither the viable cell count nor the frequency of Trp + nmtations was significantly changed. The thymine-starved cells were then centrifuged, and resuspended in I/~ o volume of unsuppiemented minimal medium to concentrate the suspension t~ about 2.IO" cells per ml. Aliquots (o.2 ml) of the concentrated suspension were then spread on the surface of supplemented minimal agar, solidified with 1.5% Difco Purified Agar, and containing glucose, Casamino Acids and tryptophane as described above, ~md either 5o yg/ml thymine (control plates) or 50 y g / m l BU (BU plates). BU and contrail plates were incubated for periods ranging froln o to 0o rain. The bacteria were harvested by pipetting 2 ml ~>fsterile saline solution (o.9% NaC1) onto the surface of each plate, and spreading thoroughly with a sterile glass rod. The wash fluids from txxo identical plates were pooled and assayed for viable titer (by plating appropriate dilutions) and for frequency of Trp~ mutants (by plating undiluted o.I-ml aliquots) on minimal agar supplemented with 2.5~}?, (v/v) Difco Nutrient Broth and 5 ° f~gzml thymine (GEM agar). Colonies were counted after two days. All incubations were :~t 37'. Yell~w light Io3 K

,~

i0 z

l-cD

5 10I

k io C

I00

m 107

I 15

I 30

I 45

I 60

I 75

9~0

MINUTES ON BU PLATES

Fig. t. V i a b l e cell t i t e r a n d f r e q u e n c y of T r p ¢ m u t a n t s i n p o p u l a t i o n s h a r v e s t e d f r o m B U p l a t e s a f t e r i n c u b a t i o n p e r i o d s of o t o 90 rain. Z e r o p o i n t v a l u e s a r e b a s e d o n p o p u l a t i o n s p l a t e d oll cont r o l p l a t e s , c o n t a i n i n g t h y m i n e i n s t e a d of B U , a n d h a r v e s t e d i m l n e d i a t e l y a f t e r p l a t i n g . •, \ V P 2 s ( u v r A ) ; O, V q P s o ( u v r d lexA) ; m, W P z o o ( u v r d recd); 0 , \ ¥ P I (uvrA + lexA ~ ztvrd ~) ; ~ , a v e r a g e of d a t a f r o m a l l f o u r s t r a i n s . P o i n t s a r e a v e r a g e s a n d v e r t i c a l l i n e s s h o w r a n g e of v a r i a b i l i t y of d a t a o b t a i n e d in t h r e e e x p e r i m e n t s . L i n e s a r e d r a w n t h r o u g h a v e r a g e v a l u e s for all f o u r s t r a i n s .

SHORT COMMUNICATIONS

409

was used to illuminate operations involving BU plates, which were otherwise stored and incubated in the dark. Results of platings on BU plates are shown in Fig. I. Viable cell counts on the BU plates remained nearly constant for about 60 min, and increased no more than twofold by 90 min. The frequency of Trp + mutants, in all four strains, rose rapidly, especially during the period from 15 to 30 min on BU agar, reaching a maximum level at about 45 rain, and maintaining a frequency close to this level for as long as 9 ° min. Populations harvested from control plates (data not shown) showed an earlier and greater increase in viable cell count (up to twofold by 60 rain and fourfold by 9 ° rain), but showed no significant increase in the frequency of Trp ~ mutants during the 9 ° min of incubation on thymine-containing agar. This frequency did not exceed 8 mutants per lO 7 bacteria harvested from control plates at any time, in any of the four strains. The response to BU is very similar in all four strains, each of which produces about 5O-lOO times the number of Trp ÷ mutants after 60 min on BU agar that it produces after incubation tor the same length of time on control plates. Since maxim u m Trp + mutant frequencies are obtained after incubations on BU agar which permit little or no cell division, the mutant frequencies in these populations (corrected by subtraction of frequencies in control populations) probably correspond to the frequencies of BU-induced mutations. The similar responses of these four strains to BU as a mutagen contrasts sharply with their radically different susceptibilities to UV mutagenesis (see Table I). We conclude that BU mutagenesis in E. coli genes is largely independent of uvrA, recA and lexA gene products, and, by implication, of the dark repair mechanisms which require these gene products. Our results are compatible with a model of BU mutagenesis as due primarily to mispairing of the base analogue in DNA replication. They do not support the hypothesis that BU produces a type of DNA damage that is recognized by uvr gene products as a target for excision repair. We have no explanation for the apparently different pathways of BU mutagenesis operating in the DNA of E. coli and in the DNA of lambda bacteriophage.

Department of Biological Sciences, Douglass College, Rutgers University, New Brunswick, N. J. o89o3 (U.S.A.)

EVELYN M. W I T K I N ELIZABETH C. PARISI

I PIETRZYKOWSKA, I., O n t h e m e c h a n i s m of b r o m o u r a c i l - i n d u c e d m u t a g e n e s i s , Mutation Res., 19 (1973) 1-9. 2 STACY, K. A., AND E. SIMSON, I m p r o v e d m e t h o d for t h e isolation of t h y m i n e r e q u i r i n g m u t a n t s of Escherichia coli, J. Bacteriol, 9o (1965) 554-555. 3 VOGEL, H. J., AND D. M. BONNER, A c e t y l o r n i t h i n a s e of Escherichia coli : p a r t i a l purification a n d s o m e properties, J. Biol. Chem., 218 (1956) 97-1o6. 4 *VVITKIN, E. M., U l t r a v i o l e t - i n d u c e d m u t a t i o n a n d D N A repair, Ann. Rev. Microbiol., 23 (1968) 525-552 .

Received July 3oth, 1974