Bacteriophage T4 particles are refractory to bisulfite mutagenesis

Mutation Research, 129 (1984) 149-152 Elsevier

149

MTR 03940

Bacteriophage T4 particles are refractory to bisulfite mutagenesis Lynn S. Ripley and John W. Drake * Laboratory of Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709 (U.S.A.) (Received 12 April 1984) (Revision received 25 June 1984) (Accepted 20 July 1984)

Summary Bisulfite-induced deamination of cytosine produces uracil, a thymine analog reported to be mutagenic both in vitro and in vivo. Although deamination of cytosine in DNA should produce G : C ~ A : T transitions, treating bacteriophage T4 particles with 0.9 M bisulfite at pH 5 at 37 o C produced no more mutations than did the equivalent buffer without bisulfite. Lack of bisulfite mutagenicity is fully consistent with the reported resistance of 5-substituted cytosines to bisulfite-induced deamination, since T4 DNA contains glucosylated 5-hydroxymethylcytosine. However, bisulfite also failed to induce mutations in T4 particles whose DNA contained unmodified cytosine. The lack of mutagenesis persisted in E. coli hosts deficient in uracil glycosylase, an enzyme expected to participate in the repair of the putative bisulfite-generated uracil. Cytosine in T4 DNA may be largely protected from bisulfite attack within phage particles.

Bisulfite was reported to be mutagenic to bacteriophage T4 particles and to specifically induce G : C ~ A : T transitions (Summers and Drake, 1971). This mutagenic specificity appeared consistent with the ability of bisulfite to deaminate cytosine, producing uracil. However, subsequent chemical studies (Hayatsu and Shiragami, 1979; Wang et al., 1980) demonstrated that 5-substituted cytosines are not deaminated by bisulfite, although they are weakly subject to other alterations. Since T4 DNA contains glucosylated 5-hydroxymethylcytosine (G5HMC), its DNA might be expected to be protected against bisulfite-induced deamination. Moreover, personal communications from others indicated their inability to reproduce bisulfite mutagenesis in T4 particles. Thus, a re-investigation of bisulfite mutagenesis was undertaken. We first attempted to confirm the measurements reported in 1971 by measuring bisulfite-in* To whom correspondence should be addressed. 0027-5107/84/$03.00 © 1984 Elsevier Science Publishers B.V.

duced reversion of some of the same rlI mutants used previously. These mutants revert by G : C A : T transitions. Table 1 compares the revertant frequencies of rUV7 and rUV48 reported previously and measured here. The results of exposures to bisulfite in the two studies superficially appear similar, and clearly demonstrate increases in revertant frequencies after treating with 0.9 M bisulfite at pH 5 for 4 h at 37°C. The data sets diverge, however, when the critical buffer-control values are examined: in our hands, incubation in acetate buffer at pH 5 for 4 h at 37 °C produced increases in revertant frequencies indistinguishable from those produced by the bisulfite treatment. This result strongly suggests that it was not bisulfite that produced the observed mutagenesis, but merely exposure to low pH at 37 o C, a result fully consistent with the previously described phenomenon of 'heat' mutagenesis (Baltz et al., 1976). We have explored a number of aspects of the treatment procedures, seeking an explanation for the earlier failure to detect increased revertant

150 TABLE 1 REVERSION RESPONSES OF T 4 r l l MUTANTS TO BISULFITE TREATMENTS Mutant rUV7

rUV48

Protocol a

Revertants/10 7 survivors Untreated

Acetate

Bisulfite

1971 Current

3

1.1 23

10 16

1971 Current

4

0.8 11

14 12

a The '1971' protocol is that of Summers and Drake (1971), whose values are then listed in the table. Freshly prepared 1 M sodium bisulfite was adjusted to pH 5 with NaOH, 0.1 M sodium acetate was also adjusted to pH 5, and the reactions were terminated by 10-fold dilutions into D broth (0.2% Bacto tryptone, 0.5% NaCI). In the current protocol (which was, as far as we can tell, identical to the 1971 protocol), phage stocks were grown in E. coli BB cells at 37 o C (usually in M9 medium supplemented with casamino acids), prediluted to 1011/ml in D broth, diluted 10-fold in acetate or bisulfite solution, incubated 4 h at 37 o C in stoppered tubes, diluted 10-fold further in D broth, and assayed at low dilutions on KB cells (to score r l l ÷ revertants) and at high dilutions on BB cells (to score total survivors). Surviving fractions were typically close to 1 in acetate and about 0.5 in bisulfite.

frequencies in the a c e t a t e - b u f f e r controls. Because of d a y - t o - d a y variations in final rev e r t a n t frequencies, the p H values of b u f f e r a n d bisulfite solutions were d e t e r m i n e d , n o t o n l y at the time of p r e p a r a t i o n b u t also i m m e d i a t e l y after a d d i n g the p h a g e particles a n d after c o m p l e t i n g incubation. Differences on the o r d e r of 0 . 1 - 0 . 2 p H units were observed. L o w e r p H values t e n d e d to b e associated with higher r e v e r t a n t frequencies, a result expected if ' h e a t ' mutagenesis was responsib l e for the o b s e r v e d mutagenesis. I n o r d e r to c o n t r o l this p H variability, a d d i t i o n a l measurem e n t s were p e r f o r m e d in which the bisulfite solution was b u f f e r e d b y the inclusion of acetate. F o r each p h a g e tested, the p H of the acetate c o n t r o l was a d j u s t e d to equal that of the bisulfite solution (all values b e i n g close to p H 5.0). T h e results are shown in T a b l e 2; they c o n f i r m the i n a b i l i t y of bisulfite to increase revertant frequencies a b o v e those increased i n d u c e d b y the acetate buffer alone. H e a t - i n d u c e d m u t a g e n e s i s in T4 is d e p r e s s e d b y high salt c o n c e n t r a t i o n s (Baltz et al., 1976). It was therefore p o s s i b l e that the 0.9 M N a + i n t r o d u c e d

with the bisulfite t r e a t m e n t r e d u c e d heat m u t a g e n esis b y an a m o u n t that o b s c u r e d an increase ind u c e d b y bisulfite. However, this was not the case: i n c o r p o r a t i o n of 0.9 M NaC1 into the acetate buffer control lowered m u t a t i o n frequencies only slightly (Table 2). Thus the excess of mutagenesis a t t r i b u t a b l e to bisulfite t r e a t m e n t was only marginally d e t e c t a b l e (an average of 2-fold greater than the N a C 1 / a c e t a t e control). T h e p H d e p e n d e n c e of bisulfite t r e a t m e n t was then e x p l o r e d in an a t t e m p t to find a p H that w o u l d p e r m i t d e t e c t i o n of bisulfite-induced m u t a genesis a b o v e the b u f f e r - i n d u c e d b a c k g r o u n d . However, bisulfite t r e a t m e n t s at higher p H s (6, 7 a n d 8) i n d u c e d no reversion of r U V 7 , and treatm e n t s at p H 4.2 increased rates of reversion in b o t h acetate a n d bisulfite similarly ( d a t a not shown). E a c h of several sources of bisulfite was equally ineffective. These i n c l u d e d samples from different c o m p a n i e s (e.g., M a l i n c k r o d t a n d Sigma) a n d different lots. N e w l y p u r c h a s e d versus old, stored s a m p l e s (including one b o t t l e unsealed m o r e t h a n a y e a r previously) were u n i f o r m l y u n a b l e to i n d u c e mutagenesis. Bisulfite a d d u c t s at the 6 p o s i t i o n of cytosine constitute the d e a m i n a t i o n - p r o n e species; the resuiting 6 - s u b s t i t u t e d uracil can be cleaved to uracil b y e x p o s u r e to high p H ( H a y a t s u , 1976). Therefore, several s a m p l e s of bisulfite-treated r l I m u t a n t s were s u b s e q u e n t l y i n c u b a t e d for 30 min

TABLE 2 REVERSION RESPONSES OF T 4 r I I MUTANTS TO BUFFERED-BISULFITE TREATEMENTS Mutant

rUV7 rUV13 rUV48

Revertants/107 survivors a Untreated

Acetate

Acetate + Acetate + 0.9 M NaC1 bisulfite

2 2 3

13 11 9

4

9 12 10

a The protocol was as described in Table 1 except that the phage stocks were predihited in acetate buffer instead of D broth, the bisulfite solution also contained 0.1 M acetate, and pH values were closely monitored, The rUV7 entry is the average of 4 Expts., each of which exhibited the same trends. The rUVI3 and rUV48 entries represent single but typical experiments.

151

at pH 8 in 0.1 M Tris buffer. Revertant frequencies were not further increased. This test may, however, have limited relevance if bisulfite adducts do not form at the 6 position of G5HMC. Despite the inability of bisulfite to unequivocally mutate ordinary T4 particles, we expected that T4 particles containing plain cytosine instead of G5HMC would be mutated. Multiply mutant T4 strains whose DNA contained cytosine - - the d e n A - d e n B - 5 6 - a l c - strain of Snyder et al. (1976) grown on the B834 host strain - - plus an r l I mutation capable of reverting by a G : C A : T transition were therefore examined. Because the uracil expected to be produced in such T4 DNA is subject to removal by the host DNA-uracil N-glycosylase (Warner and Duncan, 1978), the phages were plated on an ung- host lacking the glycosylase. Typical results appear in Table 3. TABLE 3 REVERSION RESPONSES OF C Y T O S I N E - C O N T A I N I N G T 4 r l l M U T A N T S TO BISULFITE rUV7

Protocol a Host uracil- Revertants/107 survivors b

DNA

DNA Untreated Acetate Bisulfite glycosylase

G5HMC I II

+ +

9 12

29 23,25 21, 26

34 21,17 19, 20

13 16

18 35,15 27,17

15 18,58 18,32

-

Cytosine I

+ -

a Stocks of the multiple "1"4 mutant d e n A - d e n B - 5 6 - a l c r U V 7 were grown in M9 casamino acid medium and concentrated by differential centrifugation. Phages whose D N A contained G 5 H M C were grown on K803 cells, whose amber suppressor overcomes the block to 5 H M C production. Phages whose D N A contained cytosine were grown on B834 cells, which are s u - and do not restrict cytosine-containing T4 D N A . Spontaneous r l I + backgrounds and r l frequencies were always somewhat higher in stocks grown in K803 or B834 than in stocks grown in BB cells. In protocol I, the phages were prediluted to 1011/ml in D broth, treated with acetate or bisulfite as described in Table 1, diluted 10-fold into 0.1 M Tris buffer (pH 8.0) for 30 min at 37 o C, and assayed on K803(X) cells for r l l + revertants and on K803 cells for total survivors. In protocol II, the treated samples were passaged through one cycle of growth before being assayed as above; the passaging host was either BDl126 ung ÷ (proficient for uracil-DNA glycosylase) or B D l l 2 1 u n g - (lacking the glycosylase) (Duncan and Weiss, 1982). b Multiple entries are from 2 different Expts.

Although heat-induced mutagenesis was again observed, there was no significant increment of mutagenesis over the control in the bisulfite-treated samples. The cytosine content of these T4 stocks was confirmed by subjecting their DNA to digestion by EcoRI, H i n d l I I and H a e l I I restriction endonucleases, which are unable to act on T4 DNA containing G5HMC (A. Sugino, personal communication). The glycosylase deficiency of the host was confirmed by demonstrating that T4 particles (kindly provided by H. Warner), containing cytosine in place of G5HMC plus a substantial proportion of uracil residues (approximately 30%; see Warner and Duncan, 1978) in place of thymines, were restricted on the ung ÷ host but grew well on the ung- host. It should be noted that heat-induced mutagenesis was not enchanced by plating on the ung- host, although the r l I mutants used are believed to revert by cytosine deamination to produce uracil (Baltz et al., 1976), which should be subject to glycosylase attack in the ung ÷ host. This result suggest that, at least in the circumstances surrounding infection of the cell by T4, the glycosylase does not act sufficiently rapidly to remove a significant fraction of uracil residues before the first round of DNA replication can convert G : U base pairs to A : U base pairs. However, the very high levels of uracil in the phages used to confirm the properties of the host strain would be expected to produce lethality even with a slowly acting glycosylase. The possibility that carry-over of bisulfite (to an initial concentration in the top plating agar of about 0.003 M) or acetate inhibits glycosylase action was rendered unlikely by reconstruction controls that revealed no effect of such carry-over upon the low (ca. 0.1%) plating efficiency of the uracil-rich test phages on the ung ÷ host cell. We can offer no fully satisfactory explanation for the apparent immutability of T4 particles by bisulfite. The immutability of ordinary, G5HMCcontaining T4 is now expected because of the resistance of 5-substituted cytosines to bisulfite-induced deamination, but the immutability of cytosine-containing T4 must be explained in some other way. (Subsequent removal of uracil by the host glycosylase is clearly not the explanation, although other repair systems might achieve the same result.) One possibility is simply that T4

152 D N A m a y b e p r o t e c t e d b y virtue o f its packaging. This n o t i o n is s u p p o r t e d b y the o b s e r v a t i o n of H a y a t s u a n d S h i r a g a m i (1979) that the D N A of the closely related p h a g e T2 is susceptible to bisulfite a t t a c k when u n p a c k a g e d , b u t is resistant when resident in the intact particle. (The 5 H M C s of T2 D N A are n o t fully glycosylated, a n d their 5 - h y d r o x y m e t h y l g r o u p s react with bisulfite to generate - C H 3 S O 3 moieties.) Should T4 D N A be similarly p r o t e c t e d b y its packaging, then even c y t o s i n e - c o n t a i n i n g T4 D N A c o u l d be resistant to bisulfite mutagenesis. In vitro bisulfite t r e a t m e n t of s i n g l e - s t r a n d e d D N A followed b y its i n t r o d u c t i o n into a suitable cell results in mutagenesis whose specificity is fully consistent with a d e a m i n a t i o n m e c h a n i s m (Shortle a n d N a t h a n s , 1978). It is far less clear, however, that the in vivo bisulfite mutagenesis r e p o r t e d in a n u m b e r of o r g a n i s m s ( H a y a t s u , 1976; Shapiro, 1983) is e n g e n d e r e d b y d e a m i n a t i o n . F o r example, a l t h o u g h p h a g e ~ is m u t a g e n i z e d in vitro b y bisulfite t r e a t m e n t s ( H a y a t s u a n d Miura, 1970; I i d a et al., 1974; D u n c a n a n d Weiss, 1982), the m u t a genic specificity has not been d e t e r m i n e d , a n d the presence or a b s e n c e of host uracil glycosylase does n o t reveal a p a t t e r n that is s i m p l y consistent with a m u t a g e n i c m e c h a n i s m b a s e d on c y t o s i n e d e a m i n a t i o n . The efficacy of bisulfite as a m u t a gen in E. coli also r e m a i n s clouded: s o m e strains a p p e a r to r e s p o n d ( M u k a i et al., 1970) while others d o n o t ( K u n z a n d G l i c k m a n , 1983). Thus, the m e c h a n i s m a n d even the occurrence of bisulfite mutagenesis r e m a i n in question.

References Baltz, R.H., P.M. Bingham and J.W. Drake (1976) Heat mutagenesis in bacteriophage T4: the transition pathway, Proc. Natl. Acad. Sci. (U.S.A.), 73, 1269-1273.

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