495

Biochimica et Biophysica Acta, 564 (1979) 4 9 5 - - 5 0 6 © E l s e v i e r / N o r t h - H o l l a n d Biomedical Press

BBA 9 9 5 3 8

HOST-CELL REACTIVATION OF ALKYLATED T7 BACTERIOPHAGE *

D E N I S L A N E , M A R G A R E T D. M A M E T - B R A T L E Y a n d B A R B A R A K A R S K A - W Y S O C K I

Ddpartement de Biochimie, Facultd de Mddicine, Universitd de Montrdal, C.P. 6128, Succ. A, Montrdal, Qudbec H3C 3J7 (Canada) ( R e c e i v e d March 2 8 t h , 1979)

Key words: Alkylation; Host-cell reactivation; Base excision repair; DNA repair; (Phage T7)

Summary Purified T7 phage, treated with methyl methanesulfonate, was assayed on Escherichia coli K-12 host cells deficient in base excision repair. Phage survival, measured immediately after alkylation or following incubation to induce depurination, was lowest on a mutant defective in the polymerase activity of DNA polymerase I (p3478). Strains defective in endonuclease for apurinic sites (AB3027, BW2001) gave a significantly higher level of phage survival, as did the strain defective in the 5'--3' exonuclease activity of DNA polymerase I (RS5065). Highest survival of alkylated T7 phage was observed on the two wild-type strains (ABl157, W3110). These results show that alkylated T7 phage is subject to repair via the base excision repair pathway.

Introduction The base excision repair pathway for damaged DNA [1] starts with removal of the damaged or unusual base and simultaneous generation of an apurinic or apyrimidinic site. This first step may be accomplished enzymatically by a DNA N-glycosylase in the case of DNA containing uracil or 3-methyladenine [2] or non-enzymatically in the case of 7-methylguanine [3]. The site lacking a base is then recognized by an endonuclease which makes an incision (nick) in the polynucleotide chain, close to the lesion. Subsequent repair involves exonucleolytic transformation of the nick into a single-strand gap, followed by repair replication and excision of the apurinic site and finally ligation of the nick [4]. In Escherichia coli, both endonuclease IV [5] and endonuclease VI [4] can

* A preliminary r e p o r t o f t h e s e results was m a d e at t h e ICN-UCLA Symposium on DNA Repair Mechanisms, February, 1978 [17].

496

act on apurinic sites; the major of these two endonuclease activities, which we shall call endonuclease VI in accordance with the suggestion of Gossard and Verly [4], is inseparable biochemically and genetically from exonuclease III [6--9] (see also Ref. 10). In their model for repair, by E. coli enzymes, of apurinic sites in DNA, Gossard and Verly [4] propose that endonuclease VI introduces a nick on the 5'-side of the apurinic site. Exonuclease III, working in a 3'--5' direction from the nick, transforms it into a gap. DNA polymerase I then fills in the gap, excises the apurinic site in a di- or trinucleotide and translates the nick until it is finally closed by polynucleotide ligase. T7 bacteriophage has been used extensively as a biological model for study of the toxic action of chemicals on DNA [11--16]. However, possible repair o f chemical damage to phage DNA during the phage replicative cycle was not taken into account in these studies. Since E. coli mutants, defective in various enzymes in the base excision repair pathway, are n o w available, it should be possible to determine whether or not repair, mediated by host enzymes, does in fact occur in chemically damaged phage. In the present study, we have looked for repair of alkylated T7 phage by comparing phage survival on wildt y p e E. coli and on various mutants defective in endonuclease VI or in DNA polymerase I. Materials and Methods

Bacterial strains and growth conditions. All experiments were carried o u t with derivatives of E. coli K-12. Strains A B l 1 5 7 and W3110, thy- were used as wild-type strains. Table I presents a list of strains used, their relevant genotype, source and literature reference. Strains were stored in 20% glycerol at --15°C [22]. F o r the experiments reported here, the strains were grown, at 30°C, in broth (10 g Bacto-tryptone, 2.5 g yeast extract, 5 g NaC1, 0.12 g MgSO4, per liter, final pH adjusted to 7.2--7.3). Broth was supplemented with 20 pg/ml thymine for strains W3110, thy- and p3478. Measurement o f bacterial sensitivity to methyl methanesulfonate. These experiments were carried o u t by a method slightly different from that of Ljungquist et al. [19]. Cultures in the logarithmic growth phase were serially diluted in broth and plated on methyl methanesulfonate-containing plates. The plates contained broth supplemented with 1.5% agar and different concentra-

TABLE I S T R A I N S OF E. C O L I K-12 Strain

G e n o t y p e with respect to repair

Source

Reference

ABl157 BW2001 AB3027 W3110 thyp3478 RS5065

polA + polA + polA polA + polA

xthA + x thA xthA

polA

ex 2

B. Weiss B. Weiss E. c o l i G e n e t i c S t o c k C e n t e r E. c o l i G e n e t i c S t o c k C e n t e r E. coli Genetic Stock Center D. D e n h a r d t

18 6 19 18 20 21

497 tions of methyl methanesulfonate. The methyl methanesulfonate was added to the medium at 50°C just before the plates were poured. The plates were left at room temperature for 3 h before the bacteria were plated. After incubation of the plates at 30°C for 18 h, visible colonies were counted. Bacteriophage. Wild-type T7S (a gift from F.W. Studier) was multiplied on E. coli B grown in broth, was concentrated with polyethylene glycol/dextran sulfate, and was purified by CsC1 density gradient centrifugation, as previously described [23]. Phage suspensions were stored in 0.01 M Tris, 0.01 M MgCl2, 1 M NaC1, pH 7.5 (phage storage buffer). Phage assay. All phage titers were determined in broth medium by the agar layer method [24]. Petri dishes were incubated for 3--4 h at 30°C and visible plaques were counted. Titers were calculated as the average of plaque counts for three Petri dishes containing the same phage dilution. Alkylation of T7 phage and incubation of alkylated phage. Purified phage at a concentration of 5 • 1011--1 • 1012/ml was alkylated for 2 h at 37°C, in phosphate buffer (0.3 M Na2HPO4, pH 7.2) containing various concentrations of methyl methanesulfonate. At the end of the incubation, the phage suspension was cooled to 4°C. Then, it was either titered immediately (immediate inactivation) or dialyzed overnight versus phage storage buffer. Aliquots of the dialyzed phage suspension, distributed in small screw-top vials, were incubated for various times at 30°C and were titered immediately at the end of this incubation (delayed inactivation). Controls for all experiments were prepared simultaneously; all conditions were identical except for the absence of methyl methanesulfonate. Results

Sensitivity of bacterial strains to methyl methanesulfonate The different mutants listed in, Table I were either originally isolated on the basis of their methyl methanesulfonate sensitivity or were subsequently found to be methyl methanesulfonate sensitive. ~In order to have quantitative data on this sensitivity for all strains, we measured colony survival on freshly prepared plates containing various concentrations of methyl methanesulfonate. The results are illustrated in Fig. 1. The two wild-type strains, A B l 1 5 7 and W3110, thy-, were the most resistant to methyl methanesulfonate; A B l 1 5 7 showed 50% plating efficiency at 4.6 mM methyl methanesulfonate, in full agreement with Ljungquist et al. [19], while W3110, thy-, considerably more sensitive, had a 50% plating efficiency at 1.5 mM methyl methanesulfonate. Intermediate sensitivities were shown by BW2001 (50% plating efficiency, 1.1 mM) and RS5065 (50% plating efficiency, 0.9 mM). The two most sensitive strains were p3478 and AB3027. With a 50% plating efficiency at 0.42 mM, p3478 was very comparable to polA strains studied by Ljungquist et al. [19]. Strain AB3027, with defects in both endonuclease VI and DNA polymerase I, is extremely methyl methanesulfonate sensitive (50% plating efficiency at 0.17 mM methyl methanesulfonate, compared with 0.19 mM obtained by Ljungquist et al. [19]). The relative order of sensitivities is also in good agreement with results of Da Roza et al. [25].

498

4o

( 10 Concentration

(raM)

Fig. 1. M e t h y l m e t h a n e s u l f o n a t e s e n s i t i v i t y of d i f f e r e n t E. coli K - 1 2 strains. B a c t e r i a w e r e g r o w n o n freshly prepared plates containing different concentrations of m e t h y l m e t h a n e s u l f o n a t e and colonies were c o u n t e d . R e s u l t s are p r e s e n t e d as S/So, w h e r e S r e p r e s e n t s t h e n u m b e r o f c o l o n i e s d e v e l o p e d in p r e s e n c e o f m e t h y l m e t h a n e s u l f o n a t e a n d S O r e p r e s e n t s the n u m b e r o f c o l o n i e s in t h e a b s e n c e o f m e t h y l m e t h a n e s u l f o n a t e , as a f u n c t i o n of m e t h y l m e t h a n e s u l f o n a t e c o n c e n t r a t i o n , o, A B l 1 5 7 ; o, W 3 1 1 0 t hy -; A BW2001;A RSS065;.,p3478;D AB3027.

Efficiency of plating of bacterial strains for T7 phage Interpretation of phage survival curves after alkylation is facilitated if all strains plate untreated T7 phage with similar efficiencies. Results presented in Table II show that the strains used here are, in fact, very similar in their

T A B L E II E F F I C I E N C Y O F P L A T I N G O F B A C T E R I A L S T R A I N S F O R T7 P H A G E F o r p h a g e titers, f r e s h o v e r n i g h t c u l t u r e s o f e a c h b a c t e r i a l s t r a i n w e r e used. 0.5 m l o f b a c t e r i a l c u l t u r e at a c o n c e n t r a t i o n o f 5 • 1 0 8 / m l was m i x e d w i t h 1 m l o f t h e p h a g e d i l u t i o n a n d 3 m l o f soft agax; t h e mixt u r e w a s p l a t e d a c c o r d i n g t o t h e a g a r l a y e r m e t h o d [ 2 4 ] . E x p e r i m e n t a l e r r o r o f p h a g e t i t e r s w a s ± 10%. V a l u e s f o r t h e relative p h a g e t i t e r h a v e b e e n n o r m a l i z e d r e l a t i v e t o t h e t i t e r s o b t a i n e d o n s t r a i n A B l 1 5 7 . T h e s e c o n d c o l u m n gives t h e a v e r a g e v a l u e f o r r e l a t i v e p h a g e titer, b a s e d o n t h e n u m b e r o f d e t e r m i n a t i o n s i n d i c a t e d in t h e t h i r d c o l u m n . Strain

Relative phage titer

N u m b e r of d e t e r m i n a t i o n s

ABl157 BW2001 AB3027 W3110 thyp3478 RS5065

1 0.96 0.83 1.1 0.96 1.0

29 18 18 11 29 11

499 efficiency of plating T7 phage. The data in Table II represent values obtained from titrations of non-alkylated samples which served as controls in the experiments reported below. Inclusion of all these samples in the averages was possible because incubation of non-alkylated phage for as long as 72 h at 30°C had no measurable effect on phage titer. Values have been normalized relative to titers obtained on strain A B l 1 5 7 . In another series of control experiments, we compared the titer of alkylated phage on strain A B l 1 5 7 and on a related mutant defective in rec genes (strain JC5547 (rec A rec B rec C) [26] obtained from E. coli Genetic Stock Center). We found no significant differences in immediate or delayed inactivation. These results show that, in our experimental conditions of low multiplicity of infection, recombination mediated by host enzymes does n o t play a significant role in repair of alkylated phage. Immediate inactivation o f T 7 phage treated with m e t h y l methanesulfonate In a first set of experiments, phage survival was measured immediately after alkylation. Phage, at this moment, contains alkyl groups on bases and a limited number of apurinic sites and nicks [15]. Fig. 2 presents survival curves obtained using different host cells; log S/So, where S is the titer of alkylated

0

log S/So

-2

-3

-

0

4

~

10 Concentration (mM)

20

Fig. 2. I m m e d i a t e i n a c t i v a t i o n of T 7 p h a g e t r e a t e d w i t h m e t h y l m e t h a n e s u l f o n a t e . Phage titers w e r e d e t e r m i n e d , o n v a r i o u s b a c t e r i a l strains, i m m e d i a t e l y a f t e r t r e a t m e n t w i t h m e t h y l m e t h a n e s u l f o n a t e . T h e r e s u l t s a r e p r e s e n t e d as log S/SO, w h e r e S is p h a g e t i t e r a f t e r a l k y l a t i o n an~l S O is p h a g e t i t e r f o r t h e n o n a l k y l a t e d c o n t r o l , as a f u n c t i o n o f m e t h y l m e t h a n e s u l f o n a t e c o n c e n t r a t i o n . E a c h e x p e r i m e n t a l p o i n t r e p r e s e n t s t h e a v e r a g e o f t w o s e p a r a t e e x p e r i m e n t s ; a v e r a g e e x p e r i m e n t a l e r r o r i n log S/S 0 was +0.07. D i f f e r e n t s y m b o l s i n d i c a t e t h e v a r i o u s b a c t e r i a l h o s t s u s e d f o r t l t e r i n g p h a g e : e , A B l 1 5 7 ; ~, A B 3 0 2 7 ; A B W 2 0 0 1 ; a, p 3 4 7 8 .

500 phage and So, the titer of control phage, is plotted versus concentration of methyl methanesulfonate for each bacterial strain. It is clear from these curves that host-cell genotype affects survival. Survival (host-cell reactivation) is highest on wild-type strain A B l 1 5 7 and lowest {about 20 times less at the highest dose) on strain p3478, deficient in DNA polymerase I. Intermediate values were obtained for BW2001, deficient in endonuclease VI, and for AB3027, deficient in both endonuclease VI and DNA polymerase I. It is particularly striking that strain AB3027, itself very methyl methanesulfonate sensitive (Fig. 1), is less deficient than strain p 3 4 7 8 in host-cell reactivation of alkylated T7 phage. All survival curves on this figure show a discontinuity. Such a discontinuity has also been observed for ultraviolet-irradiated T7 phage [27] but its explanation remains obscure.

Delayed inactivation of T7 phage treated with methyl methanesulfonate Incubation of alkylated phage, which produces depurination, reduces phage survival [11,12]. We incubated dialyzed, alkylated phage suspensions for 24, 48 and 72 h at 30°C before titering them on the various host cells used in the preceding experiments. Fig. 3 shows typical results for this kind of experiment;

IogS/s

4

-5

-8

10 C o n c e n t r a t i o n (raM)

20

Fig. 3. I n a c t i v a t i o n o f T7 p h a g e t r e a t e d w i t h m e t h y l m e t h a n e s u l f o n a t e a n d i n c u b a t e d 48 h at 3 0 ° C . A t the end of the incubation period, phage titers were d e t e r m i n e d on various bacterial strains, identified by t h e s a m e s y m b o l s u s e d in Fig. 2. S r e p r e s e n t s t h e t i t e r o f p h a g e w h i c h w a s a l k y l a t e d a n d t h e n i n c u b a t e d w h i l e S O r e p r e s e n t s t h e t i t e r o f c o n t r o l p h a g e , t r e a t e d s i m u l t a n e o u s l y b u t in t h e a b s e n c e o f m e t h y l methanesulfonate. Each point represents the average of three different experiments; average experimental e r r o r in l o g S / S o w a s -+0.32.

501 this particular graph presents survival curves for alkylated phage, measured after 48 h of incubation. It is clear, from comparison with Fig. 2, that phage survival has decreased because of incubation subsequent to alkylation; apurinic sites, produced by hydrolysis Of the alkylated bases, are thus more toxic than the alkylated bases themselves. Phage survival is also a function of host-cell genotype. Once again, survival is highest on strain ABl157. Intermediate survival is observed on BW2001 and AB3027, while survival is lowest on strain p3478. Similar families of curves were obtained for the various times of incubation following alkylation. Although the relative order of host-cell proficiency for phage reactivation remained the same, absolute differences among the various host cells increased as a function of increasing incubation time. This is clearly seen in Fig. 4, which presents complete delayed inactivation curves for the wild-type host ABl157 and mutant p3478, deficient in DNA polymerase I and least proficient for hostcell reactivation of alkylated-depurinated T7 phage. Curves for strains BW2001 and AB3027 fall between these two extremes (data not shown). In the experiments of Fig. 4, as well as in those described below, difficulty was encountered in measuring phage titer for phage alkylated at the highest dose (0.02 M) and incubated for 48 or 72 h; this is probably due to the high concentration of inactivated phage particles in the dilutions needed to measure the phage titer of these samples.

I

or

20mM

-sit0

A

,

,

24

48

,

72 0 24 Hours at 30 ° after alkytation

48

72

Fig. 4. D e l a y e d i n a c t i v a t i o n o f T 7 p h a g e t r e a t e d w i t h m e t h y l m e t h a n e s u l f o n a t e . ( A ) Phage titers c a r r i e d o u t w i t h strain A B l 1 5 7 . (B) Phage t i t e r s c a r r i e d o u t w i t h s t r a i n p 3 4 7 8 . D i f f e r e n t c u r v e s c o r r e s p o n d t o d i f f e r e n t d o s e s of m e t h y l m e t h a n e s u l f o n a t e used f o r a l k y l a t i o n o f T 7 p h a g e . A l k y l a t e d p h a g e was dialy z e d a n d t h e n i n c u b a t e d f o r 0, 24, 4 8 or 72 h b e f o r e being titered; c o n t r o l phage u n d e r w e n t s i m u l t a n e ous a n d i d e n t i c a l t r e a t m e n t e x c e p t f o r t h e a b s e n c e of m e t h y l m e t h a n e s u l f o n a t e . E a c h p o i n t r e p r e s e n t s the a v e r a g e o f t h r e e d i f f e r e n t e x p e r i m e n t s e x c e p t f o r t h e p o i n t s at a dose o f 0 . 0 2 M, 72 h; f o r t h e s e p o i n t s , t w o e x p e r i m e n t s are i n c l u d e d f o r strain A B l 1 5 7 ( A ) a n d o n e e x p e r i m e n t f o r strain p 3 4 7 8 (B).

502

Importance of genetic background for host-cell reactivation of alkylated and depurinated T7 phage Although all host cells used in the preceding sections are strains of E. coli K-12, the mutants do not all derive from the same parent strain (see Table I); strains BW2001 and AB3027 derive from A B l 1 5 7 while p 3 4 7 8 derives from W3110, thy-. In addition, the two wild-type parent cells ( A B l 1 5 7 , W3110, thy-) have differing sensitivities to methyl methanesulfonate (Fig. 1). We thus felt it necessary to verify that the differences in phage survival observed for strains A B l 1 5 7 and p3478 were due to the lack of DNA polymerase I in the latter strain and n o t to other repair-unrelated differences in genetic background between the strains. Our verification consisted in comparing alkylated phage survival on strains A B l 1 5 7 and W3110, thy-, wild-type parent of strain p3478. Fig. 5 shows the survival curves for alkylated, depurinated T7 phage. It is clear that the two host cells support phage survival in a very similar way, despite their own differing sensitivities to methyl methanesulfonate.

Role of DNA polymerase I in survival of alkylated phage Strain p 3 4 7 8 bears a defect in the polymerizing activity of DNA polymerase I b u t the 5'--3' exonuclease activity associated with the enzyme appears to be normal [ 2 8 ] . Konrad [21] and Konrad and Lehman [29] have isolated

0[

I

I

24 48 Hours at 30 ° after a l k y l a t i o n

72

Fig. 5. C o m p a r i s o n o f d e l a y e d i n a c t i v a t i o n c u r v e s o f a l k y l a t e d T 7 p h a g e , d e t e r m i n e d o n t w o d i f f e r e n t w i l d - t y p e s t r a i n s o f E. coli K - 1 2 . e , A B I I 5 7 ~ ©, W 3 3 1 0 t h y - ; o t h e r d e t a i l s a n d u n i t s are e x p l a i n e d in F i g . 4. E a c h p o i n t r e p r e s e n t s t h e average o f t w o e x p e r i m e n t s e x c e p t f o r t h e p o i n t s at a d o s e o f 0 . 0 2 M m e t h y l m e t h a n e s u l f o n a t e , 72 h, w h i c h are t h e r e s u l t s o f o n e e x p e r i m e n t .

503

°l

2o r

-2 I

-8

I

~ ,

24 48 Hours at 30° after alkylotion

20mM 72

Fig. 6. I n f l u e n c e of m u t a t i o n s a t t h e p o I A l o c u s in E. co|i K - 1 2 o n d e l a y e d i n a c t i v a t i o n o f a l k y l a t e d T 7 p h a g e . P h a g e was t r e a t e d as d e s c r i b e d in t h e l e g e n d o f Fig. 4. S y m b o l s : f o r b a c t e r i a l strains, o, W 3 1 1 0 t h y - ; ~, R S 5 0 6 5 ; m, p 3 4 7 8 . E a c h g r o u p o f c u r v e s c o r r e s p o n d s to a different dose of m e t h y l m e t h a n e s u l f o n a t e . All p o i n t s r e p r e s e n t t h e a v e r a g e o f t w o d i f f e r e n t e x p e r i m e n t s w i t h t h e f o l l o w i n g exception: p o i n t s at 0 . 0 2 M, 4 8 h, f o r strains R S 5 0 6 5 a n d p 3 4 7 8 are t h e results o f o n e experiment.

mutants defective in the 5'--3' exonuclease activity but retaining normal polymerizing activity. Strain RS5065 is one of these mutants [29] (see also Ref. 30); it has low residual 5'--3' exonuclease activity at 30°C (less than 3% of normal activity, as tested in vitro) but normal polymerizing activity. In order to study the relative importance of the two activities of DNA polymerase I, we compared phage survival on strains W3110 thy-, RS5065 and p3478. The results are shown in Fig. 6. The absence of the polymerizing activity is clearly more detrimental to phage survival than the absence of the 5'--3' exonuclease activity. Discussion T7 phage treated with methyl methanesulfonate constitutes a well-characterized repair substrate. Its DNA contains, as the two major lesions, the alkylated purine bases, 3-methyladenine and 7-methylguanine; it has very few phosphotriesters and only a small number of apurinic sites and single-stranded breaks [15]. One would expect these DNA lesions to be repaired by the base excision pathway [1 ]. Our results suggest this repair pathway is indeed operative in methyl

504 methanesulfonate-treated T7 phage. When alkylated phage is titered on bacterial strains defective in endonuclease Vl or DNA polymerase I, its survival is diminished. The absence of these enzymes presumably prevents normal repair from occurring and thus lowers phage survival. Earlier claims that alkylated T7 phage does not undergo repair [13] are therefore unfounded. Having shown that repair via host-cell enzymes (host-cell reactivation) does occur, we may examine the various pathways available for base excision repair in alkylated phage. It is well-established, at least for ultraviolet-irradiated bacteria, that multiple pathways exist for repair of lesions; for example, DNA polymerase I can be replaced by other polymerases for repair replication [31] and various 5'--3' exonucleases apparently function in excision [30]. A comparison of host-cell survival in the presence of methyl methanesulfonate and alkylated phage survival measured on these host cells should indicate similarities or differences in the repair pathways used. The two wild-type hosts, A B l 1 5 7 and W3110, have different sensitivities to methyl methanesulfonate b u t the survival of methyl methanesulfonate-alkylated phage on these two hosts is comparable. Host cell AB3027 (polA, xthA), bearing two repairrelated mutations, is extremely methyl methanesulfonate sensitive; however alkylated T7 phage survival is higher on this strain than on strain p3478 (polA) which itself.is less methyl methanesulfonate sensitive than AB3027. These results suggest that alkylated T7 phage may be repaired in a less complex way than alkylated E. coli, i.e. through use of fewer repair pathways. Thus T7 phage may be a valuable probe for study of certain repair pathways in E. coll. Our studies show that endonuclease VI plays a role in repair of alkylated phage DNA. Few apurinic sites exist in the alkylated phage at the time of injection [15]. However, their number presumably increases rapidly after injection, due to enzymic removal of 3-methyladenine. These newly produced apurinic sites u n d o u b t e d l y explain the importance of endonuclease VI for phage survival immediately after alkylation. This point could be further verified when mutants in 3-methyladenine-DNA glycosylase [32] become available. Due to non-enzymic depurination of both 3-methyladenine and 7-methylguanine, the number of apurinic sites continually increases during incubation of alkylated phage at 30°C. This results in lower phage survival and enhances the differences in phage survival measured on wild-type and endonuclease VI-deficient hosts. Lack of endonuclease VI appears to affect phage survival less than the lack of DNA polymerase I. Perhaps, in xthA strains, endonuclease V! can be replaced by the minor apurinic endonuclease activity, endonuclease IV [51. E. coli DNA polymerase I apparently plays a very important role in repair of alkylated and alkylated-depurinated T7 phage. Phage survival is lowest on strain p3478, which is deficient in the polymerizing activity of this enzyme. A defect in the 5'--3' exonuclease activity of DNA polymerase I, while lowering alkylated phage survival, is less detrimental than a defect in the polymerizing activity. This relative importance, in repair, of the polymerizing activity compared to the 5'--3' exonuclease activity of DNA polymerase I has also been observed for ultraviolet-irradiated bacteria [31] and nitrous acid-treated bacteria [25]. Surprisingly, the double mutant AB3027 (polA, xthA) supports T7 survival better than strain p3478. This may simply show that endonuclease

505 VI and DNA polymerase I are involved in the same repair pathway for alkylated T7 phage; once the endonuclease is lacking, the loss of polymerase activity confers no additional sensitivity (for reasoning, see Ref. 33). Another possible explanation relates to the residual level of polymerizing activity (as detected by in vitro assay) present in these two polA strains; strain AB3027 has a higher residual activity than does strain p3478 and it also better supports survival of alkylated T7 phage. One might ask if T7 DNA polymerase, the product of gene 5, could be active in repair. While results of Kuemmerle and Masker [27] suggest that the T7 gene 5 product can substitute for DNA polymerase I, at least in an in vitro repair system for ultraviolet-induced damages, our results for alkylated phage suggest that DNA polymerase I must be more effective than the phage polymerase. Lawley et al. [12] and Brakier and Verly [13] have shown that delayed inactivation can be correlated with the production of 6--8 apurinic sites in the phage DNA. Taking into account the repair of lesions which we have shown here, would decrease this figure. Thus it is clear that a small number of lesions inactivates the phage. This could be due to the fact that the phage replicative cycle is relatively short and that repair, to be effective, must take place prior to replication when phage infection occurs at low multiplicities; indeed, in these conditions, repair by recombination should not occur for alkylated phage and our results with the mutant JC5547 (recA, recB, recC) support this idea. We know however, that alkylation of phage interferes with DNA synthesis [34] and greatly retards the phage infective cycle [35]; there should thus be adequate time for repair. Alternatively, the high activity of endonuclease VI could produce such extensive DNA degradation in cases of highly alkylated and depurinated DNA [36] that repair becomes impossible. Analysis of intracellular phage DNA should provide insight into this problem.

Acknowledgements This work was supported by the Natural Sciences and Engineering Research Council of Canada, La Fondation Jos. RhSaume, and l'Universit~ de Montreal. D.L. was the recipient of a scholarship from the Minist~re de l'Education (Quebec).

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