Mu~.alion Research, 28 (1975) 3o5-3o8 © Elsevier Scientific Publishing Company, A m s t e r d a m - - P r i n t e d in The Netherlands

305

SHORT COMMUNICATION Effects of the Escherichia coli K12 recA56, uvrB and polA mutations on U V reactivation in bacteriophage T7 Survival of UV-damaged bacteriophage ~ is increased when the bacterial host itself is irradiated with small doses of UV light 28. The increased phage survival is known as UV reactivation, although it can be initiated by several other agents or treatments, including X-rays ~1, nitrogen mustard 28, mitomycin C 2~, HNO2 (T. L. KERR, unpublished), thymine starvation 1~ and presence of the tif mutation 8. Phage damage by a number of different agents, including UV, HNO221, NH~OH 2~, nitrogen mustard (T. L. K E ~ , unpublished) and 5-bromouracil incorporation before UV damage ~,1~ is also reactivated. The reactivation process occurs in several phages including ;~28, T I °-6, T3 ~, p i l l P2 '~, P229, H P I 1., ~3R-~ and the Serratia phage Kappa ~°. Attempts to identify the bacterial function involved by studying the reactivating capacity of different bacterial mutants have not given simple results b~cause the mutants vary in their effect on different phages and with the type of damage used. The experiments reported below show major differences between the effects of the bacterial recA, uvrB and polA mutations on UV reactivation in phage T7 and those reported for phage ;~. We conclude that either the recA or the uvrB function is needed to initiate UV reactivation in the bacterium or as a part of the basic reactivation mechanism itself. UV survival of phage T 7 is the same in the uvrB mutant as in the double mutant recA uvrB. Our results confirm this and also show reduced survival on the polA strain (Fig. Ia). Repair of UV damaged T 7 is therefore restricted to excision and differs from that in phage lambda in which both the recA and uvrB mutations reduce survival independently 2~. As shown in Fig. Ib UV reactivation of phage T 7 occurs in recA mutants but does not in uvrB, polA, or recA uvrB bacteria. UV-reactivation of UV damaged phage ). does not occur in recA ~° or reck4 uvr~, ~8 mutants. In wild type strains UV reactivation reaches a m a x i m u m at a UV dose of about 300 erg/mm ~ for phages ;~ and T7 (unpublished data). Fig. IC shows the survival of phage T 7 treated with HNO~ and plated on the different host bacteria. No significant differences in survival were apparent. Repair of HNO., damaged phage T 7 by recA, uvrB or polA gene products was absent. The UV reactivation curves for phage T7 HNO~ damage are shown in Fig. Id. The wild type strain showed a dose-reduction effect with HNO~ damage, reaching m a x i m u m reactivation at a UV dose of only 50 erg/mm ~. Neither the racA nor the uvrB mutations eliminated reactivation separately, but the recA uvrB combination did so. UV reactivation was absent in the polA mutant. We have also shown that UV reactivation of HN02 damaged phage ;~ is blocked in recA uvrB double mutants. In phage T 7 the uvrB gene product is thus essential for UV reactivation of UV damage but not HNO~ damage. In phage ;~ UV reactivation occurs in uvrB ~ mutants, but at a lower UV dose for maximal reactivation. The reactivation mechanism m a y act directly on lesions generated by HNO2 but be unable to repair pyrimidine dimers until an excision gap is produced. In phage ;~ such repairable gaps m a y be generated by replication ~ or by the recA and red gene products, known to affect ;~ survival ~8,~.

306

SHORT COMMUNICATION

i

. , N., . ~

-50 -52 -54

-~ -6 ~ ~ ~ # -1 ~

~ -56 150 300 4~0 600 50 1~ 150 200 25~ Uvdoseinerg/mm2 lc~J ~ q - 4.6 -47

~ =

= ~ - - - 1

~

~

-

,

,

5

~

-2

-4.8

.~ ~

-3

-49

I -4

~ I x ~~ " "

"

.j-50

-6

-52 o

2

~

e

Hno2(min)

s

,o

o

~o *~ ~go 2~o2go Uv dose in erg/mm2

Fig. Ia. UV survival of p h a g e T 7. Buffer suspensions of b a c t e r i o p h a g e T 7 were UV irradiated in I m m layers a n d plated directly b y the agar layer m e t h o d ~ on the following Escherichia coli strains: ~, A B I i57~ ; ~, JC5o88 recA 56~; ~, J G I I2(a) polA ( F r o m Dr. J. D. G~oss) ; ~, ABI885 uvrBta; X, QB248 recAuvrB (a r e c o m b i n a n t derived flora JC5o88 recA H f r and ABI885 uvrB F ) Fig. I b. UV reactivation of U V - d a m a g e d (I o -~ survival) bacteriophage T 7. Log fraction survival of UV irradiated p h a g e T 7 adsorbed to bacteria UV irradiated for different periods before adsorption ~a. S y m b o l s and Escherichia coli strains as in Fig. ia. Fig. ic. Survival of HNO~-treated bacteriophage T 7. I.O ml. of phage suspension was diluted iofold in o.o5 M NaNO~ in o.2 M acetate buffer, p H 4.8, at 37 °. The t r e a t m e n t was t e r m i n a t e d by diluting ioo-fold into chilled p h o s p h a t e buffer, p H 7.2. Results are s h o w n for strains AB1157 and QB248 recAuvrB. SurvivM of all other strains listed u n d e r Fig. I a fell within this range. S y m b o l s as in Fig. i a. Fig. id. UV reactivation of HNO~-damaged (io s survival) bacteriophage T 7. Log fraction s u r v i v a l of HNO~-damaged phage adsorbed to bacteria UV irradiated for different periods. Escherichia coli strains and s y m b o l s as in Fig. ia. E x p e r i m e n t s were done three times. The H a n o v i a Chromatolite UV l a m p was calibrated using a B l a c k - R a y UV m e t e r (Ultra Violet P r o d u c t s Inc., San Gabriel, Calif., U.S.A.).

Several authors have suggestedS,11, 22 that U V reactivation is a new repair process induced by the host treatment. Since no single m u t a n t has yet been discovered which inhibits U V reactivation under all conditions, the process is unlikely to be confined to the repair of D N A damage and is probably a part of essential host D N A

SHORT COMMUNICATION TABLE

307

I

U V REACTIVATION OF PHAGES T 7 AND ~

Phage

Phage damage T7

HNO~

UV

HNO~

÷

÷ --

÷~ _~

÷ ÷~ + a

Strain ~IJr~

rec~ polA uvrB recA wild type

2

UV

__

__

+

_

__a

_

÷

÷

--

16~18

__~

÷

+ , UV reactivation; --, no UV reactivation aUnpublished data. m e t a b o l i s m in which m u t a t i o n s would be lethal. The fact t h a t no b a c t e r i a l m u t a n t has been f o u n d to reduce s u r v i v a l of H N O ~ - d a m a g e d p h a g e T 7 {Fig. lC) suggests t h a t rep a i r of this t y p e of p h a g e d a m a g e does not occur in u n i r r a d i a t e d b a c t e r i a . UV reactiv a t i o n of H N 0 2 d a m a g e in p h a g e T 7 is therefore n o t a redirection of a k n o w n r e p a i r mechanism. The so far universal effect of t h e recA uvrB double m u t a t i o n in blocking UV r e a c t i v a t i o n suggests t h a t one of the p r o d u c t s of these two genes m u s t be p r e s e n t for its i n d u c t i o n or as p a r t of t h e basic mechanism. Since the p r o d u c t s of b o t h genes are k n o w n to act on b a c t e r i a l D N A one m u s t conclude t h a t UV r e a c t i v a t i o n involves b a c t e r i a l D N A , at least when it is i n i t i a t e d b y UV i r r a d i a t i o n of the host b a c t e r i u m . A c o m p a r i s o n of the UV r e a c t i v a t i o n of p h a g e s T 7 a n d ~ is given in Table I. This p a p e r contains the following o b s e r v a t i o n s : (I} UV r e a c t i v a t i o n of UVd a m a g e d p h a g e T 7 is b l o c k e d b y the uvrB m u t a t i o n (this is n o t so in p h a g e X) a n d m a y be a process of D N A g a p repair. {2} UV r e a c t i v a t i o n of U V - d a m a g e d p h a g e T 7 can occur in recA Eseheriehia coli (recA p r e v e n t s UV r e a c t i v a t i o n of U V - d a m a g e d ~t refs. 8, 14, i 6 a n d 20). (3) The polA m u t a t i o n blocks UV r e a c t i v a t i o n of UV a n d HNO~ d a m a g e in p h a g e T 7 (polA does n o t do so in p h a g e ).4}. (4) E. eoli m u t a t i o n s uvrB, polA, reeA a n d reeA uvrB do n o t alter the s e n s i t i v i t y of p h a g e T 7 infecting t h e m to HNO~ d a m a g e . {~} P h a g e T 7 differs from p h a g e ~ in showing a lower UV d o s e - r e s p o n s e for m a x i m u m UV r e a c t i v a t i o n of HNO~ d a m a g e . (6} Double m u t a n t reeH uvrB b a c t e r i a do not UV r e a c t i v a t e p h a g e T 7 d a m a g e d w i t h UV or HNO~. These m u t a n t s h a v e t h u s been u n a b l e to UV r e a c t i v a t e u n d e r all conditions so far tested, a n d are the only class of m u t a n t s k n o w n to have this p r o p e r t y . The a u t h o r s t h a n k Mr. A. J. JONES for technical assistance. R. A. McKEE acknowledges an a w a r d from t h e N o r t h e r n I r e l a n d Ministry of E d u c a t i o n .

Sub-Department of Genetics, R . A . MCKEE* The Queen's University, M . G . R . HART Belfast (Northern Ireland) I Ar)AMS, M. I7I., in The Bacteriophages, Interscience, New York, 1959 2 BACHMAN,B. J . , Pedigrees of some mutant strains of Escherichia coli K-I2, Bacteriol. Rev., 36 (1972 )

525--557

•

3 BERTANI, L. E., Host-dependent induction of phage mutants and lysogenisation,

Virology, 12

~I96O) 553-569 • • Present address: School of Agriculture, Sutton Bonington, Loughborough (Great Britain).

308

SHORT COMMUNICATION

4 CAILLET-FAUQUET, ~P., AND M. DEFAIS, UV reactivation of phage 2 in a polA m u t a n t of E. cell, Mutation Res., 15 (1972) 353-3555 CALKINS, J., The T - N - P R model of radiation response, J. Theoret. Biol., 39 (1973) 609-622. 6 CASTELLAZZI, M., J. GEORGE AND G. BUTTIN, l°rophage induction and cell division in E. coli, I. F u r t h e r characterisation of the t h e r m o s e n s i t i v e m u t a t i o n tif-I whose expressiml mimics the effect of UV irradiation, 1Viol. Gen. Genet., 119 (1972) 139 152. 7 CLARK, A. J., The beginning of a genetic analysis of r e c o m b i n a t i o n proficiency J. Cell. Physiol.. 7 ° Suppl. i (1967) i65-18o. 8 DEFAIS, M., P. FAUQUET, M. RADMAN AND M. ERRERA, Ultraviolet reactivation and ultraviolet mutagenesis of 2 in different genetic systems, Virology, 43 (1971) 495-503 • 9 GAREN, A., AND ~NT.D. ZINDER, Radiological evidence for partial genetic h o m o l o g y between bacteriophage and h o s t bacteria, Virology, i (1955) 347-376. io GEISSLER, E., Differences in host controlled reactivation of lambdoid and T phages, Mol. Gen. Genet. lO 9 ~I97O) 257-263. I I GEORGE, J., R. DEVORET AND M. RADMAN, I n d i r e c t ultraviolet reactivation cf phage 2, Prec. Natl. -dead. Sci. ( U . S . A . ) 71 (1974) 144-147. I2 HARM, W., On t h e relationship bet~veen h o s t cell reactivatiml and UV reactivation in UV inactivated phages, Z. Vererbungsl., 94 (1963) 67-79. i5 HARM, W., AND C. S. ]~UPERT, Infection of t r a n s f o r m a b l e cells of Haemophilus influenzae by bacteriophage and bacteriophage DNA, Z. Vererbungsl., 94 (1963) 67-79. 14 HART, M. G. R., AND J. ELLISON, Ultraviolet reactivation in bacteriophage lambda. J. Gen. Virol., 8 (197 o) 197-2o8. 15 HOWARD-FLANDERS, i°., R. 1~. BOYCE AND L. THERIOT, Three loci in Escherichia cell K - I 2 t h a t control the excision of pyrimidine dinlers and certain other lnutagen p r o d u c t s from DNA, Genetics, 53 (1966) 1119 1136. 16 KERR, T. L., AND M. G. R. HART, Effects of the rec and exr m u t a t i o n s of Escherichia cell on UV reactivation of bacteriophage l a m b d a d a m a g e d b y different agents, Mutation Res., 15 (1972) 247 25817 KERR, T. L., AND M. G. R. HART, Effects of the recA, lex and exrA m u t a t i o n s on the survival of d a m a g e d 2 and P I phages in lysogenic and non-lysogenic strains of Escherichia coli K-I2, Mutation Res., 18 (1973) 113 116. 18 KNESER, H., Relationship between K-reactivation and UV-reactivation of bacteriophage lambda, Virology, 36 (1968) 303-305 . 19 KNESER, H., l(. METZGER AND W. SAUERBIER, Evidence of different m e c h a n i s m s for ultraviolet reactivation and " o r d i n a r y h o s t cell r e a c t i v a t i o n " of bacteriophage lambda, Virology, 27 (1965) 213-221. 20 OGA\VA, H., K. SHIMADA AND J. TOMIZAWA, Studies on radiation sensitive m u t a n t s of Escherichia coli, I. M u t a n t s defective in the repair synthesis, Mol. Gen. Genet. i o i (1968) 227-244. 21 ONe, J., AND Y. SHIMAZU, Ultraviolet reactivation of a bacteriophage containing a singles t r a n d e d deoxyribonucleic acid as a genetic element, Virology, 29 (1966) 295 302. 22 OTSUJI, N., AND S. OKUBO, t~eactivation of ultraviolet and nitrous acid inactivated phages by host cells, Virology, 12 (196o) 607-6o9. 23 RADMAN, M., L. CORDONE, D. I~RSMANOVIC-SIMICAND M. ERRERA, C o m p l e m e n t a r y action of r e c o m b i n a t i o n and excision in t h e repair of ultraviolet irradiation d a m a g e to DNA, J. Mol. Biol. 49 (197 o) 2o3-212. 24 RUPP, W. D., AND t ). HOWARD-FLANDERS, Discontinuities in the D N A synthesised in an excision-defective strain of Escherichia cell following ultraviolet irradiation, J. ~Ilol. Biol. 31 (1968) 291-3o4 • 25 SIGNER, E. R., AND J. WEIL, R e c o m b i n a t i o n in bacteriophage 2, I. M u t a n t s deficient in general recombination, J. Mol. Biol. 34 (1968) 261-271. 26 TESSMAN, E. S., G r o w t h and m u t a t i o n of p h a g e T I on ultraviolet-irradiated host cells, Virology 2 (1956) 679-688. 27 VIZDALOVA, M., The inactivating effect of h y d r o x y l a m i n e on E. cell phages and the possibility of repair of the r e s u l t a n t damage, Int. J. Radiat. Biol. 16 (1969) 147 155. 28 WEIGLE, J. J., I n d u c t i o n of m u t a t i o n s in a bacterial virus, Prec. Natl. Acad. Sci. ( U . S . A ) . , 39 (19.53) 628-636. 29 WEIGLE, J. J., AND R. DULBECCO, I n d u c t i o n of m u t a t i o n s in bacteriophage T 3 b y ultraviolet light, Experientia, 9 (1953) 372 373. 3 ° W'INKLER, V. U., Studien fiber die UV-induzierte m u t a b i l i t a t des Serratia-phagen K a p p a d u t c h versuche mit U V - b e s t r a h l t e m indikator u n d p h a g e n k r e u z u n g e n , Z. Naturforsch. iSb (1963) 118 123 . R e c e i v e d O c t o b e r 8th, 1974 Revision received January

8 t h , 1975