J. Mol. Biol. (1976) 108, 67-81

Stimulation of the Synthesis of Bacteriophage T4 Gene 32 Protein by Ultraviolet Light Irradiation H. M. KRISCH

AND

G. VAN

HOUW~.

D~partement de Biologic Mol~culaire Universitd de Gen~ve, Geneva, Switzerland (Received 6 February 1976, and in revised form 11 August 1976) The synthesis of bacteriophage T4 gene 32 product, P32, has been followed by gel electrophoresis of lysates of infected cells which have been irradiated with ultraviolet light. In wild-type infections irradiation after the commencement of late gene expression results in a rapid stimulation of the rate of P32 synthesis. Within four minutes after irradiation P32 is synthesized at 11 times the rate of the unirradiated control infection. P32 seems to be the only T4 protein which exhibits such u.v. inducibility. This inducibility is dependent on the function of genes d6 and 47 and to a lesser extent on several other T4 genes thought to be involved in repair (P43, w and y). An infection defective in both P43 and P46 shows essentially no stimulation of the rate of P32 synthesis after irradiation. In the absence of DNA replication the parental DNA is degraded after irradiation in a dose-dependent manner. The extent of P32 induction in such an infection is also proportional to the dose. I t is suggested that the production of gaps during repair of u.v.-irradiated DNA is responsible for the stimulation of P32 synthesis. A model is proposed in which such regions of single-stranded DNA compete for P32 by binding it nonspecifically, thus reducing the amount of P32 free to block the expression of gene 32. Because the expression of gene 32 is self-regulatory this would result in increased P32 synthesis. The possible role of P32 in the repair of u.v.-damaged DNA is discussed.

1. Introduction There appear to be at least two genetically defined u.v. repair pathways in bacteriophage T4-infected Escherichia coli (Maynard-Smith & Symonds, 1973). Excision repair in T4 is mediated by a phage-encoded (gene v) thymine dimer-specific endonuclease (Yasuda & Sekiguchi, 1970). The mechanism of post-endonucleolytic dimer excision is unknown as no phage m u t a n t (other than v) has been shown to be specifically defective in dimer excision (Mortelmans & Friedberg, 1972). There is evidence which suggests, however, t h a t host functions are capable of excising the dimer after the incision step has been carried out b y the v gene endonuelease (Friedberg & King, 1971). The second repair p a t h w a y resembles, at least superficially, the post-replication repair in E. coll. T4 mutants in this pathway are sensitive to a variety of agents in addition to u.v. (X-rays, alkylating agents, mitomycin C and ~,-rays (Mortelmans & Friedberg, 1972)) and affect the level of genetic recombination (Symonds et al., 1973). I t has been known for a long time that u.v.-induced lesions increase the probability of a genetic exchange occurring between D N A molecules in infected cells (Jacob & 67

68

H . M . K R I S C H AND G. VAN H O U W E

Wollman, 1955; Hershey et al., 1958). I t is unclear, however, whether such recombination is an essential p a r t of a repair process or if an intermediate in the repair p a t h w a y is frequently shunted into the recombination pathway. The role of P32 in u.v. repair has not been precisely defined, although it has been demonstrated t h a t m u t a n t s in this gene are u.v.-sensitive under semi-permissive conditions (Baldy, 1970; Wallace & l~Ielamede, 1972). I t is thought t h a t the function P32 plays in repair is essentially the same, as its presumed role in D N A synthesis (Alberts & Frey, 1970; Delius et al., 1972). The ability of P32 to catalyse the denaturation of D N A and to stabilize single-stranded regions of D N A could facilitate the association of repair system proteins with their substrates and might enhance their activity (Huberman et al., 1971). P32 m a y have an additional crucial role in repair in t h a t when it binds to single-stranded regions (for example to gaps generated b y repair) it protects them from being attacked b y nucleuses (Curtis & Alberts, 1976). This function m a y be essential to preserve the integrity of a gapped D N A intermediate until the repair process is complete. A question raised by our previous work on the autoregulation of the synthesis of P32 (Krisch et al., 1974) is what is the physiological function of such cont:rol in the T4 bacteriophage-infected cell? The processes of replication, recombination and repair all generate single-stranded regions of DNA to which P32 can bind and the relative importance of these processes could v a r y significantly in the different situations T4 encounters in nature. Since P32 is normally required in very large amounts (104 molecules/infected cell; Alberts & Frey, 1970), a mechanism to ensure an adequate supply of this gene product m a y be essential. The ability to increase specifically the amount of I)32 available to function in the repair of u.v. lesions, for example, could be particularly advantageous. We have explored the possible role of P32 serf-regulation in radiation repair.

2. Materials and M e t h o d s (a) Bacterial straln8 E. coli B E (sup e) from the Geneva collection was used as the host bacterium in all experiments, except where noted. E. cell AS19, a permeability mutant derived from E. cell B, was used as the host in the Rimactan experiments (Sekiguehi & Iida, 1967). E. coli CR63 (sup+l) was used to prepare phage stocks, and as the permissive host for phage containing amber mutations. (b) Bacteriophage All of the phages used in these experiments are mutants of T4D. The wild-type TdD used in this laboratory was originally obtained from A. H. Doermarm. Except for the gene t mutant, taxnA3, (Josslin, 1970,1971), the amber mutants are described by Epstein et al. (1963). The T4 radiation-sensitive mutants were all provided by N. V. Hamlett (ttamlett & Berger, 1975). Phage containing combinations of these mutations were constructed by standard cross procedures (Krisch et al., 1972). The identification of complex genotypes was accomplished following the methods described by Doermann & Boehner (1970). The identification of the u.v.-sensitive markers in complex genetic backgrounds is described by Hamlett & Berger (1975). The following mutant phages were used: 30amH39X, 41amN81, 42amN122, 43araB22, 43araB263, 44amN82, 45amE10, 46am_N130, 47amA456, tarnA3, v, w-m22, and yl00. (c) Media M9S medium (Champe & Benzer, 1962) which contains 0.2% (w/v) Casamino acids was used in the preparation of stocks and for the growth of host bacteria. Hershey broth

ULTRAVIOLET

LIGHT INDUCIBILITY

OF P32 S Y N T H E S I S

69

(Steinberg & Edgar, 1962) was used as the growth m e d i u m for the preparation of indicator bacteria. Phage and bacteria were plated on Hershey bottom agar using E H A top layer agar (Steinberg & Edgar, 1962). (d) Experimental procedure H o s t cells were grown at 37~ to 108/ml in M9S m e d i n m and then centrifuged and resuspended at a cell density of 1.4 x 108/ml in M9S.1 medium, which contains only 0"02~/o Casamino acids. Unless otherwise indicated, the entire subsequent procedure was carried out at 30~ E x p o n e n t i a l E. cell B ~ cells at a density of 1.4 x 10~ in M9S.1 medium were added to the adsorption tube and aerated for 2 m i n before the addition of phage. A 6-fold greater volume of M9S.1 m e d i u m containing phage at a concentration of 2.3 x 109/ml (a multiplicity of 10) was added to the adsorption tube to begin the infection. Adsorption under these conditions was rapid and essentially all cells were infected by 3 min after the addition of phage. A t the times indicated in the t e x t and Figure legends, 2.5 ml infected cells (2 x 108 cells/ml) were added to 0.25 ml of a m i x t u r e of 15 14C-labelled amino acids (New E n g l a n d Nuclear; N E C 445) at 10 ~Ci/ml. Then 4 min later, 0.25 ml 20~ Casamino acids was added to terminate the labelling, and after a 5-min chase, the samples were p u t in ice.

(e) Radioactive lysate preparation, gel electrophoresis and determination of protein yields The methods were essentially identical to those previously used in this laboratory (Russel, 1973; Krisch et al., 1974). Determination of relative protein yield was accomplished b y exposing X - r a y film (Kodak) to the dried gel slab for a period of 1 to 6 days. Autoradiographs were developed and then traced with a J o y c e - L o e b l recording microdensitometer. The area of the peak corresponding to a protein of interest was determined with a Numonics graphics calculator. The data were normalized by dividing the peak area by the n u m b e r of triclfloroacetic acid-precipitable cotmts added to the gel. The value of the peak area/cts per rain was taken as the average rate of synthesis during the period of the pulse labelling. All values shown in a given Figure were determined using lysates from the same experiment, analysed in the same gel. The values obtained in separate experiments h a v e not been standardized for exposure time, etc. Consequently, the numerical values in different Figures are not to be compared. All of the gels from which q u a n t i t a t i v e d a t a were taken were 10% slab gels in which the P32 b a n d and the adjacent P44 and P r I I B bands were well-separated. A discussion of the sensitivity and some sources of error in measurements of band intensities on gels is given by Russel (1973). (f) Irradiation A t the times after infection indicated in the Figure legends, 22 ml of infected cells at a density of 2 • 10S/ml were transferred to a sterile 90 m m glass Petri plate containing a magnetic stirring bar and irradiated for 0.5 to 3-0 min. A Westinghonse Sterilamp (782-L30) germicidal u.v. lamp (emitting primarily at 2537 ,~) was employed and normally the distance from the source to the plate was 88 cm where the incident dose rate was measured to be 4.18 e r g / m m 2 per s by the m e t h o d of Sauerbier (1964). The actual dose in a given experiment was varied by altering the distance from the source, the period of irradiation, or both. I m m e d i a t e l y after irradiation the infected culture was transferred to an aeration tube an d replaced in the water-bath. The unirradiated control culture was handled identically, except t h a t an aluminiLun lid was left on the Petri plate during the period of irradiation. (g) [aH]thymidine incorporation E. coli B ~"was grown as described for the preparation of host cells for protein labelling. The cells were resuspended at a cell density of 4"0 • 10S/ml in M9S.1 medium containing 100 ~g 2'-deoxyadenosine/ml. A t t = 0 an equal volume of a phage solution at 4.0 X 109/ ml was added and the infected culture was aerated at 30~ At t : 5 min, [aHJthymidine (New E n g l an d Nuclear) and cold carrier thymidine were added to the culture so t h a t the final conch was 2 ~g thymidine/ml at 0.2 ~Ci/ml. Fo r the determination of [aH]thymidine

70

H. M. K R I S C H AND G. VAN H O U W E

incorporation, samples (0.5 ml) of the infected culture were precipitated in an equal volume of ice-cold 20% (w/v) triehloroacetic acid, filtered on GF/C glass-fibre filters, and washed twice with 5 inl 5~o trichloroaeetie acid and once with 5 ml 95~/o (v/v) ethanol. Radioactivity was determined b y liquid scintillation counting in t o l u e n e / P P 0 / P O P O P . (h) [3H]thymidine-labelled phage Essentially the same procedure as that used to measure [3H]thymidine incorporation was employed; however, E. coli 0R63 was used as the host bacterial strain and the final conch of thymidine was 2.5 tLg/ml (1-25 t~Oi/ml).The infected culture (10 ml) was treated with clfloroform after 60 rain and then incubated for 1 h with 10 ~g egg white lysozyme/ml and 10 t~g pancreatic DNAase I/ml. Bacterial debris was removed by eentrifugation at 6000 g for 10 min. The phage were purified by pelleting at 29,000 g for 1 h. The pellet was washed and resuspended in M9S medium. With this purification, greater than 80~/o of the trichloroacetic acid-precipitable counts were absorbed to the bacteria b y 5 min after infection. Bacteria infected with labelled phage were pelleted 5 rain after infection and resuspended in fresh medium to remove unabsorbed phage. Samples of the infected culture were taken as described in section (g) above and assayed for triehloroaeetic acidprecipitable counts. (i) [3H]uracil incorporation E. coli AS19 was grown and then infected with phage as described in section (d) above. At the times indicated in the Figure legends, 1.0-ml samples of the infected culture were added to [3H]uraeil (Amersham, TRK241) and cold carrier uracil so that the final conen was 0.1 t~g/ml (1 t~Ci/ml). After 1 rain incubation at 30~ the pulse was terminated by the addition of 1 ml 10~/o trichloroacetic acid and the sample was placed on ice. (j) Rimactan inhibition Rimactan (rifampicin) was generously provided b y Ciba~-Geigy (Sulsse). At the times indicated in the Figure legends, an aqueous solution of Rimactan was added to the infected culture to give a final concn of 100 ~g/ml. 3. R e s u l t s (a) The ultraviolet light stimulation of P32 synthesis Figure 1 shows the p a t t e r n of P32 synthesis in a lysis-defeetive infection (at comparable times the p a t t e r n of P32 synthesis in wild-type and tamA3 (lysis-defective) infections is identical). The synthesis of P32 commences early in infection a n d continues at a high rate until after the initiation of replication a n d of late gene expression. A b o u t 10 to 15 minutes after infection at 30~ P32 synthesis begins to diminish. The u.v. irradiation of T4-infected E. cell results in a reduction in the overall rate of protein synthesis. I f the cells are irradiated at the beginning of the late period, well after the onset of D N A synthesis, there is a substantial reduction in t h e synthesis of the m a j o r structural proteins which constitute the infectious particle. I m m e d i a t e l y after irradiatioJ ~(1000 erg]mm 2) there is a m a r k e d increase in the rate of P32 synthesis (Fig. 1); no other T4-induced protein appears to have a similar response to irradiation (Fig. 2). E v e n after a considerable delay, during which a nearly n o r m a l late protein synthesis p a t t e r n can re-establish itself, there is still a high r a t e of P32 synthesis. The identification of this protein whose synthesis is u.v.-stimulated as the p r o d u c t of gene 32 is unambiguous, since a nonsense m u t a n t in this gene fails to induce the synthesis of the same protein after irradiation (data n o t shown). This unusual response of gene 32 expression to u.v. irradiation is also manifested at earlier times in the infection; for example, in Figure 3 t h e infected cells were irradiated at a b o u t the time of initiation of D N A replication. I n this ease the general

ULTRAVIOLET

LIGHT

INDUCIBILITY

OF P32

SYNTHESIS

71

2.0

,5

l.o

0

I0

20

30

Time ofler infeclian Imin}

FIG. l. R a t e of s y n t h e s i s of P 3 2 i n gone ~ (lysis-dofectivo) m u t a n t - i n f e c t e d cells w i t h a n d w i t h o u t u.v. i r r a d i a t i o n . E . cell B e a t 2 • l 0 s oells]ml were i n f e c t e d a t 30~ w i t h t a m A 3 a t a m u l t i p l i c i t y o f i n f e c t i o n o f 10. A t t h e i n d i c a t e d t i m e s , t h e i n f e c t e d cells wore labelled w i t h z4O-labelled a m i n o a c i d s for 4 m i n , followed b y a 5 - m i n c h a s e w i t h u n l a b e l l e d a m i n o acids. E a c h p o i n t in t h e F i g u r e r e p r e s e n t s t h e a v e r a g e r a t e o f s y n t h e s i s o f P 3 2 d u r i n g t h e p u l s e labelling. R a t e s are p l o t t e d a t t h e m i d p o i n t o f t h e p u l s e i n t e r v a l (see M a t e r i a l s a n d M e t h o d s , s e c t i o n (e)). --C)--C)--, t a m A 3 - i n f e c t e d cells, n o i r r a d i a t i o n ; - - A - - A - - , t a m A 3 - i n f e c t e d cells u . v . i r r a d i a t e d f r o m 16 m i n to 20 rain a f t e r i n f e c t i o n ( d i s t a n c e = 88 c m ; dose = 1000 erg[mm2).

effect of irradiation is to retard the normal developmental sequence of phage infection. Irradiation reduces the level of protein synthesis immediately by 60% and the vast majority of early proteins are produced at lower rates. As has been reported by Hercules & Sauerbier (1974), the sensitivity of the synthesis of different early proteins to radiation varies widely. It is to be noted in this respect that the relative rate of synthesis of P32 is stimulated (approx. 2-5-fold) by the irradiation. The absolute amount of P32 made in the 22 minutes after irradiation in this experiment represents nearly twice the amount made by un-irradiated control infection (see the legend to Fig. 3). This occurs even though irradiation was prior to the time when P32 synthesis is maximal in a wild-type infection, and hence the transcriptiontranslation machinery may not yet be completely capable of expressing gone 32 with the same rate possible later in the infectious cycle. (b) A potential mechanism for the ultraviolet light induction of P32 A possible mechanism for the u.v. induction of P32 synthesis is suggested by our previous work (Kriseh ct al., 1974) on the control of P32 synthesis. In the absence of functional DNA polymerase (gone 43) and polynucleotide ligase (gone 30) the unreplicated parental DNA acquires numerous endonuclease-generated nicks which can be converted to gaps. This conversion has been shown to require the products of genes 46 and 47 (putative exonucleases); the triple mutant 43amB263-30amH39X46amN130 fails to convert the nicked parental DNA into gapped DNA because it lacks the gone 46 function (Riva et al., 1970; Prashad & Hosoda, 1972). Thus in infections with the double mutant 43 - -30- the DNA contains gaps capable of binding P32, while the triple mutant DNA contains only nicks which are incapable of binding P32 (Alberts & Frey, 1970). The double mutant synthesizes P32 at an increased rate,

0

I

(al tomA3 ZI to 25 rain + u.v.

2

(b) lamA3 27 to 31 rain 0

3

4- u.v.

4

F l o . 2. A u t o r a d i o g r a m s of s o d i u m d o d e c y l s u l p h a t e 1 0 % (w/v) p o l y a e r y l a m i d e gels of cells i n f e c t e d w i t h lysis-defective T4. E. coli B E a t 2'0 • 10 s eells/ml were i n f e c t e d a t 30~ w i t h a m u l t i p l i c i t y of infection of 10 of t a m A 3 . T h e i n f e c t e d cells were i r r a d i a t e d w i t h u.v. b e t w e e n 16 a n d 19 rain a f t e r infection (750 e r g / m m 2 ) . A t e i t h e r 21 rain (a) or 27 rain (b) t h e cells were labelled w i t h 14C-labelled a m i n o acids for 4 rain, followed b y a 5 - m i n c h a s e w i t h e x c e s s u n l a h e l l e d a m i n o acids. E q u a l v o l u m e s of r a d i o a c t i v e l y s a t e s were l a y e r e d on t h e gel. ( c t s / m i n : slot 1 = 12,580; slot 2 = 4160; slot 3 = 22,990; slot 4 = 5880.) T h e a b s o l u t e a m o u n t o f P 3 2 s y n t h e s i s is 2.7-fold g r e a t e r in t h e i r r a d i a t e d infection t h a n in t h e u n - i r r a d i a t e d infection a t 21 rain ; a t 27 rain t h i s difference increases to 4.7-fold. A r r o w s i n d i c a t e t h e p o s i t i o n of P32.

ULTRAVIOLET

LIGHT

INDUCIBILITY

OF P32

SYNTHESIS

73

2.0

I-5

c.

t.C

o

2 0.5

i

i

20 Time oiler infection (rain) I0

50

F r o . 3. R a t e o f synthesis o f P32 in gene t (lysis defective) m u t a n t - i n f e c t e d cells u.v. i r r a d i a t e d prior to D N A replication. E. cell B E a t 2 X 10 e cells/ml were infected a t 30~ w i t h tamA3 a t a multiplicity of infection of 10. A t t h e indicated times, the infected cells were labelled w i t h 14Clabelled a m i n o acids for 4 min, followed b y a 5-rain chase w i t h unlabelled a m i n e acids. R a t e s are p l o t t e d as described in the legend to Fig. 1. The a m o u n t of p r o t e i n s y n t h e s i s in the first pulse after irradiation w a s 4 0 % of t h a t of the u n i r r a d i a t e d control infection. I n the 2rid pulse this value w a s 64%, b y the 3rd it w a s 94%, a n d in the 4th it w a s reduced to 45%. --O--O--, tamA3-infectcd cells, no irradiation; - - I I - - I 1 - - , tamA3-infected cells u.v. irradiated (750 e r g / m m 2) fl'om 5"3 m i n to 8"3 m i n after infection.

while the triple m u t a n t fails to produce P32 at elevated rates (Krisch et al., 1974). At very late times this difference between the two infections becomes more appreciable. Prashad & Hosoda (1972) have demonstrated, at these times after infection, that there is a significant amount of gapping in the unreplicated parental 4 3 - - 3 0 DNA while the 4 3 - - 3 0 - - 4 6 - DNA has much less. It is very likely that the immediate result of irradiation is an analogous increase in gapped DNA. Nicks would be generated at or near the u.v. lesion by endonucleases and subsequently repair of the lesion mediated by exonueleases would generate, at least transiently, a gap and a potential binding site for P32. Alternatively, replication of a DNA molecule containing a lesion (Rupp & Howard-Flanders, 1968; Clark, 1973) could result in the opposite strand having a small unreplieated region near the lesion. Such regions would also be susceptible to exonucleolytic action and hence could become a binding site for significant quantities of P32. Thus the "induction" of P32 synthesis m a y be explained as a result of the u.v. lesions generating binding sites for P32. Because P32 is self regulating, the intraceUular concentration of free P32 could be reduced below the threshold level required to block further synthesis of this protein. (c) The effect of various mutations on ultraviolet light inducibility of P32 When phage-specific DNA synthesis is blocked by mutations in genes essential for replication, the normal shut-off of many "early" proteins does not occur (Wiberg et al., 1962). Such an infection results in an extended period of P32 synthesis at nearly the maximal rate observed in a wild-type infection (Krisch et al., 1974). Irradiation of cells infected with a DNA synthesis-defective m u t a n t allows the examination of the response of P32 synthesis without the additional complication of DNA replication and its potential interaction with the radiation damage. Figure

74

H.M.

KRISOH

AND

G, VAN

3'o 4oo

,b

HOUWE

O.

"6

o

,E~ 2b

2~

3&

40

Time after infection (rain) (a) (b)

Fzo. 4. C o m p a r i s o n of t h e r a t e o f P 3 2 s y n t h e s i s in different D N A s y n t h e s i s - d e f e c t i v e infections a f t e r u.v. irradiation. E. cell B E a t 2 • 108 eolls]ml were i n f e c t e d a t 30~ w i t h e i t h e r 4 1 - 4 5 a m X 5 (a) ( 4 1 a m N S 1 - 4 2 a m N 1 2 2 - 4 3 a m B 2 2 - 4 4 a m N 8 2 - 4 5 a m E 10) or 4 3 a r a B 2 6 3 (b) a t a m u l t i p l i c i t y of i n f e c t i o n of 10. A t t h e i n d i c a t e d t i m e s i n f e c t e d cells were labelled a n d c h a s e d as d e s c r i b e d in t h e l e g e n d to Fig. 1. (a) - - O - - O - - , 4 1 - 4 5 a m X 5 - i n f e c t e d cells, no i r r a d i a t i o n ; - - O - - O - - , 41-45amX5-infeeted cells i r r a d i a t e d 16 to 19 m i n a f t e r infection (750 e r g / m m ~ ) ; (b) - - A - - A - - , 4 3 a m B 2 6 3 - i n f e c t e d cells, no i r r a d i a t i o n ; - - A - - / k - - , 4 3 a m B 2 6 3 - i n f e c t e d cells i r r a d i a t e d 16 to 19 m i n a f t e r infection (750 e r g / m m 2 ) .

4 demonstrates that there is a rapid and pronounced increase in P32 synthesis following irradiation in the absence of DNA replication. The rate of P32 synthesis is stimulated five- to sixfold in an infection with multiple DNA replication mutant 41-45amX5. Hence DNA synthesis does not seem essential to the increase in P32 expression following irradiation. Figure 4 reveals, however, that there are significant differences in the magnitude of the response depending on the exact genotype of the infecting phage, even in the cases where DNA synthesis is stringently blocked. With the same dose of irradiation the DNA polymerase m u t a n t infection gives a twofold stimulation in comparison to the greater than sixfold effect with 41-45amX5. A m u t a n t in yet another gene essential to DNA replication (gene 44) results in a response essentially identical to that of the wild-type (Table 1). Such variation in the response might be expected if the mutation is in a gene which is involved in the generation or removal of P32 binding sites following irradiation. With this hypothesis in mind we have screened a number of mutants in genes thought to be involved in DNA replication, recombination and repair. The results of these experiments are presented in Table 1. Examination of these data in detail reveals several features of the phenomenon of u.v. induction of P32 synthesis. No single mutation abolishes completely the stimulation of P32 synthesis following irradiation. Nevertheless, mutants in either gene 46 or 47 do markedly reduce it. These genes could code for the exonuclease which is involved in the exposure of single-stranded DNA in the region of the radiation lesion. The phenotype of a mutation in either of these genes is a lack of exonucleolytie activity (Wiberg, 1966), a reduction of recombination (Bernstein, 1968; Berger et al., 1969) and a greatly increased sensitivity to u.v. (Baldy, 1968). Figure 5 shows in more detail that a gene 47 m u t a n t infection (the same results were obtained for a gene 46 mutant) is defective in the induction of P32 synthesis following exposure to u.v. The residual level of P32 synthesis following irradiation might be explained by the leakiness of mutants in gene 46 and 47, or by other exonucleases being capable of generating single-stranded regions in

ULTRAVIOLET

LIGHT

INDUCIBILITY

OF P32

SYNTHESIS

75

TABLE ]

Ultraviolet light stimulation of P32 synthesis in infections with various mutants

Infection genotype

tamA3 44amN82 v1 43araB263 47areA456 43--46w-m22 yl00 41 --42 --43 - - 4 4 - -45-

Stimulation t 2 to 6 m i n a f t e r u.v.

Stimulationt 8 to 12 m i n after u.v.

3.17~t 2.80 2.63 1.85 1.28 1.31 1.71w 2.10 4.78

3.08~t 2.56 3.60 1'34 1.36 0.85 1.79w 1"51 5.04

R a t e of P32 s y n t h e s i s 2 to 6 m i n or 8 to 12 m i n after irradiation c o m p a r e d to the rate of s y n t h e s i s a t time of irradiation. The procedure w a s identical to t h a t described in the legend to Fig. 5. T h e r a t e of s y n t h e s i s a t t h e time of irradiation w a s the a v e r a g e of t h e v a l u e s o b t a i n e d in the u n i r r a d i a t e d c u l t u r e i m m e d i a t e l y before a n d after the time of irradiation. :~ The m e a n of 5 s e p a r a t e d e t e r m i n a t i o n s , the coefficient of v a r i a t i o n of these values w a s less t h a n 15%. wI n a w - infection, as in a 5 8 - infection, the r a t e of P32 s y n t h e s i s is high even in t h e absence of irradiation. 2.0

9_..~. o-'*~

1-5

/

$ =_ I-0 (3_

(P

0.5

'

J'o

'

2b

'

30

Time ofler infection (rain)

FIG. 5. C o m p a r i s o n of the r a t e of P32 s y n t h e s i s in gene t-defective a n d gene d7-defective infection after u.v. irradiation. E. coli B z a t 2 • 10 a cells/ml were infected a t 30~ w i t h either tamA3 or 47areA456 a t a m u l t i p l i c i t y of infection of 10. A t t h e indicated times the infected cells were labelled a n d chased as described in the legend to Fig. 1. --O--Q--, tamA3-infected cells, no i r r a d i a t i o n ; - - O - - O - - , tamA3-infected cells u.v. irr a d i a t e d 16 to 19 rain after infection (750 erg]mm2); - - I - - ' - - , 47amA456-infected cells, no i r r a d i a t i o n ; - - "- - [ 3 - - " - - [ : ] - - "- - , 47 amA456-infected cells u.v. irradiated 16 to 19 rain a f t e r infection (750 erg]mm2).

76

H. M. KRISCH AND G. VAN HOUWE

irradiated DNA. For example, the gene 43 mutants are also somewhat reduced in the ability to induce P32 synthesis, perhaps because the polymerase-assoeiated exonuclease (Kornberg, 1969) has a contributory role in this process. Table 1 indicates that the u.v. inducibility of P32 synthesis is largely abolished in infections with the double mutant (43amB263-46amN130). The fact that a mutation in the v gene endonuclease does not significantly inhibit the u.v. induction of P32 is compatible with the suggestion that P32 is not involved in v gene-mediated repair (Maynard-Smith & Symonds, 1973). Excision repair is thought to be rather efficient, at least in E. coli, removing on the average only a very small number of nucleotides in the vicinity of the lesion (Hanawalt, 1975}. Such a short gap is presumably not large enough to bind a sufficient quantity of P32 to affect the rate of P32 synthesis. It seems more likely that the T4 analogue of long gap repair (removal of 103 to l04 nueleotides) in E. coli (Cooper & Hanawalt, 1972) would be the source of the vast majority of P32 binding sites after exposure to u.v. The result with the v mutant would then suggest that the v gene product is not essential for such repair. Additionally, since blocking DNA synthesis does not affect the u.v. inducibility of P32 synthesis, we would suggest that replication is also not a prerequisite of such long gap repair in T4-infected cells. In addition to the v gene mutant we have also examined u.v.-sensitive mutants in two other genes of T4; yl00 (Boyle & Symonds, 1969) and w-m22 (Hamlett & Berger, 1975). Both mutants map in the late region of the T4 chromosome between genes 24 and 25. Mutations in both genes have similar effects on u.v. sensitivity and recombination; nevertheless, genetic tests indicate that they represent separate complementation groups (Hamlett & Berger, 1975). Each mutant shows a somewhat reduced ability to induce P32 synthesis after irradiation (Table 1) when compared to a wild-type infection. I t is striking, however, that gene w like gene 58 (also u.v.sensitive) and unlike gene y mutants produce higher than normal quantities of P32, even in the absence of irradiation. The meaning of this observation remains obscure at the moment; it is interesting, however, that these gene products are all implicated in the same repair pathway (Hamlett & Berger, 1975) and yet their phenotypes are quite different with regard to P32 synthesis. (d) Ultraviolet light-stimulated hydrolysis of unreplicated parental D N A Further support for the hypothesis that single-strand regions of DNA generated by repair are responsible for the increased P32 synthesis is to be found in Figure 6. In these experiments the same r~gime of irradiation was followed as in the previous experiments, but the infecting phage were labelled with [3H]thymidine and the hydrolysis of the um'eplicated parental DNA was monitored. The results indicate that in both the 41-45amX5 and the 43amB263 infections there is a u.v.-stimulated hydrolysis of parental DNA immediately following irradiation. The amount of hydrolysis appears to be roughly proportional to dose and is clearly dependent on the genotype of the infecting phage. With the same radiation dose, a significantly greater proportion of the parental label is hydrolysed in the 41-45amX5 infection than in the gene 43 mutant infection. These results parallel those concerning the rates of P32 synthesis in the two infections (Fig. 4). The irradiated 41-45amX5 infection synthesizes P32 at a significantly higher rate than the gene 43 infection (also see Table 1), as would be expected on the basis of our hypothesis on the mechanism of P32 overproduction.

ULTRAVIOLET

LIGHT

INDUCIBILITY

O F P32 S Y N T H E S I S

77

I00 A

75

e,,.

-p_

"5

f ._o

5O 25

o

2b 3b 400

ib

,b

2b

3'o

4o

Time after infection (rain) (a)

(b)

Fzo. 6. Ultraviolet light mediated hydrolysis of unrepUoated parental phage DNA. E . colt B e a t 2 • 10 e cells/ml were infected a t 30~ with either 43amB263 or 41-45amX5 at a multiplicity of infection of 10. The infecting phage were labelled in their DNA with [aH]thymidine as described in Materials and Methods. Samples of the infected culture were taken at the indicated times and assayed for trichloroacetic acid-precipitable counts. (a) - - C ) - - O - - , 43amB263-infected cells, no irradiation; - - @ - - @ - - , 43amB263-infected cells u.v. irradiated from 16 to 19 min after infection (750 erg/mm2) ; - - [3--1-]--, 41-45amX5-infectod cells, no n.radiation; - - m - - m - - , 41-45amX5-infected cells u.v. irradiated from 16 to 19 min after infection (750 erg/mm2). (b) The same as (a), except the irradiated cultures received approx, twice the effective dose in the interval between 16 to 19 min after infection (]500 erg/mm2). 2O

~

D ~

c

r ~

5

0

i

Ib

'

2'0

I

30

T,me after refection (rain)

FIe. 7. Rate of P32 synthesis in a DNA synthesis defective infection with various doses of u.v. irradiation. E . colt B ~ at 2 • 108 eells/ml wore infected at 30~ with 43araB263 at a multiplicity of infection of 10. At the indicated times the infected cells were labelled and chased as described in the legend to Fig. 1. The distance from the u.v. lamp was 44 cm. - - O - - O - - , 43amB263.infected cells, no irradiation; - - O - - C ) - - , 43amB263-infected cells u.v. irradiated 16 to 16.5 min after infection (425 erg/mm2); - - / ~ - - / k - - , 43amB263-infetced cells u.v. irradiated 16 to 17 rain after infection (850 erg/mm2); - - [=]-- D - - , 43amB263-infeeted cells o.v. irradiated 16 to 18 min after infection (1700 erg/mm~). The amount

of parental DNA

h y d r o l y s e d ~dth this dose o f u.v. in t h e a m X 5 -

i n f e c t e d cells is a p p r o x i m a t e l y 2 0 % o r (for t h i s m u l t i p l i c i t y o f i n f e c t i o n ) t w o p h a g e e q u i v a l e n t s o f D N A . I f all o f t h i s d e g r a d a t i o n w a s t o g e n e r a t e g a p s i n d o u b l e - s t r a n d e d molecules in preparation for repair, then this would provide substrate for the binding o f 80,000 m o l e c u l e s o f P 3 2 (8 t i m e s t h e n o r m a l c o m p l e m e n t o f P 3 2 i n a n i n f e c t e d cell).

78

H . M. K R I S C H

A N D G. V A N H O U W E

Figure 7 shows that, in addition to the degradation being proportional to the u.v. dose, the rate of P32 synthesized following irradiation in a gene 43- m u t a n t is also roughly proportional to dose. This is the expected result if the amount of P32 bound to single-stranded DNA generated by repair affects the level of P32 synthesis.

(e) Effect of ultraviolet irradiation on D N A synthesis The effect of u.v. h'radiation on the incorporation of [3H]thymidine by cells infected with the lysis-defective m u t a n t tamA3 has been examined. With a dose of radiation which reduces phage yield 50-fold (750 erg/mm2), there is an immediate tenfold reduction in incorporation. This reduction persists for at least 20 minutes, subsequently the rate of incorporation increases progressively, so that by 45 minutes after the radiation it is nearly 50% of the rate in the mdrradiated control culture (data not shown). In the 20-minute interval after irradiation a large amount of P32 is synthesized (Fig. 1). I t is doubtful that all this P32 is being used for a small amount of DNA synthesis, and therefore it seems likely that repair-mediated degradation of the previously synthesized DNA may be drawing off large amounts of P32 to bind to the single-stranded regions of DNA thus generated. (f) Effect of ultraviolet irradiation o~ R N A synthesis Sauerbier et al. (1970) have observed that one of the effects of u.v. on T4-infected cells is to lower the rate of RNA transcription by causing the production of shorter RNA chains. Such chains are presumably terminated abortively at the site of photoproducts on the DNA and not resumed between the photoproduct and the next initiation site. Figure 8(a) demonstrates that a dose of irradiation which reduces [3H]thymidine incorporation tenfold also has a marked effect on the incorporation of [3H]uracil into trichloroacetic acid-precipitable material. Within several minutes incorporation is reduced to 25% of the level prior to irradiation. Addition of the RNA synthesis inhibitor Rimactan (100 ~g rifampicin/ml) immediately after irradiation (within 10 s) reduced incorporation to less than 5% of the level in the untreated control infection (Fig. 8(a)). In spite of this severe block of RNA synthesis, a high level of P32 synthesis is still induced in the irradiated Rimactan-treated culture (Fig. 8(b)). This result is compatible with a model of control of gene 32 expression which functions at the level of translation. Such a model would propose that control of P32 operated in an analogous fashion to the regulation in RNA viruses of the synthetase by the coat proteins (Eggen & Nathans, 1969). Hence the P32 might bind to the gene 32 mRNA and hinder its translation. The level of P32 expression would thus depend on the competition for 1)32 between the two binding substrates, single-stranded DNA generated by the repair and control site(s) on the gene 32 mRNA. A necessary condition for such control is that the messenger RNA be relatively stable. We have carried out several experiments which indicate that 1)32 mRNA is indeed highly stable compared to most T4 mRNA.

4. Discussion The experiments reported here demonstrate that u.v. irradiation results in a markedly increased rate of synthesis of gene 32 protein. This u.v. "induction" of P32 is explained as a direct consequence of the self-regulation of this protein. Since it appears that the gene 32 protein plays an important role in the repair of radiation

ULTI:tAVIOLET

c

LIGHT

OF P32 SYNTHESIS

79

o

"o,

I0,000

INDUCIBILITY

"-..

u.v.

4

u~ 9

a"

o_ "5

1000

..o'"" 2OO 0

Ib

2b

sb

o

'

Rig ~b

"""'-o."-'''a

2b

sb

'

Time after infechon (rain)

[a)

(b)

F i e . 8. (a) R N A s y n t h e s i s , [H3]uracil i n c o r p o r a t i o n b y t a m A 3 i n f e c t i o n a f t e r u.v. i r r a d i a t i o n . T h e p r o c e d u r e is as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . A t 16 m i n a f t e r i n f e c t i o n t h e c u l t u r e w a s split into 2 p a r t s a n d i w a s i r r a d i a t e d w i t h t h e d i s t a n c e to t h e l a m p b e i n g 36 c m . I m m e d i a t e l y a f t e r w a r d s h a l f o f i r r a d i a t e d c u l t u r e w a s t r e a t e d w i t h R i m a c t a n (100/~g/ml) ( R I F ) . --O--O--, t a m A 3 - i n f e c t e d cells, n o i r r a d i a t i o n ; - - A - - A - - , t a m A 3 - i n f e c t e d cells u.v. i r r a d i a t e d 16.0 to 16.5 m i n a f t e r infection (600 e r g / m m 2 ) ; - - F ] - - [-]--, t a m A 3 - i n f e c t e d cells u . v . i r r a d i a t e d 16.0 to 16.5 m i n (600 e r g / m m 2) a n d t h e n t r e a t e d w i t h i n 10 s w i t h 100/zg R i m a c t a n ] m l . (b) C o m p a r i s o n o f t h e r a t e of P 3 2 s y n t h e s i s a f t e r u . v . i r r a d i a t i o n a n d a d d i t i o n o f R i m a c t a n ( R I F ) . E. cell AS19 a t 2.0 • l0 s cells/ml w a s i n f e c t e d a t 30~ w i t h t a m A 3 a t a m u l t i p l i c i t y o f i n f e c t i o n of 10. A t t h e i n d i c a t e d t i m e s infected cells were labelled a n d c h a s e d as d e s c r i b e d in t h e l e g e n d to Fig. 1. --O--O--, t a m A 3 - i n f e c t e d cells, no i r r a d i a t i o n ; - - O - - O - - , / a m A 3 - i n f e c t e d cells, no u . v . , t r e a t e d a t 16.5 rain a f t e r i n f e c t i o n w i t h R i m a c t a n (100/zg/ml) ; - - A - - A - - , t a m A 3 - i n f e c t e d cells u . v . i r r a d i a t e d 16.0 to 16.5 rain a f t e r i n f e c t i o n (600 e r g ] m m 2 ) ; - - [ 3 - - E ] - - , t a m A 3 - i n f e c t e d cells u.v. i r r a d i a t e d 16.0 to 16.5 m i n a f t e r i n f e c t i o n (600 e r g / m m 2) a n d t h e n t r e a t e d w i t h i n 10 s w i t h R i m a c t a n (100 /zg/ml).

damage (Baldy, 1970; Wallace & Melamede, 1972; Wu & Yeh, 1973), self-regulation of the synthesis of P32 would be well-adapted to this function. When there is a large requirement of this protein in DNA repair, the infected cell could shift much of its synthetic capacity to making this protein. We have demonstrated that in a number of situations (Krisch et al., 1974) in which unusually large amounts of single-stranded DNA are generated, the serf-regulation of P32 manifests itself. It seems likely to us that this regulation is physiologically significant in that it provides the organism with a degree of flexibility in its capacity for replication, recombination and. repair. Our observation (Krisch & Van Houwe, unpublished observations) that an early u.v. dose to infected cells renders them relatively less sensitive to subsequent irradiation lends support to this hypothesis. A possible explanation for this phenomenon is t h a t the initial dose of radiation expands the amount of P32 sufficiently so that the subsequent irradiation can be dealt with more effectively, resulting in an effective dose reduction. In this respect it is particulaHy interesting to note that in E. coli, where a u.v.-inducible-u.v, repair system has also been suggested (Witkin, 1974; Weigle, 1953), it has been observed that at least one of its proteins is u.v. inducible (Sedgwick, 1975). It has been known for a long time that u.v. irradiation substantially enhances recombination in the infected cell (Jacob & Wollman, 1955; Hershey et al., 1958). The current results suggest a mechanism which could explain this effect. Nicks in the DNA at or near the n.y. lesion could be generated by endonucleases or b y replication. Exonucleases, probably the products of genes 46 and 47 as well as additional host and/or phage

80

H.M.

KRISCH

AND

G. V A N

HOUWE

nucleases would subsequently excise a gap in the molecule. This single-strand gap would become coated with a large quantity of P32 t h a t could serve a dual function of protecting the D N A from endonucleolytie attack as well as maintaining the gap in a rigid configuration, which m a y facilitate either the filling of the gap b y repair polymerases or the initiation of a recombination event. The extent to which the gap is eliminated b y either of these processes cannot be precisely determined but, since u.v. stimulation of recombination is fairly large, it would seem t h a t recombination is used to a significant extent. The preceding discussion of the expression of P32 in response to u.v. irradiation is based on the observation t h a t P32 is involved in the control of its own synthesis. Such control could be at the level of either transcription or translation. The observation t h a t a severe inhibition of R N A synthesis b y rifampicin and u.v. irradiation does not block the induction of P32 b y irradiation argues in favour of translational control. Other experiments carried out in this laboratory support a model of translational control for gene 32 expression (Krisch el al., manuscript in preparation). Furthermore, a series of experiments b y Russel & Gold have led them to suggest control at the level of translation (Russel & Gold, personal communication). Control of gene expression at the level of messenger translation might he of general significance. I n particular, feedback regulation of gene 32 m R N A translation would present a rather interesting and novel approach to the problem of inducing the s~lthesis of a protein in response to massive damage to the DNA. Since such synthesis would not require R N A synthesis (provided the m R N A encoding the protein was stable), even in the extreme case where R N A synthesis was totally blocked, m a x i m u m expression of this gene could still occur. I n view of the results presented here it m a y be especially interesting to determine if similar mechanisms are operant in regulating the synthesis of repair functions in other biological systems. This research was supported by a grant fl'om the Fonds National Suisse de la Recherche Scientifique (no. 3.339.74). L3~ml Silver and Neville Sy~nonds contributed valuable discussions to this work. We thank D. Belin, W. Gibbs, L. Fretli-Grtitli, N. Hamlett and G. Gujer-Kellenberger for many helpful suggestions concerning the manuscript. We are particularly gratefttl to Marjorie Russel for sharing with us her mlpublished experiments on the mechanism of P32 regulation. Dick Epstein's participation in all aspects of this work is profoundly appreciated. REFERENCES Alberts, B. M. & Frey, L. {1970). Nature (London), 227, 1313-1318. Baldy, M. W. (1968}. Cold Spring Harbor Syrup. Quant. Biol. 33, 333-338. Baldy, M. W. {1970). Virology, 40, 272-287. Berger, H., Warren, A. J. & Fry, K. E. {1969}. J. Virol. 3, 171-175. Berustein, H. {1968}. Cold Spring Harbor Syrup. Quant. Biol. 33, 325-331. Boyle, J. M. & Symonds, N. {1969}. ~Iutat. Res. 8, 431-439. Champe, S. P. & Benzer, S. {1962}. Proc. Nat. Acad. Sci., U.S.A. 48, 533-546. Clark, A. J. (1973). A n n u . Rev. Genet. 7, 69-86. Cooper, P. K. & Hanawalt, P. C. (1972}. J. Mol. Biol. 67, 1-10. Curtis, M. J. & Alberts, B. {1976}. J. Mol. Biol. 102, 793-816. Delius, H., Mantell, M. & Alberts, B. M. (1972). J. Mol. Biol. 67, 341-350. Doermann, A. H. & Boehner, L. (1970}. Genetics, 66, 417-428. Eggen, K. & Nathans, D. (1969). J. Mol. Biol. 39, 293-305.

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Epstein, R. H., Bolle, A., Steinberg, C. M., Kellenberger, E., Boy de la Tour, E., Chevalley, R., Edgar, R. S., Sussman, M., Denbardt, G. H. & Lielausis, A. (1963). Cold Spring Harbor Syrup. Quant. Biol. 28, 375-392. Friedberg, E. C. & King, J. J. (1971). J . Bactcriol. 106, 500-507. Hamlett, N. V. & Berger, H. (1975). Virology, 63, 539-567. I-Ianawalt, P. C. (1975). Genetics, 79, 179-197. Hercules, K. & Sauerbier, W. (1974). J . Virol. 14, 344-348. Hershey, A. D., Burgi, E. & Streisinger, G. (1958). Virology, 6, 287-301. Huberman, J. A., Kornberg, A. & Alberts, B. M. (1971). J. Mol. Biol. 62, 39-52. Jacob, F. & Wollman, E. (1955). A n n . Inst. Pasteur, 88, 724-749. Josslin, R. (1970). Virology, 40, 719-726. Josslin, R. (1971). Virology, 44, 101-107. Kornberg, A. (1969). Science, 163, 1410-1418. Krisch, H. M., Hamlett, N. V. & Berger, H. (1972). Genetics, 72, 187-203. Krisch, H. M., Bolle, A. & Epstein, R. H. (1974). J. Mol. Biol. 88, 89-104. Maynard-Smith, S. & Symonds, N. (1973). J. Mol. Biol. 74, 33-44. Mortelmans, K. & Friedberg, E. C. (1972). J . Virol. 10, 730-736. Prashad, N. & Hosoda, J. (1972). J. Mol. Biol. 70, 617-635. Riva, S., Cascino, A. & Geiduschek, E. P. (1970). J . ~lol. Biol. 54, 103-119. Rupp, W. D. & Howard-Flanders, P. (1968). J. Mol. Biol. 31, 291-304. l~ussel, M. (1973). J. Mol. Biol. 79, 83-94. Sauerbier, W. (1964). Biochim. Biophys. Acta, 87, 356-358. Sauerbier, W., Millette, R. L. & Hackett, P. B. (1970). Bloc,him. Biophys. Acta, 209, 368-386. Sedgwick, S. G. (1975). Nature (London), 255, 349-350. Sekiguchi, M. & Iida, S. (1967). Proc. Nat. Acad. Sci., U.S.A. 58, 2315-2320. Steinberg, C. M. & Edgar, R. S. (1962). Gcnctics, 47, 187-208. Symonds, N., Heindl, H. & White, P. (1973). Mol. Gen. Goner. 120, 253-259. Wallace, S. S. & Melamede, R. J. (1972). J. Virol. 10, 1159-1169. Weigle, J. (1953). Proc. Nat. Acad. Sci., U.S.A. 39, 628-636. Wiberg, J. (1966). Proc. Nat. Acad. Sci., U.S.A. 55, 614-621. Wiberg, J. S., Dirksen, M. L., Epstein, R. H., Luria, S. E. & Buchanan, J. M. (1962). Proc. Nat. Acad. Sci., U.S.A. 48, 293-302. Witkin, E. (1974). Genetics, 79, 199-213. Wu, R. & Yeh, Y. (1973). J. Virol. 12, 758-765. Yasuda, S. & Sekiguchi, M. (1970). J. Mol. Biol. 47, 243-255.