Contribution of thymine dimers to the ultraviolet light inactivation of mutants of bacteriophage T4

J. Mol. Biol. (1972) 66, 97-106

Contribution o f Thymine Dimers to the Ultraviolet Light Inactivation of Mutants of Bacteriophage T4 MARVIN

L. MEISTRICII

Bell Telephone Laboratories Inc., Murray Hill, N.J. 07974, U.S.A. and Ontario Cancer Institute, Toronto 284, Canadat (Received 7 January 1971, and in revised form 18 hTovember 1971) The role of thymine dimers in bacteriophage inactivation has been investigated. Sensitized irradiation was used to selectively produce the dimers in the DNA of viable T4 phage with very low yields of other u.v. photoproducts. Measurements of the relative inactivation rates and the photoreactivable sectors, and calculations of the yield of thymine dimers per lethal hit for both u.v. and sensitized irradiation were made on four mutants of T4 with different u.v. sensitivities. Photoreaetivation, v-gene reactivation, and the yield of thymine dimers per lethal hit were all greater with sensitized irradiation than with u.v. irradiation but the extent of x-gene reactivation was the same with both irradiation methods. Since sensitized irradiation produces lower yields of photoproducts other than thymine dimers, we conclude that (1) thymine dimers act as lethal lesions in T4 phage, (2) other lethal photoproducts are also produced by u.v. irradiation, (3) the thymine dimer is the principal u.v. photoproduet repaired by both photoreactivation and v-gene reactivation, and (4) the lethal effects of thymine dimers and other u.v, lesions are both reversed by x-gene reactivation.

1. Introduction The inactivating effects of ultraviolet radiation on viruses and cells have been extensively studied, but in order to understand the mechanisms involved it is necessary to correlate specific molecular photoproducts with the lethal effects. I t has been shown, from action spectra, that absorption by nucleic acids is responsible for inactivation of most micro-organisms (TWinkler, Johns & Kellenberger, 1962; Smith & Hanawalt, 1969). I t therefore follows that inactivation results from photochemical changes involving nucleic acids, specifically DNA in the case of the DNA bacteriophages. Approximately eight distinct molecular alterations have been identified in u.v.irradiated DNA (J. K. Setlow, 1966; Smith & Hanawalt, 1969), and it is quite possible that there exist other lesions that are undetected by the experiments which have been performed. The most abundant n.y. photoproduct of DNA is T=T$. However, the presence of a molecular lesion in DNA does not necessarily indicate a role for that lesion in inactivation since there exist efficient mechanisms which enzymically repair lesions (Rupert, 1964; Setlow & Carrier, 1964) and other means by which viable progeny can be produced from damaged parental DNA (Rupp & Present address. :~Abbreviations used: T : T , thymine-thymine dimer(s) (eyclobutane type) ; C~T, cytosine-thymine dimer; C=C, eytoslne-cytosine dimer; vR, v-gene reactivation; xR, x-gene reactivation; p.l.h., phage lethal hit (l/e dose). 7 97

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M . L . MEISTRICH

Howard-Flanders, 1968). The most direct relation between T = T and inactivation has been based on experiments in which some transforming ability of u.v.-irradiated DNA is restored with short wavelength light (h -~ 240 nm) which is known to reverse the three pyrimidine dimers (J. K. Setlow, 1966). The contributions of T = T and the other pyrimidine dimers, C-----Tand C----C, have been separated (Setlow & Setlow, 1967). However, another u.v. photoproduct, an adduct of cytosine and thymine, is also reversed by u.v. light (Patrick, 1970) and might contribute to the reversible inactivation. In the cases of bacteriophage, bacteria and higher cellular organisms, the correlations are indirect. Photoreactivation reverses u.v. inactivation and under the appropriate conditions (Jagger, Stafford & Snow, 1969) repairs pyrimidine dimers without acting on other known photoproducts. The existence of other as yet unidentified photoreactivable lesions is unlikely, but cannot be ruled out. Similarly, the mechanism of excision-repair (Setlow & Carrier, 1964), which involves excision of pyrimidine dimers followed by repair replication, has been correlated with an increased survival following u.v. irradiation (Boyle & Setlow, 1970). Since excision-repair of chemically damaged nucleotides (Strauss, Coyle & Robbins, 1968) and excision of other photoproducts (Varghese & Day, 1970) occurs in a similar manner, this type of repair is not specific for pyrimidine dimers. Thus the conclusion that pyrimidine dimers are lethal photoproducts is based on a correlation between the action of two repair systems which are known to act on dimers, and concomitant increased resistance to u.v. inactivation. The only evidence that T = T is lethal to phage or cells is that it is the most abundant pyrimidine dimer (Setlow & Carrier, 1966a). In order to investigate the role of T = T in inactivation more directly, we have used a technique of sensitized irradiation to produce T = T in DNA with very low yields of other u.v. photoproduets (Lamola, 1968,1969,1970). Two charged derivatives of acetophenone, designated AcCD and Ae¢I~, can be used to sensitize the DNA within intact phage (Meistrich, Lamola & Gabbay, 1970). Irradiation of phage with light of wavelength greater than 310 rim, in the presence of AciD, produces T = T abundantly with virtually no other photoproducts formed (~eistrich & Lamola, 1972). In the present study, sensitized irradiation of various mutants of bacteriophage T4 was performed in order to answer the following questions. (1) Are T = T important lethal lesions ? (2) Are other lesions also lethal ? (3) Do the reactivation processes, photoreactivation, vR and xR, act preferentially on T = T or on other classes of lesions ? From chemical studies it is known that sensitization produces fewer photoproduets (and no pyrimidine dimers), other than T = T , than does u.v. irradiation and that both photoreactivation and vR repair T = T . The biological data presented here are used with these chemical measurements to demonstrate that (1) and (2) are true and that photoreactivation and vR act preferentially on thymine dimers but that xR does not.

2. Materials and Methods (a) Bacteriophage strains The osmotic shock-resistant mutant, T4Bol possesses active v and x genes which confer relative resistance ~o u.v. irradiation. Phages TVv1 (v-x +) and T4x (v+x-), the n.y.sensitive mutants of TdD (Harm, 1963) were obtained from Dr W. Harm. The double

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mutants, T4vol and T4xol were constructed by crossing T4Bol with the u.v.-sensitive mutants. The genetic backgrounds of these phage were made more nearly t h a t of T4B b y successive backcrosses with T4Bo~rI[ and T4Bo~. Two pairs of backcrosses were performed to obtain T4vol; one pair of backcrosses was done for T4xo~. T4vxol was produced b y crossing T4vol and T4xol. :For simplicity, T4Bol, T4vol, T4xol and T4vxol will be referred to, respectively, as T 4 + , T4v, T4x, and T4vx. (b) Bacteriophage techniques The phage techniques and media used were those of Barnett, Brenner, Crick, Shulman & Watts-Tobin (1967). All platings were done using fresh agar plates with 5 × 107 chilled log-phase Escherichia coli B in the top agar layer, as indicator bacteria. Photoreactivation was performed as described previously (Meistrich et al., 1970) b y illuminating complexes of irradiated phage and chloramphenieol-treated bacteria with light of wavelength greater t h a n 380 n m for 30 rain. The photoreactivable sector was the fraction of dose reversed, measured along the actual inactivation curve with slight corrections made for efficiency of plating of complexes and titer loss caused b y photoreactivating light. (c) Irradiation Direct u.v. irradiation at 254 nm was provided b y a germicidal lamp (2.5 erg/mmS/sec). Light of wavelengths f> 310 n m was used for the sensitized irradiation, and calibrated as in the preceding paper (Meistrich & Lamola, 1972). The cationic derivatives of acetophenone, Ac¢D and Ac¢M have been previously described (Meistrich et al., 1970). The Ac¢D used in these experiments was synthesized b y Dr A. A. Lamola. Ac¢D sensitization was performed in Mg2+-free M9 buffer, p i t 7, with a monovalent cation concentration of 0.19 M. The phage were incubated with 5 × 10 -3 M-Ac¢D at 37°C for 5 to 10 hl. before irradiation. S a m p l e s for Ac¢M sensitization were incubated with 7'2 × i0 -S 1~ of the sensitizer in either 0.19 ~ or 0'06 ~ buffer at 4°C, overnight, followed b y 30 rain at 30°C. The relative inactivation rates and photoreactivable sectors were the same with both buffers.

3. Experimental Results (a) Ultraviolet irradiation The m u t a n t s c o n s t r u c t e d for these e x p e r i m e n t s were u.v. i r r a d i a t e d to a s c e r t a i n t h a t t h e y h a d t h e s a m e p r o p e r t i e s as H a r m ' s original m u t a n t s . The u.v. i n a c t i v a t i o n of these m u t a n t s is shown in F i g u r e 1 (a). E x p o n e n t i a l curves h a v e been d r a w n t h r o u g h t h e e x p e r i m e n t a l p o i n t s using t h e e x t r a p o l a t i o n value of n ---- 1.5 which has been d e t e r m i n e d for T 4 + (Cavilla & Johns, 1964) a n d T4v (Wulff, 1963a) a n d a p p e a r s t o be similar for T4x a n d T4vx ( H a r m , 1963). (The e x t r a p o l a t i o n value, n, is t h e intercept of t h e e x p o n e n t i a l p a r t of t h e s u r v i v a l curve w i t h t h e ordinate.) The i n a c t i v a t i o n r a t e s of t h e m u t a n t s of T4Bol c o n s t r u c t e d here were t h e same as t h e original u.v.sensitive m u t a n t s , a n d t h e r e l a t i v e i n a c t i v a t i o n rates (Table 1) agree w i t h H a r m ' s (1963) values to w i t h i n 5~/o . The p h o t o r e a c t i v a b l e sectors o f t h e u , v . - i r r a d i a t e d m u t a n t s h a v e been m e a s u r e d (Table 2) a n d e x c e p t for T4x t h e results agree w i t h H a r m ' s (1963). Our m e a s u r e m e n t s on t h e T4x m u t a n t o b t a i n e d from H a r m g a v e a sector of 0.26 a n d this v a l u e is consistent w i t h a n o t h e r r e c e n t m e a s u r e m e n t (Boyle & S y m o n d s , 1969) a n d t h e sector o f 0.24 for t h e osmotic s h o c k - r e s i s t a n t d e r i v a t i v e . Thus, t h e m u t a n t s c o n s t r u c t e d here r e s p o n d to u.v. i r r a d i a t i o n in a m a n n e r i d e n t i c a l to t h e original u.v.-sensitive m u t a n t s . (b) AceD sensitization The s u r v i v a l of t h e osmotic s h o c k - r e s i s t a n t m u t a n t s following sensitized i r r a d i a t i o n in t h e presence of A c e D was m e a s u r e d (Fig. l(b)). The u.v.-sensitive m u t a n t s are

5~[. L. MEISTRICH

100 i

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IO 0

I00

200

300 0

50

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I00

150

200

Dose (sec of irradiation ) (b)

FIG. ]. Survival of four mutants of phage T4 following (a) u.v. and (b) A o ¢ D sensitized irradiation measured by the number of plaque-forming units. The data for u.v. irradiation are averages of 4 experiments. The results for AcCD sensitization are averaged from 2 experiments in w h i c h t h e p h a g e were i n c u b a t e d w i t h t h e sensitizer for 7 h r a t 37°C. TABLE

1

Relative inactivation rates of mutants of T4Bo 1 to direct ultraviolet light and sensitization Phagem~a~ + v x

vx

u.v.(254nm)

AeCDsensitization

AeCMsensitization

1.0 4.4 1.9 7.8

1'0 3.5 ---

1.0 2.2 1.6 4.1

T h e r a t e s are c o m p u t e d f r o m t h e e x p o n e n t i a l p a r t of t h e s u r v i v a l curve. T h e relative r a t e s (arbitrarily choosing T4 -{- as u n i t y ) were c o m p u t e d f r o m sets of s a m p l e s i r r a d i a t e d a n d p l a t e d on t h e s a m e d a y a n d t h e n t h e s e relative r a t e s were a v e r a g e d . All r e s u l t s are a v e r a g e s f r o m a t least t h r e e e x p e r i m e n t s . T h e e x p e r i m e n t a l s t a n d a r d error w a s less t h a n 5 % in all cases. TABLE 2

Photoreactivable sectors of the mutants inactivated by direct ultraviolet light and sensitization Phage mutant

u.v.

-~ v

0.29 0-63 0.24 0.60

vx

AcCD s e n s i t i z a t i o n 0.57 0.81 0.49 0.74

AeCM s e n s i t i z a t i o n 0.34, 0 . 4 8 t 0.77

S a m p l e s h a d b e e n i n a c t i v a t e d to s u r v i v a l levels in t h e r a n g e of 10-2 to 5 x 10-4. All r e s u l t s are a v e r a g e s of b e t w e e n 3 a n d 8 e x p e r i m e n t s . T h e e x p e r i m e n t a l s t a n d a r d d e v i a t i o n s of t h e m e a n are less t h a n 0.02 in n e a r l y all cases. t T h e two v a l u e s for AeCM indicate two series of e x p e r i m e n t s of t h r e e m e a s u r e m e n t s each. A l t h o u g h t h e p h o t o r e a c t i v a b l e sectors for all o t h e r eases were c o n s i s t e n t in t h e t w o series of e x p e r i m e n t s , t h e sectors for T 4 + w i t h AeCM s e n s i t i z a t i o n were different.

T H Y M I N E DIMERS AND U.V. I N A C T I V A T I O N

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also more susceptible to sensitized inactivation. The curves are generally exponential to survival levels of 10-3, or less, except for a slight shoulder at low doses. The inactivation curves of T4~- and T4v in the low-dose region gave approximate extrapolation values of n = 1.4 and n = 2, respectively. More careful measurements are necessary to determine whether the shoulder for AcCD-sensitization of T4v is significantly larger than t h a t for 3?4+ and the value of n = 1-5 for u.v.-irradiation of these phages. For the present we shall assume extrapolation values of n = 1.5 for T4~-, T4x and T4vx and n = 1.8 for T4v. The survival curve of T4x deviates from an exponential relation. This might represent heterogeneity in the permeability of the phage in the sample, subjective errors in counting the smaller plaques produced by this mutant, or an intrinsic property of the biology of this mutant. The deviation from exponential is small and the uncertainty of measuring the slope is less than 10%. The inactivation rate is taken from the portion of the curve above 10 -2 since this region represents the bulk of the phage inactivation. The relative rates of inactivation computed from the experiments shown in Figure l(b) and from other measurements on samples with different times of incubation with the sensitizer are presented in Table 1. Phage inactivated b y AcCD-sensitization are photoreactivable. The shape of the survival curve for T4-~ and T4v following m a x i m u m photoreactivation is approximately exponential (unpublished results). Hence the photoreactivation following sensitized irradiation can be represented b y a photoreactivable sector. We shall also interpret the photoreactivation of T4x and T4vx in terms of a photoreactivable sector. The values for the sectors given in Table 2 show t h a t the photoreactivation of phage inactivated with sensitization is always greater than with u.v. irradiation. (c) AcCM sensitization Another derivative of acetophenone, AcCM, can be used to sensitize the formation of thymine dimers (Meistrich & Lamola, 1972) and phage inactivation. The survival

E 10-1 i a~ (3__a o. v

>

-...

\ ~7

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~v

03 V

1°-3o

\

4

T

T

8 12 Irradiation time (min)

~\

16

FIG. 2. Survival curves for T4+ and T4v following AcCMsensitization. The data are the averages of 3 experiments. The dashed lines indicate the slope of the exponential part of the inactivation curve of these phages in the absence of any sensitizer.

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MEISTRICtI

curves for T 4 + and T4v (Fig. 2) are exponential to survivals of less than 10 -3 (unpublished results) except for the shoulder at low doses, which gives an extrapolation value of n ~ 2.6. The data in Figure 2 plus several experiments in which the phage were irradiated in 0.06 ~-buffcr are averaged to obtain the relative inactivation rates (Table 1). 4. D i s c u s s i o n Three irradiation procedures which produce T - - T : u.v. irradiation at 254 nm, AcCD sensitization, and AcCM sensitization have been compared with respect to the inactivation of mutants of T4 phage and the photoreactivability of that inactivation. Ultraviolet irradiation and AcCD sensitization differ in the classes of photoproducts produced; sensitization produces fewer lesions other than T = T (Lamola, 1970; Meistrich & Lamola, 1972). We expect t h a t AcCM sensitization produces a distribution of photoproducts similar to AcCD since both molecules have identical triplet-state energies (Meistrich et al., 1970). The differences in results obtained using the two sensitizers (Tables 1 and 2) might be attributable to the fact that, even under optimal conditions, AcCM increases the inactivation rate only sixfold over background (Fig. 2) and as much as 15% of the lethal effect m a y be produced directly b y the irradiation. Measurements of the yields of T----T/p.l.h. with the various irradiation methods have been made for T4-~ (Meistrich & Lamola, 1972). Since the number of T = T produced per unit dose of u.v. irradiation is the same for the different mutants (Sauerbier, 1964), the same is most likely true for sensitized irradiation. Thus values for T=T/p.l.h. for the mutants (Table 3) m a y be computed b y dividing those measured for T4~- b y the relative inactivation rates (Table 1). TABLE 3 Thymine dimers per phage lethal hit Phage mutant

u.v.

AcCD sensitization

AcCMsensitization

-~ v x vx

10.2 4.7 6.5 2.5

26 6.0 13.5 3-3

21 6.0

The values are the numbers of T=T produced per phage by a dose of one p.l.h, of u.v., or sensitized irradiation. I n the subsequent analysis, we shall assume t h a t T = T produced by the three irradiation techniques behave similarly. Although we cannot rigorously prove that the dimers produced b y the various irradiations are exactly identical with regard to distribution throughout the genome and in local sequences, the following arguments make it highly unlikely t h a t there are any major differences, t tNote added in proof: recent measurements by Brunk (1972, 16th Ann. Biophys Soc. Meeting, Abstr. p. 236a) have shown that u.v.irradiation produces a higher yield of thymine dimers in longer tracts of pyrimidines. Although these measurements were only at high doses, extrapolation indicates an appreciable effect of tract length at lower doses. Brunk's experiments using AcCD sensitization to produce dimers were performed at saturation doses. Thus it cannot be determined whether the distribution of dimers at low doses is uniform or follows the same trend as the u.v. irradiation data.

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Acetophenone- or AcCD-sensitization of E. coli DNA can dimerize 37 ~o of the total thymines, a value comparable to the theoretical maximum of 44% of thymines which are dimerizable. AcCD sensitization of intact T4 phage can dimerize 6% of the thymines and although the rate of dimerization decreases at high doses the behavior is not indicative of saturation. It is likely that regions of the DNA molecule, by virtue of the packaging within the phage head, are less accessible to the sensitizer and in these regions the rate of dimerization may be lower. Since the DNA molecules of different phage in a population are circular permutations of the genome (Streisinger, Emrich & Stahl, 1967), the DNA of each phage is probably packed in a different manner, effectively randomizing the regions of the DNA which may be more exposed to the action of the sensitizer. Furthermore, since at least 6~o of the thymines in the phage can be dimerized and the quantum yield of sensitized dimerization is at least 0.03 (Meistrich & Lamola, 1972) iris apparent that thymine dimerization is not limited to a small segment of the phage DNA. Similarly, it has been shown that u.v.irradiation (h~280 nm) can dimerize about 50% of the dimerizable thymines (Setlow & Carrier, 1963; Wulff, 1963b) a value which is most likely limited by equilibrium between formation and breakage of dimers. Furthermore, in the dose range for the biological experiments described here, less than 0.1% of the thymines are dimerized and the number of dimers is linear with dose. Thus, at low doses T = T should be formed randomly at nearly all of the available sites for thymine dimerization. In addition, the induction of mutations in two genes at the same relative frequencies by u.v. and sensitized irradiation supports the assumption that the dimers are similarly distributed (Meistrich & Shulman, 1969). The effect of local sequences on the distribution of dimers is not considered to be major. The effects of energy transfer or quenching in certain bases, which might alter the rates of dimcr formation by u.v. irradiation in certain regions, are probably not very important at room temperature (Eisinger & Lamola, 1971a,b). In the case of sensitization, sensitizer binding is unlikely to be strongly dependent on local base sequence since the interaction is predominantly ionic involving the phosphate backbone. Furthermore, the concentration of sensitizer in these experiments should be high enough to overwhelm moderate differences in binding constants, Space-filling (CPK) models of AcCD binding to DNA in the region of glucosylated hydroxymethylcytosine bases shows that the glucosyl groups present a slight, but not an important, sterie block to the approach of the sensitizer. Therefore, we may assume that T = T produced by u.v. and sensitized irradiation are similar with respect to distribution and biological behavior. The experimental results (Tables 1, 2 and 3) provide several lines of evidence which demonstrate that T = T must be a lethal lesion. I f T = T were not a lethal lesion, then the photoreactivation must be attributed to repair of lethal u.v. lesions other than T~-T. In that case we would expect little photoreactivation of phage inactivated by sensitization because chemical measurements show that very low yields of other u.v. lesions are formed and it is unlikely that any other minor products of sensitization are photoreactivable. But since sensitized phage are photoreactivable, T---T must be lethal lesions. An analogous argument may be used with vR which also repairs T ~ T (Setlow & Carrier, 1966b; Yasuda & Sekiguchi, 1970) providing further corroboration that T = T are lethal lesions. The large photoreactivable and vR sectors following sensitized irradiation indicate that. T = T are the principal"lethal lesions produced by sensitization,

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Their role in u.v. inactivation can be estimated as follows from Table 3. In the case of sensitization of T4vx there are 3.3 T=T/p.l.h. and the photoreactivation data indicate that at least 74% of the lethal effect is from T-----T.Thus 3-3 T = T account directly for at least 0.74 p.l.h, in T4vx. Hence, the 2.5 T--T produced by u.v. irradiation per p.l.h, account directly for at least (2.5 × 0.74/3.3) or 0.56 p.l.h. Thus T = T is responsible for more than one-half the u.v. inactivation of this phage and must be considered as an important lethal lesion. The same conclusions can also be reached by considering the chemical analysis which shows that AcCD sensitization produces very few photoproducts other than T--T (Meistrich & Lamola, 1972). Since T4vx has few, if any, repair mechanisms there should be a close correspondence between the number of chemical lesions and the biological effect. For three cases we can make the following predictions. (1) I f T--T were the only lethal lesion, the T-~T/p.l.h. for both irradiation methods would be equal. (2) If T = T were lethal but there were other lethal lesions, the T=T/p.l.h. would be somewhat higher (less than a factor of 2) in the case of AcCD-sensitization. (3) If T--T were not lethal but all inactivation were due to other lesions, then T=T/p.l.h. for sensitization would be much higher (eightfold higher from the chemical data) than that for u.v. irradiation. The data support case (2) confirming that T = T is a lethal lesion, and furthermore demonstrating that u.v. irradiation produces other inactivating lesions besides T----T. The data presented here also indicate the specificity of the various repair processes involved in reactivation. Sensitization produces more T=T/p.l.h. and therefore lower yields of other lesions than does u.v. irradiation. The inactivation produced by sensitization is not only photoreactivable (Table 2) and reversed by vR (Table 1) but the reactivation in both eases is greater than with u.v. irradiation. Thus these repair processes act more specifically on the lethal lesions produced by sensitized than by u.v. irradiation. The chemical analyses and the data in Table 3 show that T----T constitutes a greater proportion of lethal lesions in the case of sensitization; we conclude that photoreactivation and vR reverse T = T more effectively than the other u.v. lesions. This conclusion is consistent with the observation (Table 2; Harm, 1963) that the photoreactivable sector is lower for T4v + than T4v. The x-gene also confers u.v. resistance to T4 phage by a mechanism which is unknown but may have some aspects in common with genetic recombination (Harm, 1964). The n.y. sensitivities of the x mutants are greater than those of x + phage by factors of 1.6 and 1-9, respectively, for v + and v- phage. Similarly, upon sensitized irradiation, the inactivation rates of the x mutants are increased by 1.9 and 1.8 for phage ~4th the v + and v- alleles, respectively (Table 1). The xR is essentially the same for both irradiation techniques and both allelic states of the v-gene. Since vR preferentially removes T----T it follows the xR acts on T = T , the other n.y. lesions, and any other photoproducts produced by AcCD sensitization. This result is consistent w i t h the similar photoreactivable sectors observed for x + and x - mutants. The measurements on the inactivation produced by Ac¢lVi gave values for vR, photoreactivation and T--T/p.l.h. which in general were intermediate between those obtained with AcCD sensitization and u.v. irradiation. These observations can all be explained by the single assumption that AcCM sensitization produces more lethal lesions other than T = T than does sensitization with AcCD but fewer than with u.v. irradiation. This further supports the conclusions reached above by showing that

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another sensitizer of the thymine triplet-state can yield qualitatively the same results. I n order to obtain a complete description of the system it has been necessary to perform four types of measurements using various irradiation conditions and mutants. These are: (1) relative inactivation rates of different mutants, (2) photoreactivable sectors, (3) T = T / p . I . h . , and (4) analysis of photoproduet distribution. Previous studies have attempted to determine the role of various lesions in inactivation. Using longwavelength irradiation (A : 302 nm) Cavilla & Johns (1964) have observed an increase in the ratio of inactivation rates of T4v to T4v + with a concomitant decrease in photoreaetivable sector. I t was necessary to postulate the formation of a new u.v. lesion which could be repaired by vl~ but not by photoreactivation. Since no photoproduct analysis was done, no firm conclusions could be drawn. Haug & Sauerbier (1965), using 315-nm radiation at a p H of 3.5, have measured for T 4 v x an increased yield of T = T / p . l . h . but a lowered photoreactivable sector, a result inconsistent with the present model. However, they did not measure the photoproducts other t h a n T = T to support their contention t h a t there are important photoreactivable photoproducts other t h a n T = T . The conclusions drawn above are consistent with all of the present data and m a y be summarized as follows : T = T are lethal lesions but so are other u.v. lesions. The three irradiation techniques produce thymine dimers plus varying yields of other lethal photoproducts with the latter decreasing in the order: u.v. irradiation, A c ¢ ~ sensitization, AcCD sensitization. Photoreactivation and vl~ repair T = T preferentially to other u.v. lesions; xl~ acts approximately equally on T = T and other photoproducts. Using the data obtained in this study, we have on the basis of relatively few assumptions computed quantitative values for the contributions to inactivation and the amount of repair of various classes of lesions (M. Meistrich, manuscript in preparation). I thank Drs A. A. Lamola Dr 1%. G. Shulman for many

and W. 1%. Bruce for critically reviewing the manuscript, useful discussions, and Miss Patrieia Muglia, Mr Thomas

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