Deoxyribonucleic acid synthesis induced with ultraviolet light in Brij 58-treated Bacillus subtilis spores germinated in the presence of chloramphenicol

35

Biochimica et Biophysica Acta, 378 (1975) 35--43 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 98165 D E O X Y R I B O N U C L E I C ACID SYNTHESIS INDUCED WITH U L T R A V I O L E T LIGHT IN B R I J 58-TREATED BA CILL US S UBTILIS SPORES GERMINATED IN THE PRESENCE OF C H L O R A M P H E N I C O L

YASUTARO FUJITA and TOHRU KOMANO Laboratory of Biochemistry, Department of Agricultural Chemistry, Kyoto University, Kyoto (Japan) (Received June 24th, 1974)

Summary The direct measurement of ultraviolet light-stimulated DNA synthesis in the permeable Bacillus subtilis cells was performed. Bacillus subtilis spores germinated in the presence of chloramphenicol were treated with Brij 58 and irradiated with ultraviolet light, and [3 H ] d T T P was incorporated into these cells by the DNA polymerase assay system. Characteristics of the incorporation were distinct from those into spores germinated in the absence of chloramphenicol and treated with Brij 58, in the respect that the former incorporation did not require ATP and only partially depended on the presence of all four deoxyribonucleoside triphosphates. The incorporation of [3 H] dTTP into DNA was confirmed by CsC1 density gradient centrifugation. A DNA polymerase I-deficient strain, JB1 49(59) had no [ 3 H ] d T T P incorporating activity induced by ultraviolet light irradiation when the germinated spores were treated with Brij 58. Analysis of alkaline sucrose gradient centrifugation revealed that fragmented DNA caused by ultraviolet light irradiation was rejoined to the size of DNA of non-irradiated cells by incubating irradiated cells in the DNA polymerase assay mixture containing NAD*. The results also suggested that a machinery of DNA repair probably pre-existed in the spore.

Introduction A number of laboratories has shown that D N A damages induced by radiation and by chemical agents can be repaired in vivo [1]. Recently, several workers have attempted to investigate DNA repair in vitro in order to understand the biochemical mechanism involved in it [2--5]. DNA repair in vegetative cells of Bacillus subtilis has been investigated not only in the semi-in vitro b u t also in the in vitro system. Noguti and Kada [4] have demonstrated that the transforming activity in the lysate of 7-ray-irradiated and toluene-treated

36 cells increased remarkably in the presence of four deoxyribonucleoside triphosphates and nicotinamide adenine dinucleotide, and the 7-ray-induced DNA single-strand breaks are rejoined to a significant extent. Laipis and Ganesan [3] have reported that DNA extracted from X-ray-irradiated cells deficient in DNA polymerase I is repaired in vitro by adding purified DNA polymerase I and DNA ligase, or the lysate from wild-type cells. In the previous paper [6], we have reported an increasing activity of [3 H] dTTP incorporation into DNA in the various stages of germinating spores which are treated with Brij 58, and this DNA synthesizing activity is regarded as that of DNA replication. This activity is not observed in spores germinated in the presence of chloramphenicol, but [3 H] dTTP incorporation into these cells is induced by ultraviolet light irradiation. This report evidences that [3 H] dTTP incorporation induced by ultraviolet light irradiation reflects repair replication, that DNA polymerase I is responsible for the repair, and that DNA damaged by ultraviolet light irradiation in Brij-treated cells is repaired in the presence of substrates for DNA polymerase and a cofactor, NAD ÷, for DNA ligase. Materials and Methods

Bacterial strains. B. subtilis Marburg 168 ( t h y - t r p ~ ) [ 7 ] and a polymerase I-deficient (pol-) strain, JB1 49(59) (ind-his-cys-) [8], which was a gift from Dr H. Terano, were used. Spore preparation. The procedure for spore preparation was the same as described in the previous paper [6]. Vegetative cells of the bacteria were incubated in liquid Schaeffer m e d i u m [9] for 25 h at 37°C with vigorous shaking. Spores thus formed were collected and cleaned with lysozyme (EC 3.2.1.7) and 1% sodium laurylsulfate, and then washed with distilled water. The refractile appearance of the spores was confirmed with a phase-contrast microscope. Spores containing [3 H] DNA were prepared in the same medium containing 3 pCi/ml of [3 H ] t h y m i n e (The Daiichi Pure Chemical Co., 13.5 Ci/mM). Germination. The germination conditions w e r e the same as described in the previous paper [6]. Demain medium [10], supplemented with 20 pg/ml of L-tryptophan and 10 pg/ml of t h y m i n e for 168 or 20 ttg/ml of L-tryptophan, L-histidine, and L-cysteine for JB1 49(59), was used throughout the germination experiments. Spores were suspended in the medium and incubated at 37°C with shaking. Initiation of germination was observed by the decrease in turbidity of the spore suspension. Morphological changes were observed with a phase-contrast microscope at a magnification of 2 000. Brij treatment and assay o f [3H] dTTP incorporation induced by ultraviolet light irradiation. The conditions of Brij treatment, ultraviolet light irradiation, and assay of [ 3 H ] d T T P incorporation were almost the same as described in the previous paper [6]. 2.5 • 10 s spores/ml were germinated in the m e d i u m containing chloramphenicol (100 ttg/ml) for 2 h at 37°C. Cells were collected and suspended in 0.1 M potassium phosphate buffer (pH 7.5) containing 6.6 mM MgC12. After adding Brij 58 to a final concentration of 0.5%, the suspension was incubated for 15 min at 37°C (1.3 • 109 cells/ml). Cells were

37 collected and resuspended in the same buffer ( 1 . 7 . 1 0 9 cells/ml). The cell suspension (1 ml) in a petri dish (9 cm in diameter) was exposed to ultraviolet light at a distance of 19 cm (a dose rate of 37 ergs • m m -2 • s-~ ). As the ultraviolet source, a 15-W germicidal lamp (National Co., peak wave-length 253.7 nm) was used. [ 3 H ] d T T P incorporation activity per 5 . l 0 s cells was measured by the slightly modified m e t h o d of Okazaki and Kornberg [11] for the DNA polymerase (EC 2.7.7.7) assay. The reaction mixture (total volume 0.5 ml) contained 20 pmoles of Tris--HC1 buffer (pH 8.0), 2 pmoles of MgCl:, I pmole of ~-mercaptoethanol, 14 nmoles of 4 deoxyribonucleoside triphosphates, 0.5 pCi [ 3 H ] d T T P (The Radiochemical Centre, 22.5 Ci/mM), and 5 - 1 0 8 cells. The product was precipitated by adding 0.5 ml of cold 10% trichloroacetic acid. The resulting acid-insoluble fraction was collected on a Whatman GF/C glass filter paper, washed with 5% trichloroacetic acid five times (total volume 30 ml), and dried. Radioactivity was counted in a toluene scintillation system. Analysis o f the product. Analytical conditions were almost the same as described in the previous paper [6]. Spores were germinated for 2 h at 37°C with chloramphenicol and treated with Brij 58. After [ 3 HI dTTP incorporation, cells (6 • 108 ) were incubated with 200 pg/ml lysozyme for 15 min at 37°C. Sodium laurylsulfate (1%), NaOH (0.1 M) and 32 P-labeled B. subtilis DNA were added to the suspension, which was then incubated for 5 min at 60°C. Unlysed cells were removed b y centrifugation. The resulting lysate (1.2 ml) was brought to equilibrium sedimentation in neutral CsC1 at 76 000 × g for 24 h at 20°C in an RPS 40 rotor o f a Hitachi 65P ultracentrifuge. After centrifugation, the ingredients were fractionated and the acid-precipitable radioactivity was measured in a toluene scintillation system. Chemicals. The chemicals used were purchased as follows: chloramphenicol from K y o w a h a k k o Co., p-chloromercuribenzoate from T o k y o Kasei Co., Brij 58 from Atlas Industries. Measurement of the size o f DNA. Spores containing [3 H] thymine-labeled DNA were germinated at 37°C for 2 h in the presence of chloramphenicol and treated with Brij 58, and were divided into three portions. One portion was used as a non-irradiated control, another was irradiated with ultraviolet light, and the other was irradiated with ultraviolet light and incubated for 1 h at 37°C in the DNA polymerase assay mixture, which contained NAD ÷ (50 pM) b u t dit n o t contain [3 H ] d T T P in order to repair ultraviolet light-damaged DNA. Then, the lysate was prepared from each portion in the following manner. The cells were incubated with lysozyme (200 pg/ml) for 15 min at 37°C and lysed with 1% sodium laurylsulfate and 0.1 M NaOH for 5 min at 60°C. Unlysed cells were removed b y centrifugation. The lysate was layered on the top of a 5--20% alkaline sucrose gradient containing 0.1 M NaOH, 0.9 M NaC1 and 1 mM EDTA, and centrifuged for 120 min at 81 000 X g in an RPS 40 rotor of a Hitachi ultracentrifuge at 15°C. After fractionation, 100 gg R N A was added to each fraction as carrier, and then trichloroacetic acid was added to 5%. The resulting acid-insoluble material was collected on a Whatman GF/C glass filter paper, washed twice with 5% trichloroacetic acid and dried. Radioactivity was counted in a toluene scintillation system.

38

Results

[3 H] dTTP incorporation induced by ultraviolet light irradiation In order to confirm the evidence that permeable germinated spores incorporated [3 HI dTTP after irradiation with ultraviolet light, the kinetics of the [3 H] dTTP incorporation were studied. Fig. 1A shows the relation between the ultraviolet light dose and the [3 H] dTTP incorporation activity in spores germinated in the presence of chloramphenicol for 2 h, treated with Brij 58, and irradiated with ultraviolet light. [3 H] dTTP incorporation increased linearly up to a dose of 1 000 ergs/mm 2 but the rate of incorporation was gradually reduced at higher doses than 1 000 ergs/mm 2 . Without the Brij treatment, the [3H]dTTP incorporation observed was fairly low. Fig. 1B shows the time course of [ 3 H ] d T T P incorporation into cells after irradiation at 2 220 ergs/ m m 2 . The [3 H] dTTP incorporation increased sharply and linearly for 20 min and then reached the saturation point. It is evident that [3 HI dTTP is incorporated into spores germinated in the presence of chloramphenicol and treated with Brij 58 in proportion to the doses of ultraviolet light.

Requirements for [3 H] dTTP incorporation induced by ultraviolet light irradiation As reported previously, Brij-treated vegetative cells incorporated [3 H] dTTP and this incorporating activity was regarded as the activity of DNA replication [6]. So that the [3 H] dTTP incorporating activity induced with the ultraviolet light of spores germinated in the presence of chloramphenicol and treated with Brij 58 was compared with that of Brij-treated vegetative cells in respect to the requirements for [ 3 H ] d T T P incorporation. Table I shows the

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Fig. 1. [3HI d T T P incorporation induced with various doses of ultraviolet light (A) and time course of the incorporation induced with a dose of 2 2 2 0 e r g s / m m 2 (B). Spores were germinated for 120 rain in the presence of chloramphenicol and treated with Bzij 58, and the [ 3 H ] d T T P incorporating activity was assayed (o). T h e details were described in the text. ~ A [3H] d T T P incorporation into germinating spores untreated with Brij 58.

39 TABLE I R E Q U I R E M E N T S FOR [3H] d T T P I N C O R P O R A T I O N S p o r e s w e r e g e r m i n a t e d w i t h c h l o r a m p h e n i c o l , t r e a t e d w i t h Brij 58 a n d i r r a d i a t e d w i t h a dose o f 2 2 2 0 e r g s / m m 2. T h e r e a c t i o n m i x t u r e w a s i n c u b a t e d for 3 0 m i n a t 3 7 ° C . T h e d e t a i l s w e r e d e s c r i b e d in the t e x t . 100% a c t i v i t y c o r r e s p o n d s to a n i n c o r p o r a t i n g a c t i v i t y o f 179 p m o l e s o f T T P p e r 1 . 1 0 9 cells. Germinated, irradiated spores

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100 82 67 48 3 0

C o m p l e t e (+ A T P ) - ATP - dATP, - dGTP, - dCTP + p-Chloromercuribenzoate N o cell

100 51 14 38 0

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requirements for the [3H] dTTP incorporation into the above treated spores induced by ultraviolet light irradiation in comparison with those into Brijtreated vegetative cells [6]. The incorporation induced by ultraviolet light irradiation did not require ATP, while that into vegetative cells required ATP. This incorporation was partially inhibited by p-chloromercuribenzoate. This incorporation was partially dependent on the presence of all four deoxyribonucleoside triphosphates, while they were essential to the incorporation into vegetative cells. This fact indicated that the uptake of [3H]dTTP in the absence of other triphosphates was very high, and would suggest that dTMP was incorporated exclusively into the DNA gap introduced by the excision of thymine dimer. Therefore, the [3 H]dTTP incorporation into the above treated spores was distinguished from that into Brij-treated vegetative cells, especially with respect to ATP dependency. It has been reported that replicative synthesis requires ATP, whereas repair synthesis does not [2]. The [3 H] dTTP incorporating activity induced with ultraviolet light appears to reflect repair synthesis, while that into Brijtreated vegetative cells is the result of replicative synthesis as discussed in the previous paper [6].

[3H] dTTP incorporation into DNA To investigate whether or not 3H label was incorporated into DNA, the following experiment was performed. Spores were germinated in the presence of chloramphenicol, treated with Brij 58, and irradiated with ultraviolet light. After incorporating [3H]dTTP into the above treated spores, DNA was extracted, and equilibrium centrifugation in neutral CsC1 together with 32 P-labeled DNA of vegetative B. subtilis cells was performed. As shown in Fig. 2, the 3 H label coincided with the 32 p label. This result inomated that [3 H] dTTP was incorporated into the DNA of the cells and that the DNA had the same density as that ofB. subtilis DNA. DNA rejoining in Bri]-treated, ultraviolet light-irradiated cells To investigate whether or not strand breaks of DNA induced by ultra-

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Fig. 2. T h e CsC1 i s o p y c n i c b a n d i n g p a t t e r n o f t h e [ 3 H ] d T T P i n c o r p o r a t i o n p r o d u c t . [ 3 H ] d T T P i n c o r p o r a t i o n a n d e x t r a c t i o n o f D N A w e r e d e s c r i b e d in t h e t e x t . T h e d e n s i t y w a s a d j u s t e d t o 1 . 7 0 5 b e f o r e c e n t r i f u g a t i o n ( 7 6 0 0 0 X g, 2 4 h a t 2 0 ° C ) . T h e d e n s i t y i n c r e a s e s f r o m r i g h t t o left. e , i n c o r p o r a t e d 3 H label; o, p a t t e r n o f 3 2 p - l a b e l e d D N A o f v e g e t a t i v e cells is s h o w n f o r c o m p a r i s o n . Fig. 3. S e d i m e n t a t i o n p a t t e r n o f D N A in a l k a l i n e s u c r o s e d e n s i t y g r a d i e n t . C o n d i t i o n s o f a n a l y s e s w e r e d e s c r i b e d i n t h e t e x t . D N A s w e r e e x t r a c t e d f r o m t h e n o n - i r r a d i a t e d c o n t r o l (o) a n d i r r a d i a t e d cells (1 1 1 0 e r g s / m m 2 ; e ) . D N A w a s also p r e p a r e d f r o m cells i r r a d i a t e d w i t h u l t r a v i o l e t l i g h t (1 1 1 0 e r g s / m m 2 ) a n d i n c u b a t e d f o r 6 0 r n i n a t 3 7 ° C ( A ) in t h e D N A p o l y m e r a s e assay m i x t u r e w h i c h c o n t a i n e d N A D + ( 5 0 # M ) b u t d i d n o t c o n t a i n [ 3 H ] d T T P (w); (B) i n t h e p o l y m e r a s e a s s a y m i x t u r e w h i c h d i d n o t c o n t a i n [ 3 H I d T T P (m), o r (C) in N A D + ( 5 0 # M ) ( i ) . R e p l l c a t i v e f o r m I o f ~ X 1 7 4 D N A , 5 4 . 1 S U : ~bX 1 7 4 D N A ( s i n g l e stranded), 18 S |.

violet light irradiation were rejoined in this system, the following experiments were performed. The spores containing [3 H] thymine-labeled D N A were germinated in the presence of chloramphenicol, and treated with Brij 58. And then, the lysates were prepared, respectively, from non-irradiated cells, irradiated cells, and another portion of irradiated cells which was incubated in the D N A polymerase assay mixture, which contained N A D ÷ but did not contain [ 3H]dTTP. Each of the lysates was subjected to an alkaline sucrose gradient analysis. The zone sedimentation patterns of DNAs, shown in Fig. 3A demonstrated that the strand breaks of D N A caused by ultraviolet light irradiation were rejoined by incubating irradiated cells in the reaction mixture. Fig. 3B or 3C shows that in the absence of deoxyribonucleoside triphosphates or N A D ÷

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Fig. 4. [3H] d T T P incorporation induced with ultraviolet light ( A ) a n d ATP-stimuiated [3H] d T T P incorporation (B). Conditions of m e a s u r e m e n t of the incorporation induced with ultraviolet light were described in the text. ATP-stimttiated [ 3 H ] d T T P incorporation into non-irradiated vegetative cells was assayed b y the m e t h o d described in the previous paper [6]. Spores were germinated u p to the indicated times, treated with Brij 58 and incubated in the D N A polymerase assay mixture containing 0.4 m M A T P . o, experiments using a m u t a n t strain, JBI 49(59); e, control, using a wild-type strain, 168.

one half to one quarter of the DNA seems to rejoin to control size b u t the rest breaks into smaller fragments. The slight differences in t h e position of nonirradiated DNA are probably n o t significant since the size of DNA depends on the shearing force during the preparation of the lysate. Ultraviolet light irradiation itself merely forms pyrimidine dimers [12] and cannot produce strand breaks. Nuclease activity specifically on DNA possessing damages caused with ultraviolet light is probably functioning during the preparation of lysates. These experiments were performed at a dose of 1 110 ergs/mm 2. We failed to rejoin fragmented DNA formed at a dose of 2 220 ergs/mm 2 in this system. It was considered that DNA damaged t o o much was no longer repaired in Brij-treated cells. From these results, we concluded that ultraviolet light damage in Brijtreated cells was repaired in the presence of substrates for DNA polymerase and a cofactor for DNA ligase.

[3 H] dTTP incorporation in a polymerase I-deficient mutant, JB149(59) To examine whether or n o t DNA polymerase I participates in this repair synthesis, we performed the following experiment in which a polymerase I-deficient mutant, JB1 49{59) is used. Strain JB1 49(59) is sensitive to both methyl methane sulfonate and ultraviolet light, and contains only 10% of the DNA polymerase activity of the wild-type strain [13]. Spores of this strain were germinated in the presence of chloramphenicol and treated with Brij 58. [ 3 H ] d T T P incorporation was n o t induced by ultraviolet light irradiation in these spores (Fig. 4A). On the other hand, an increasing rate of [3 H] dTTP incorporation into DNA in spores germinated up to various stages and treated with Brij 58 [6] was observed at the same level as the wild-type strain, as shown in Fig. 4B. Pol- mutation has no effect on the ATP-stimulated incorporation (replication synthesis [6] ), b u t is related to the incorporation induced with ultraviolet light which is considered to reflect repair synthesis.

42 Discussion E. coli DNA polymerase I and B. subtilis. DNA polymerase I have some properties in c o m m o n , but they are strikingly different in nuclease activity: B. subtilis e n z y m e lacks detectable nuclease [11,19]. A comparison of the properties of a E. coli polA1 m u t a n t (W3110 polA1) and those of a B. subtilis polA1 m u t a n t (JB1 49(59)) reveals m a n y similarities in repair function in vivo, though the remaining DNA polymerase activity in the B. subtilis m u t a n t is relatively high [8,13,16,20,21]. Both polymerases appear to participate in a similar process of repair. Several investigators have suggested that in ultraviolet lightexposed E. coli, DNA polymerase I and DNA ligase are involved in DNA repair [14--18]. Laipis and Ganesan [3] have reported that X-ray-irradiated DNA extracted from B. subtilis deficient in DNA polymerase I is repaired in vitro with the lysate of wild-type cells. Noguti and Kada [4] have suggested that DNA polymerase and DNA ligase participate in 7-ray-irradiated DNA repair in toluenized B. subtilis cells. The experiments we reported here elucidate that ultraviolet light-stimulated DNA synthesis is first directly measured in permeable B. subtilis cells and that this synthesis is ATP independent and attributed to DNA polymerase I. ATP-independent DNA synthesis mentioned in this paper m a y be the result of repair replication in ultraviolet light-irradiated cells. This DNA synthesis is distinct from ATP-dependent DNA synthesis as reported in the previous paper [6]. Fig. 3 shows that fragmented DNA of irradiated cells is rejoined to the size of DNA of non-irradiated cells in the presence of substrates for DNA polymerase and NAD ÷ which is the cofactor of DNA ligase in B. subtilis as it is in E. coli [22]. This result suggests that DNA polymerase and DNA ligase may participate in the rejoining of fragmented DNA. The fact that DNA synthesizing activity is not detected in cells of JB1 49(59), a m u t a n t of DNA polymerase I-deficient strain, shows that DNA polymerase I takes part in the repair replication, or gap filling following the removal of the t h y m i n e dimer. These results indicate that the process of the repair of ultraviolet light-induced damage, which involves excision, repair replication by DNA polymerase I, and rejoining by DNA ligase, can take place in these permeable spores, that is, in the semi-in vitro system. Using spores germinated in the presence of chloramphenicol is more advantageous than spores germinated up to other stages in the following reasons. When spores are germinated in the presence of chloramphenicol, these cells have no enzyme which is newly synthesized during germination, though there is a possibility that enzymes are synthesized in the presence of chloramphenicol (100 pg/ml). Therefore, excision t y p e repair of ultraviolet lightinduced damage may be carried out by a pre-existing repair machinery in spores. The [ 3 H ] d T T P incorporation activity into these cells without ultraviolet light irradiation is fairly low so that the ultraviolet light-induced activity is not disturbed by the u n k n o w n incorporation activities, including that due to DNA replication which is not observed in this stage of germinating spores [6]. Then, it is possible to measure and analyse with ease the activity of repair replication functioning in these cells. Since the mode of excision repair in these cells is considered to be the same as that in vegetative cells, the present system

43

is thought to be an excellent tool to examine the enzymatic basis of excision repair of B. subtilis. Recently, Masker and Hanawalt [5] have reported that ultraviolet lightstimulated DNA synthesis in toluenized E. coli deficient in DNA polymerase I requires the presence of four deoxyribonucleoside triphosphates and ATP. This mode of DNA synthesis appears to be different from the present ATP-independent DNA synthesis attributed to DNA polymerase I. Segev et al. [23] have suggested that the ATP-independent repair process in permeable E. coli cells irradiated with ultraviolet light involves the participation of DNA polymerase I. There is a possibility that ATP-independent repair replication, including the repair induced with ultraviolet light, may be attributed to DNA polymerase I. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Howard-Flanders, P. (1968) Annu. Rev. Biochem. 37, 175--200 Moses, R.E. and Richardson, C.C. (1970) Proc. Natl. Acad. Sci. U.S. 67,674---681 Laipis, P.J. and Ganesan, A.T. (1972) Proc. Natl. Acad. Sei. U.S. 69, 3 2 1 1 - - 3 2 1 4 Noguti, T. and Kada, T. (1972) J. Mol. Biol. 67, 507--512 Masker, W.E. and Hanawalt, P.C. (1973) Froc. Natl. Acad. Sci. U.S. 70, 129--133 Fujita, Y., Koman o, T. and Tanooka, H. (1973) J. Bacteriol. 113, 558--564 Farmer, J.L. and R o t h m a n , F. (1965) J. Bacteriol. 89, 262--263 Searashi, T. and Straus, B. (1965) Biochem. Biophys. Res. C ommun. 20, 680--687 Takahashi, I. (1965) J. Bacteriol. 89, 294--298 Demain, A. (1958) J. Bacteriol. 75, 517--522 Okazaki, T. and Kornberg, A. (1964) J. Biol. Chem. 239, 259--268 Smith, K.C. and Yoshikawa~ H. (1966) Photochem. Photobiol. 5, 777--786 Gass, K.B., Hill, T.C., Goulian, M., Straus, B.S. and Cozzarelli, N.R. (1971) J. Bacteriol. 108, 364-374 Pettijohn, A.R. and Hanawalt, P.C. (1964) J. Mol. Biol. 9 , 3 9 5 - - 4 1 0 Setlow, R.B. and Carrier, W. (1964) Proc. Natl. Acad. Sci. U.S. 5 1 , 2 2 6 - - 2 3 1 Delucia, P. and Cairns, J. ( 1 9 6 9 ) Nature 224, 1164---1166 Kelly, R.B., Atkinson, M.R., Huberman, J.A. and Kornberg, A. (1969) Nature 224, 495--501 Kelly, R.B., Cozzarelli, N.R., Deutscher, M.P.° Lehman, I.R. and Kornberg, A. (1970) J. Biol. Chem. 245, 39--45 England, P.T., Deutscher, M.P., Jovin, T.M., Kelly, R.B., Cozzarelli, N.R. and Kornberg, A. (1968) Cold Spring Harbor Symp. Quant. Biol. 33, 1--9 Gross, J. and Gross, M. (1969) Nature 224, 1166--1168 Klein, A. and Niebch, U. ( 1 9 7 1 ) N a t . New Biol. 229, 82--84 Laipis, P.J., Olivera, B.M. and Ganesan, A.T. (1969) Proc. Natl. Acad. Sci. U.S. 62, 289--296 Segev, N., Miller, C., Sharon, R. and Ben-lshai, R. (1973) Biochem. Biophys. Res. C ommun. 52, 1241--1245