Double-strand scission of DNA involved in thymineless death of Escherichia coli 15 TAU

Biochimica et Biophysica Acta, 294 (1973) 2o4-213 Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - P r i n t e d in T h e N e t h e r l a n d s BBA 97486

DOUBLE-STRAND SCISSION OF DNA INVOLVED IN T H Y M I N E L E S S D E A T H OF E S C H E R I C H I A C O L I 15 TAU KOICHI YOSHINAGA

Chemical Institute, Faculty of Science, Shizuoha University, Ooya, Shizuoka (Japan) ( R e c e i v e d J u l y 24th, i972 )

SUMMARY

Double-strand DNA scissions occurring during thymineless death of Escherichia coli 15 TAU were assayed by neutral sucrose gradient sedimentation of E3H]thymidine-labelled DNA isolated from cells lysed under mild conditions. It was found that, under thymineless conditions, the sedimentation rate of DNA started to decrease only after the cells began to lose their viability. When thymine was added to thyminestarved cultures, the progress of thymineless death was stopped and viable cells began to increase. However, the sedimentation rate of the labelled DNA was not restored to that of the original DNA. These results indicate (I) that thymineless death is accompanied by double-strand scission of DNA, (2) that thymine starvation does not per se cause the DNA scissions but only when cell death ensues, and (3) that the broken DNA cannot be converted back into its original form. It is suggested that double-strand scission of DNA is the principal cause for thymineless death in this case.

INTRODUCTION

Thymineless death, the loss of ability to multiply after a period of thymine starvation, was first described by Cohen and Barner 1. This phenomenon has been investigated in thymine auxotrophs of a number of bacterial species, but its chemical nature remains poorly understood. Menningmann and Szybalski 2 first reported that thymineless death is preceded by single-strand breaks in the DNA of Bacillus subtilis and a number of observations s-8 have indicated some correlation between thymineless death and lesions of DNA. Furthermore, thymineless death was found to be enhanced greatly by concomitant RNA synthesis 9-12. Hanawalt et al. 13 suggested that transcription might sometimes involve the introduction of repairable single-strand breaks into the bacterial DNA and that exonuclease action might enlarge the resulting gaps in the absence of the thymine required for its repair. Escherichia coli 15 TAU was used in the present study. This strain showed resistant-type thymineless death 14 and no recovery from thymineless death was observed. The cells, which had completed the cycle of DNA replication 15, rapidly lost viability during thymine starvation under conditions which allowed protein and RNA synthesis to occud% The finding of irreversible breakage of DNA supports the previous hypothesis 16 that double-strand scission of DNA is the principal cause for the thymineless death of E. coli 15 TAU.

DNA

SCISSIONS AND THYMINELESS DEATH

205

MATERIALS AND METHODS

Treatment and labelling o[ the cells The thymine-requiring E. coli 15 TAU (see ref. 17) was grown at 37 °C in various synthetic media as described previously 16. Glucose-salt medium (containing per l: 2 g NH4C1, 15 g Na2HPO4 • 12 H20, 3 g KH2POa, 3 g NaC1, o.I g Na2SO 4, 0.02 g MgC12 and IO g glucose) was supplemented with thymine (Thy, 2 rag/l), with arginine (Arg, 40 rag/l) or with uridine (Urd 20 rag/l) singly or in combination. Thus, Thymedium was glucose-salt medium supplemented only with thymine, and (Thy + A r g + Urd)-medium was glucose-salt medium containing thymine, arginine and uridine. E. coli 15 TAU was grown overnight in IO ml of (Thy -+- A r g + Urd)-medium at 37 °C. I ml of this culture was transferred to 9 ml of fresh (Thy + Arg + Urd)medium, and incubation was continued for a further 12o min. During this growth period of 2-3 generations, the cellular DNA was labelled uniformly b y use of a medium which contained E6-3Hlthymidine (1.o-1. 5 #Ci/ml), thymine (0.2/~g/ml) and all the other supplements. A portion of labelled cells in the log phase of growth was used in control experiments. The specific activity of the cells was about I • lO4 cpm per I • lO T cells. The remaining portion was washed by centrifugation, and incubated in Thy-medium for a further 9 ° min. The incubation in Thy-medium is required for the completion of the DNA replication cycle 15. The cells treated in Thy-medium were washed, resuspended in the various media at concentrations of not more than I • lO 7 cells/ml, and incubated. Viable counts were determined by plating on Nutrient Broth agar. Dilution was made in water at room temperature, and o.I-ml portions which contained 50IOOO viable cells were spread on plates of Nutrient Broth agar (containing per l; IO g Nutrient Broth, 5 g NaC1, and 15 g a g a r ) before incubation at 37 °C overnight. Radioactivity of the cells was measured b y the filter paper disk method described previously le,ls. Portions (o.I ml) of a culture were added to the disks and dried. The disks were then washed with cold IO and 5 % trichloroacetic acid solutions, ethanol-ether mixture, and ether. They were then dried, immersed in a toluene scintillation fluid, and counted in a liquid-scintillation spectrometer (CPM/DPM-Ioo. Beckman Instruments Inc.). Sedimentation analysis o/radioactive DNA The cells, which had a total radioactivity of 2 • lO3-2 • lO 5 cpm, were collected by centrifugation, and washed with 0.2 M NaC1 at 4 °C. They were then frozen rapidly using solid CO 2 and acetone, and stored at --20 °C until required. The frozen cells were suspended in 0.2 ml of a lysozyme-EDTA solution containing IO mM Tris-HC1 (pH 7.6), IO mM EDTA, and 2 mg/ml lysozyme (EC 3.2.1.17, Seikagaku Kogyo Co., Ltd), and converted into spheroplasts b y incubation at 37 °C for 30 rain. Sedimentation analysis of DNA was carried out under either neutral or alkaline conditions. The neutral sucrose solution contained IO mM Tris-HC1 (pH 7.6), IO mM E D T A and 1 % sodium dodecyl sulphate, and the alkaline solution contained o.I M NaOH. In both cases, o.I ml of labelled cell suspension, which contained 2. lO 79 " lO7 cells in most of the experiments and 6 • IOe-9 • lO 6 cells in the others, was layered on top of 4.4 ml of a 5-20 % sucrose gradient and centrifuged at approximately

206

K. Y O S H I N A G A

2o °C using an RPS-4o rotor in a Hitachi ultracentrifuge. After centrifugation at 29 ooo rev./min for 8o rain, the contents were collected in fractions of one or two drops each on disks of filter paper, which were then washed and counted as described above. The spheroplasts seemed to be disrupted in the sedimentation tubes, and the recovery of radioactivity was found to be more than 7 ° °/o.

Estimation o/ s20,w and molecular weight o] DNA s20,w of DNA was estimated b y the equation and tables proposed by McEwen ~9. The relative sedimentation distance from the meniscus to each fraction was measured for each sedimentation profile. The density of DNA and the temperature were taken to be 1.7o and 20 °C, respectively. The molecular weight in neutral solution was calculated according to the equation proposed by Burgi and Hershey 2°, where s20,~ 0.080 M °,35, and that in alkaline solution according to the equation, s~0,~~ ~ 0.0528 M °'4°° (see ref. 21).

RESULTS

Thymineless death and recovery When cells which had been incubated in Thy-medium were transferred to glucose-salt medium, thymineless death was not observed as reported previously15,1~. However, when the cells were transferred from Thy-medium to (Arg + Urd)medium, the surviving population density was rapidly reduced after a I h lag period, and, as shown in Fig. I (Curve AU0), it was about I/IOO of the original density after 3 h. In order to study the fundamental nature of the cells which are losing their ability to multiply, the cells cultured for various periods in (Arg + Urd)-mediunl were transferred to several other media (Fig. I). In the glucose-salt medium, the reduction of viability continued in cells which had been treated for more than I h in (Arg + Urd)-medium (Fig. i, Curves GS l and GS2). However, the reduction in viabilitywas stopped after transfer to the Thy-medium, and the viability subsequently remained unchanged (Fig. I, Curves T 1, T~ and Ta). When the cells were transferred from (Arg + Urd)-medium to (Thy + Arg + Urd)medium, the surviving population regained the ability to multiply at the normal rate after a lag period of about I h (Fig. I, Curves TAU1, TAU2 and TAU3). Donachie and Hobbs z~ reported that thynlineless death is completely reversible in E. col, 15 T -, J G 151, and Cummings and Kusy 14 also observed a rapid and almost complete recovery of viability in E. coli 13 on the addition of both chloramphenicol and thymine. However, such a rapid and complete recovery of viability was not observed in E. eoli 15 TAU even in the absence of the required amino acid. The difference in the recovery m a y be caused b y the use of different strains. Degradation o/ DNA after thymine starvation The cells, which had been labelled uniformly with F3Hlthymidine and then incubated in Thy-medium, were transferred to (Arg + Urd)-medium. After 3 h incubation in (Arg + Urd)-medium, thymine was added and incubation was continued. During the incubation in (Arg + Urd)- or (Thy + Arg + Urd)-medium, o.I-ml samples of the cultures were taken at various times and placed on filter paper disks.

DNA SCISSIONS AND THYMINELESS DEATH .... I

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Fig. I. Viability of E. coli 15 T A U a f t e r t h y m i n e s t a r v a t i o n . Cells growing e x p o n e n t i a l l y in (Thy + A r g + U r d ) - m e d i u m were t r a n s f e r r e d to, a n d i n c u b a t e d in, T h y - m e d i n m for 9o rain, a n d t h e n t r a n s f e r r e d to ( A r g + U r d ) - m e d i u m ( O , C u r v e AU0). A f t e r i n c u b a t i o n in ( A r g + U r d ) - m e d i u m for o, I, 2 or 3 h, t h e y were t r a n s f e r r e d to, a n d i n c u b a t e d in, ( T h y + A r g + U r d ) - m e d i u m (@, Curves TAU0, T A U 1, T A U : a n d TAUs), T h y - m e d i u m (rN, Curves T1, T 2 a n d T:~) or g l u c o s e - s a l t m e d i u m (/x, c u r v e s GSo, GS t a n d GS,). T h e fraction of cells s u r v i v i n g d u r i n g t h e i n c u b a t i o n w a s p l o t t e d as a p e r c e n t a g e of t h e n u m b e r of cells initially t r a n s f e r r e d to ( A r g + U r d ) - m e d i u m . Fig. 2. D e g r a d a t i o n of E.coli I5 T A U D N A after t h y m i n e s t a r v a t i o n . T h e D N A w a s first labelled u n i f o r m l y w i t h [3H]thymidine, a n d t h e labelled cells were i n c u b a t e d in T h y - m e d i u m for 9 ° min. T h e y were t h e n t r a n s f e r r e d to ( A r g + U r d ) - m e d i u m a n d i n c u b a t e d ( Q ) . A f t e r 3 h i n c u b a t i o n , t h y m i n e was a d d e d a n d t h e i n c u b a t i o n c o n t i n u e d (@). o.i ml of t h e c u l t u r e was a d d e d to a filter p a p e r disk a t t h e i n d i c a t e d t i m e . T h e r a d i o a c t i v i t y of t h e acid-insoluble fraction was c o u n t e d a n d p l o t t e d as a p r o p o r t i o n of t h e original radioactivity.

The disks were then washed in cold trichloroacetic acid, and the radioactivity of acidinsoluble materials on the disks was counted (Fig. 2). The results showed that about 15 % of the total radioactivity was changed from an acid-insoluble to an acid-soluble form during about I h of incubation in (Thy + Arg + Urd)-medium. However, such solubilization of cellular DNA was not observed in (Arg + Urd)-medium for at least 3 h. Reiter and Ramareddy ~3 obtained similar results with a B . subtilis mutant, that is, some proportion of labelled cellular DNA was released into the medium when thymine was added to a thymine-starved culture, but the cellular label was not lost during thymine starvation. In order to study the process of breakdown of DNA in the thymine-starved cells, ~H-labelled DNA was extracted from cells suffering thymineless death in (Arg + Urd)-medium, and was analyzed in neutral and alkaline sucrose gradients (Figs 3 and 4)- Fig. 3a shows a typical sedimentation pattern of labelled DNA from

208

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Fig. 3. N e u t r a l sucrose gradient centrifugation of the D N A from the cells incubated in ( A r g U r d ) - m e d i n m . The cells, which h a d been labelled with [3H]thymidine and i n c u b a t e d in Thym e d i u m for 9o rain, were transferred to ( A r g + U r d ) - m e d i u m . After incubation for o (a), 2 (b) and 3 h (c) in this m e d i u m , the cells were collected, w a s h e d b y centrifugation, and then treated with l y s o z y m e - E D T A . The t r e a t e d cells (2-9 • lO 7 cells) were layered on the t o p of a 5-2o ~o sucrose g r a d i e n t (pH 7.6) containing s o d i u m dodecyl sulphate, and centrifuged at 29 ooo r e v . / m i n at a p p r o x i m a t e l y 2o °C for 8o min. F r a c t i o n s were collected and acid-insoluble radioactivity was counted. Fig. 4. Alkaline sucrose gradient centrifugation of the D N A from the cells incubated in ( A r g + U r d ) - m e d i u m . After incubation for o (a), 2 (b) and 3 h (e) in ( A r g + U r d ) - m e d i u m , labelled D N A was centrifuged in alkaline solution. The conditions were identical to those described in the legend to Fig. 3, except t h a t the sucrose g r a d i e n t contained o. I M N a O H .

control cells. The main fraction, which was the heaviest, was found to have a sedimentation constant of 117 ~- 7 S, corresponding to the molecular weight of i . I • lO 9. This molecular weight, which was determined from 5 analyses, is slightly larger than those reported by McGrath and Williams 24, and Baker and Hewitt 25. However, using autoradiography, Cairns 26 observed that the chromosome of E. coli is 7oo 9oo # m long. This length is equivalent to 1. 4 • lO9-1.8 • lO9 daltons, a value close to ours. Therefore, the principal peak seems to represent intact DNA which has completed its replication cycle. Accessory peaks, which were found above the main peak, had variable positions, and are considered to be artifacts formed during the experiments. There was a reduction in the proportion of the heaviest DNA and an irregular sedimentation pattern when the cells were incubated for 2 h in (Arg + Urd)-medium (Fig. 3b). However, the pattern was not as reproducible as that in control experiments. The variable nature of the pattern seems to reflect the instability of DNA in those cells which are rapidly losing their multiplication activity. However, as Fig. 3b shows, two or three peaks were observed in the region where DNA of a sedimentation constant between ioo and 12o S should be found. The heaviest peak is probably due to the DNA which has completed its replication cycle (Fig. 3a). The other two peaks

DNA

209

SClSSIONS AND THYMINELESS DEATH

had similar S values to those of the DNA of log-phase cells (Fig. 5), although some of the DNA in these peaks seems to originate from dead cells. The smaller sedimentation value of replicating DNA seems to be due to strand scission at the growing point or at the unusually shear-sensitive part ~. I t was alsG observed in alkaline solution that the ratio of slowly sedimenting to rapidly sedimenting materials was smaller in the case of DNA which had completed its replication than with DNA which was still replicating (Figs 4 a and 5b).

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medium. The labelled cells were taken from log-phase culture and analyzed in neutral (a) and alkaline sucrose gradients (b) as described in the legends to Figs 3 and 4. Fig. 6. Sucrose gradient centrifugation of the DNA from the cells incubated in glucose-salt medium. The labelled cells which had been incubated in Thy-medium were incubated in glucosesalt medium for 3 h, and analyzed in neutral (a) and alkaline sucrose gradients (b) as described in the legends to Figs 3 and 4. During the 3-h incubation in (Arg + Urd)-medium, the surviving population was reduced to i % of its original level. The neutral sucrose gradient analysis of labelled DNA from these cells showed the almost complete disappearance of the three peaks described above, together with the appearance of a broad, irregularly shaped peak, as shown in Fig. 3c. The sedimentation constant of the main fraction of this broad peak ranged from 40 to IOO S, and the molecular weight from 5 " IO~ to 7 " z°8 • The reduction in the sedimentation rate of the DNA in neutral sucrose gradient appears to be a result of double-strand breakage of the DNA molecule. Under conditions of thymine starvation, the repair synthesis of DNA molecule is expected to be almost stopped, although m R N A synthesis m a y be accompanied by endonucleolytic cleavage of DNA molecules, and the exonucleolytic removal of nucleotides from DNA molecules m a y also proceed is. These would lead to overlapping breakages of the double strand. However, the possibility that double-strand scission might be produced at the sites of single-strand breakage in DNA b y enzymic action or by hydrodynamic shearing during experimental operation is not excluded, as has been discussed b y Dean et al. ~8. Therefore, the nature of the breakage in DNA produced during thymine starvation was studied further in an alkaline sucrose gradient.

2 io

K. YOSHINAGA

When the labelled DNA from control cells of E. coli 15 TAU was analyzed by alkaline sucrose gradient (Figs 4a and 5b), a broad and heterogeneous peak appeared, which is in contrast to the results obtained by McGrath and Williams 24 and b y Ringrose 29. The difference between these observations m a y be due mainly to the differences in lysis procedures. Fig. 4 shows typical sedimentation patterns in alkaline sucrose gradients of labelled DNAs extracted from cells which had been incubated in (Arg + Urd)-medium for various periods. The sedimentation value was drastically reduced during the first 2 h of incubation in (Arg + Urd)-medium, but no significant reduction was observed thereafter. Since the reduction of the sedimentation rate in alkaline sucrose gradients can be explained in terms of single-strand breaks in the DNA, these results indicate that single-strand scission of DNA took place extensively during the first 2 h, but only to a small extent thereafter, even though the prolonged incubation caused a drastic reduction in the viability (Fig. I, Curve AU0). The discovery of parallelism between the decrease in the amount of DNA having large sedimentation constants (larger than IOO S) in neutral sucrose gradients and the decrease in the viability of the cells supports the hypothesis t6 that thymineless death is caused by double-strand scission of cellular DNA, even though the progress of DNA degradation during thymine starvation can be recognized by means of alkaline sucrose gradient centrifugation even before the onset of thymineless death. When the cells which had been labelled with [aH]thymidine and then incubated in Thy-medium were transferred to glucose-salt medium, no reduction in the viability was observed for at least 3 h (see refs 15 and 16). Fig. 6 shows the sedimentation patterns of labelled DNA taken from the cells which had been incubated in glucose salt medium for 3 h. The patterns are almost identical with those shown in Figs 3a and 4 a. These results indicate that the structure of DNA was not changed to any significant extent during the incubation in glucose-salt medium. It m a y be concluded, therefore, that the double-strand breakage is related to thymineless death but not to thymine starvation.

Repair o/damaged DNA I t was assumed in our previous paper 16 that the DNA which underwent doublestrand scission during thymine starvation would not be repaired after the addition of thymine. This assumption was examined in the following experiments. The cells of E. coli 15 TAU which had been labelled with ~aHJthymidine were incubated in Thymedium for 9 ° rain and then in (Arg + Urd)-mediunl for 2 or 3 h. These cells were incubated in Thy- or (Thy + Arg + Urd)-medium for another 3 h, and the DNA was then analyzed by neutral and alkaline sucrose gradient centrifugations. As can be seen, the patterns in Figs 7 a and 7 c are very similar to those in Figs 3b and 3c, respectively. These results, which were obtained with neutral sucrose, indicate that double-strand breakages formed in (Arg + Urd)-medium are not repaired even in the presence of thymine. However, the pattern in Fig. 7 b, which was obtained in alkaline solution, is somewhat different from that in Fig. 4 b. The molecular weight calculated for the m a x i m u m of the peak in Fig. 7 b is 3.o" lO T, while that in Fig. 4 b is 2.o • lO 7, suggesting that some of the single-strand breakages are repaired in the presence of thymine. The pattern in Fig. 7 d is different from that in Fig. 4c. The molecular weight

DNA

SCISSIONS AND THYMINELESS DEATH

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calculated for the m a x i m u m of the main peak in Fig. 7d is 9 " IOe, while that in Fig. 4 c is 2.0 • IO~. This difference indicates that single-strand scission continued even during the incubation in Thy-medium after the cells had been incubated in (Arg + Urd)medium for 3 h. This continuation of single-strand scission seems to explain the appearance of the acid-soluble material shown in Fig. 2, since the sedimentation behaviour shown in Figs 7 c and 7d was also observed with cells which were incubated in (Arg + Urd)-medium for 3 h and then in (Thy + Arg + Urd)-medium for 3 h (unpublished data). When the cells which had been incubated in (Arg + Urd)-medium for 2 or 3 h were transferred to, and incubated in, (Thy + Arg + Urd)-medium instead of Thy-medium, the results were the same as those described above (unpublished data). These results strongly indicate that the fragments produced b y double-strand scission of DNA during thymine starvation are not utilized directly in the synthesis of complete cellular DNA even in the presence ot thymine. This finding also supports the hypothesis that thymineless death is caused by double-strand scission of DNA.

DISCUSSION

In the alkaline sucrose sedimentation analyses with E . coli, single-strand breaks were not detected in the DNA after thymine starvation "5,3°. Reiter and Ramareddy ~'~ analyzed uniformly-labelled DNA from thymine-starved cells of B . subtilis in neutral sucrose gradients. They observed that almost all of the radioactivity which remained in the cells banded at the same positions as in the control experiment with non-starved cells. However, they observed a loss of DNA from the cells and concluded that DNA degradation parallels cell death. Walker ~ and Reichenbach et al. s found that singlestrand breaks in the DNA of E . coZi occurred during thymine starvation. These different observations m a y be due to the different properties of the strains used and to differences in the physiological conditions of the starved cells. Reichenbaeh et al. 8

212

K. YOSHINAGA

reported that the chromosomal damage in the strain JG 151 was not irreversible, since rejoining of the breaks occurred under conditions which allowed spontaneous recovery of replicating ability. This fact suggests that, under suitable conditions, dividing ability can be restored to the cells in which the DNA breaks are reversible, as with J G 151. Tile recovery of dividing ability cannot be expected in the cells in which the DNA breaks are irreversible, as with 15 TAU. Cummings and Kusy 14 studied recovery from thymineless death and suggested that there are two types of E. coli; those which form filamentous cells after an inactivating event, and those which do not. The tormer were able to recover from the inactivating event, but the latter never recovered. Filamentous cells were observed to be formed in strain 15 TAU during the treatment in (Arg -b Urd)-medium 16. The process of recovery, therefore, does not seem to be related to cell elongation in this case. However, there is a possibility that recovery m a y occur in our strain if chloramphenicol is added. Kogoma and Lark al observed that DNA is synthesized for a long time after a period of thymine starvation if protein synthesis is arrested by chloramphenicol, and they suggested that chloramphenicol permits the synthesis of DNA b y preventing its degradation in strains carrying the 15 restriction locus. It was reported that derivatives of strain 15 contained defective prophages a2,'~3. They emerged and caused cell lysis only when thymine was added to the culture, but did not emerge during thymine starvation 34. Therefore, phages would not be expected to emerge in 15 TAU when DNA breaks occur during thymine starvation. I t was also reported that the phage particles from 15 TAU were tailless and did not exert colicinogenic activity on the strains of E. coli 15 (see ref. 35). Therefore, it does not seem likely that the DNA break observed in 15 TAU was brought about by the induction of phage. As described in the previous report 16, two types of thymineless death were recognized in E. coil I5 TAU. One type of death proceeds slowly and is observed when the log-phase cells are incubated in glucose-salt medium. The other type of thymineless death is observed in (Arg + Urd)-medium, in which viability rapidly decreases. In ( A r g - Urd)-medium, RNA and protein synthesis are known to continue. Synthesis of m R N A was found to be particularly important in the killing event in thymine-deficient culture 9-13'16, and is expected to be accompanied by singlestrand scission at various points on both strands of the DNA molecule. This would give ample opportunity for double-strand scission in the absence of the repair synthesis. In the present investigation, the former type of thynlineless death was eliminated b y using the cells which had completed their DNA replication cycle (see ref. 15). The appearance of low-molecular weight DNA was observed in relation to the progress of thymineless death. Kaplan a6 observed that X - r a y irradiation of E. coli induces double-strand scission in DNA, and concluded that the scission is principally responsible for the lethal effect of ionizing radiation. From the results of the present experiments, it is suggested that the principal lethal events would be caused by the production of double-strand breaks in DNA molecules, which would result in an irreversible blockage of normal DNA replication and cell division.

DNA SCISSIONSAND THYMINELESS DEATH

213

ACKNOWLEDGMENTS T h e a u t h o r w i s h e s t o t h a n k P r o f . M. S h i m o m u r a f o r h i s c o n t i n u e d a d v i c e d u r i n g t h e c o u r s e of t h i s i n v e s t i g a t i o n a n d f o r h i s c r i t i c i s m of t h e m a n u s c r i p t , a n d D r S. M a t s u m o t o , N a t i o n a l I n s t i t u t e of R a d i o l o g i c a l S c i e n c e s , C h i b a , f o r h e l p f u l d i s c u s s i o n a n d also f o r p r o v i d i n g t h e s t r a i n of E. coli.

REFERENCES i S. S. Cohen and H. D. Barner, Proc. Natl. Acad. Sci. U.S., 4° (1954) 885.

H. D. Menningmann and W. Szybalski, Biochem. Biophys. Res. Commun., 9 (1962) 398. B. J. Smith and K. Burton, Biochem. J., 97 (I965) 240. D. Freifelder, J. Mol. Biol., 45 (1969) I. G. Ramareddy and H. Reiter, J. Mol. Biol., 5 ° (i97 o) 525. F. Brunel, A. M. Sicard and N. Sicard, J. Bacteriol., lO6 (i971) 904. J. R. Walker, J. Bacteriol., io 4 (I97O) I39I. D. L. Reichenbach, G. E. Schaiberger and B. Sallman, Biochem. Biophys. Res. Commun., 42 (I97I) 23. 9 J. Gallant and S. R. Suskind, Biochim. Biophys, Acta, 55 (i962) 627. io P. C. Hanawalt, Nature, I98 (I963) 286. i I R . 1Rolfe, Proc. Natl. Acad. Sci. U.S., 57 (I967) II4. 12 D. W. Smith and P. C. Hanawalt, J. Bacteriol., 96 (1968) 2o66. 13 P. C. Hanawalt, D, E. Pettijohn, E. C. Pauling, C. F. Brnnk, D. W. Smith, L. C. Kanner and J. L. Couch, Cold Spring Harbor Syrup. Quant. Biol., 33 (1968) I87. 14 D. J. Cummings and A. R. Kusy, J. Bacteriol., 99 (I969) 558. 15 O. Maal0e and P. C. Hanawalt, J. Mol. Biol., 3 (I96I) 144. 16 K. Yoshinaga, M. Fusaya and M. Shimomura, Reports of Faculty of Science, Shizuoka University, 6 (I97 I) 41. 17 D. Kanazir, H. D. Barner, J. G. Flaks and S. S. Cohen, Biochim. Biophys. Acta, 34 (1959) 34I. i8 R. J. Mans and G. D. Novelli, Arch. Biochem. Biophys., 94 (1961) 48. 19 C. R. McEwen, Anal. Biochem., 20 (1967) II4. 20 E, Burgi and A. D. Hershey, Biophys. J., 3 (I963) 309. 21 F. W. Studier, J. Mol. Biol., II (1965) 373. 22 W. D. Donachie and D. G. Hobbs, Biochem. Biophys. Res. Commun., 29 (1967) I72. 23 H. Reiter and G. Ramareddy, J. Mol. Biol., 5 ° (197 o) 533. 24 R. A. McGrath and R. W. Williams, Nature, 212 (1966) 534. 25 M. L. Baker and R. R. Hewitt, J. Bacteriol., io 5 (i971) 73326 J. Cairns, J. Mol. Biol., 6 (I963) 208. 27 B. H. Rosenberg and L. F. Cavalieri, Cold Spring Harbor Symp. Quant. Biol., 33 (I968) 65. 28 C. J. Dean, P. Feldschreiber and J. T. Lett, Nature, 209 (1966) 49. 29 P. Ringrose, Biochim. Biophys. Acta, 213 (197 o) 320. 3 ° S. G. Sedgwick and ]3. A. Bridges, J. Bacteriol., lO8 (1971) i422. 31 T. Kogoma and K. G. Lark, J. Mol. Biol., 52 (I97 o) 143. 32 H. D. Mennigmann, J. Gen. Microbiol., 41 (1965) I5I. 33 H. Endo, K. Ayabc, K. Amako and K. Takeya, Virology, 25 (1965) 469. 34 A. Yudelevich and M. Gold, J. Mol. Biol., 4° (1969) 77. 35 C. S. Lee, R. W. Davis and N. Davidson, J. Mol. Biol., 48 (i97 o) I. 36 H. S. Kaplan, Proc. Natl. Acad. Sci. U.S., 55 (1966) I442. 2 3 4 5 6 7 8