Abnormal metabolism of thymidine nucleotides in ultraviolet-irradiated dTDPG pyrophosphorylase-deficient mutants of Escherichia coli K12

190

Biochimica et Biophysica Acta, 476 (1977) 190--202 © Elsevier/North-Holland Biomedical Press

BBA 98930 ABNORMAL METABOLISM O F THYMIDINE N U CL E O T ID E S IN U L T R A V I O L E T - I R R A D I A T E D dTDPG P Y R O P H O S P H O R Y L A S E D E F I C I E N T MUTANTS OF E S C H E R I C H I A C O L I K12

TSUTOMU OHKAWA Department of Biochemistry, School of Medicine, Kanazawa University, Kanazawa, Ishikawa 920 (Japan)

(Received October 8th, 1976) (Revised manuscript received February 7th, 1977)

Summary T h e dTDPG p y r o p h o s p h o r y l a s e - d e f i c i e n t strains (Ter-15 and Ter-21) are m o r e sensitive to ultraviolet-irradiation than are wild-type strains (parent strains). One m u t a n t (Ter-15) forms a long filament, the ot her (Ter-21) assumes a spherical form, and b o t h are lysed within 90 min of incubation after ultraviolet-irradiation while wild-type strains f or m only filaments w i t h o u t lysing after ultraviolet-irradiation. The c o n c e n t r a t i o n of d T T P after ultraviolet-irradiation of Ter-15 within 30 min reaches a level of 8--9 times higher than that of unirradiated Ter-15 or wild-type cells, although Ter-21 maintains the same dTTP c o n c e n t r a t i o n as t hat in unirradiated cells for 75 min after ultraviolet-irradiation. In cell cultures of wild-type strains after ultraviolet-irradiation, dTDP level increases to 8-fold, dTMP increases to 5--7-fold, and dTDP-sugar increases t o 4fold th at in the unirradiated cells. In Ter-15 strain, dTDP, dTMP and dTDPsugar co n cen tr ations increase to twice higher levels for 75 min c o m p a r e d with those o f unirradiated cells. Also, in the Ter-21 strain, t h y m i d i n e nucleotides and nucleotide-sugar are of the same c onc e n t rat i ons as those in unirradiated cells. These fluctuations in the syntheses of t h y m i d i n e nucleotides appear to be correlated with cell shape, filament or spherical form, after ultraviolet-irradiation. RNA synthesis is not correlated with cell shape. Both wild-type strains and m u t a n t strains (Ter-15 and Ter-21) synthesize RNA at a rate half or a third t hat in an unirradiated control. In wild-type strains, DNA degrades at a greater rate after ultraviolet irradiation than th at o f m u t a n t strains (Ter-15 and Ter-21), b u t also t he wild-type strains recover within a shorter time than do the m u t a n t strains f r o m t he inhibit i o n of DNA synthesis after ultraviolet-irradiation. Pyrimidine dimers in DNA

191 are decreased to a b o u t 50% within 60 min after ultraviolet-irradiation in the wild-type, b u t mutant strains cannot excise dimers from DNA.

Introduction Ultraviolet irradiation of cells produces intrastrand pyrimidine dimers in DNA [1,2]. These pyrimidine dimers block post-DNA synthesis in vitro [3] and in vivo [4]. Elimination of the dimers from DNA appears to be necessary for recovery of organisms from ultraviolet-damage. Some bacterial organisms which possess the ability to repair ultraviolet-induced lesions in DNA in the dark are more resistant to ultraviolet killing than the organisms which lack the dark-repair mechanism [5,6]. Also, Boyce and Howard-Flanders [5] and Setlow and Carrier [6] have demonstrated that dimers are excised in vivo in acid-soluble oligonucleotides from the DNA of wild t y p e strains, b u t not by some ultraviolet-sensitive mutants of Escherichia coli [5,6]. Moreover, it has been shown that small numbers of bases are inserted into DNA molecules as replacement for damaged bases that have been excised [7]. After ultravioletirradiation of organisms which lack the dark-repair mechanism, the inhibition of DNA synthesis is permanent [4,8] and DNA breakdown follows [9]. In the organisms that have the dark-repair mechanism, DNA synthesis is transiently inhibited by pyrimidine dimers produced by ultraviolet-irradiation and resumes after a time [4]. Also, mutation of either the Cap R (lon), Cap S, or Cap T gene causes overproduction of capsular polysaccharide in E. coli K12 leading to a mucoid p h e n o t y p e [10--12]. Synthesis of many of the enzymes involved in capsular polysaccharide synthesis is derepressed in all three Cap mutant strains. Among these Cap mutants, only Cap R (Ion) mutants are sensitive to ultraviolet light [10,13--15]. Mucoid mutants of E. coli K12 isolated after exposure to ozone retain the phenotypic properties of the ultraviolet-induced Ion strain of E. coli K12 and are abnormally sensitive to irradiation [16]. These mucoid mutant cells tend to grow in long filaments after ultraviolet irradiation. Markovitz and Baker [17] and Bush and Markovitz [18] reported in genetic studies that ultraviolet sensitivity and capsular polysaccharide synthesis are controlled by the protein p r o d u c t of a single cistron, the Cap R (lon) cistron. The mutant strains (Ter-15 and Ter-21) isolated in a previous report were mucoid and had 3--4 times higher dTTP concentration than did the wild-type [19]. The question we wish to approach in this paper is h o w the cell growth and macromolecular synthesis of wild type and mutant strains are effected b y ultraviolet radiation. To answer this question, we analyzed, after ultraviolet irradiation, cell growth in solid and liquid medium, total cell numbers, thymidine nucleotide concentration, RNA synthesis, DNA degradation and postDNA synthesis. Materials and Methods

Bacteria Escherichia coli K12 W2252-11U- (thy-, ura-, met-) RC str (wild-type I) and

192

E. coli K12 W2252-11U- (thy-, ura-, met-) RC tel (wild-type II) were used in these experiments as parent strains. They were non-isogenic. The m u t a n t strain, deficient in dTDPG pyrophosphorylase derived from wild-type I was named Ter-15, and the one from wild-type II was called Ter-21. Ter means T-even phage-resistant cells [19]. Materials Thymidine, thymine, dTMP, dTDP, dTTP and dTDP-glucose were obtained from the Sigma Chemical Company. [2-~4C]Thymine (specific activity 59 Ci/ mol), [6-3H]thymine (specific activity 26 Ci/mmol) and [2-~4C]uracil (specific activity 59 Ci/mol) were purchased from the Nippon Isotope association. Other chemicals were from Wako-Pure Chemical company, Ltd. Cell culture and ultraviolet irradiation An overnight culture grown in synthetic medium [19] supplemented with requirements was diluted 1 : 20 into the same fresh medium, and grown about three generations to an absorbance level of 0.2--0.3 (A660; 2.5 • 10s--3.0 • 108 cells/ml) for wild-type and Ter-21 m u t a n t cells and to 0.3 (A6~0; 2.2 • l 0 s cells/ ml) for the Ter-15 m u t a n t at 37°C. This cell culture (10 ml) was poured into a 100 mm diameter glass dish in a layer 2--3 mm deep and then was exposed to ultraviolet light at a distance of approximately 50 cm from a 15 W germicidal lamp (Toshiba). The dose rate, 0.48 J / m 2 per s, was measured with a Toshiba germicidal UV-meter. To prevent photoreactivation, the irradiation and subsequent handling of the cells were carried out under yellow lights or in the dark. For the measurement of the number of viable cells after ultraviolet-irradiation, the irradiated cell culture was diluted with dilution fluid (1.0 g polypeptone, 3.0 g NaC1 and 0.1 g MgSO4 per 1) and plated on nutrient-broth agar supplemented with requirements. The approximate dose (irradiation for 60 s) was 29 J / m 2 which resulted in a viable concentration of 106/ml for the wild type cells, 5 • 102/ml for the Ter-15 cells, and 102/ml for the Ter-21 cells. For the experiments on cell growth and total cell numbers in liquid medium after ultraviolet-irradiation for 60 s at room temperature, the irradiated cells were collected by centrifugation, washed with fresh synthetic medium w i t h o u t glucose, and resuspended in the same growth medium supplemented with requirements to an absorbance of 0.2 A660 nm. This irradiated cell suspension was aerated at 37°C in the dark. The absorbance was measured at A660 nm at the times indicated with a Bausch & Lomb spectrophotometer. Also, the irradiated cell suspension (2 • 108--3 • l 0 s cells/ml for both parents and mutants) was aerated at 37°C in the dark and the total cell numbers were counted at the times indicated with a Petroff-Hauser Counting Chamber by phase-contrast microscopy. Measurements o f thymidine nucleotide concentration after ultraviolet irradiation Irradiated cells (29 J / m 2) were suspended in fresh synthetic medium supplemented with requirements containing 20 nmol/ml [6-all]thymine (15 pCi/ml; specific activity 192 • 10 a cpm/nmol). After this cell suspension was aerated at 37°C in the dark at the times indicated, 3.0-ml samples were withdrawn and

193 filtered immediately with membrane filters. These filters were immediately placed in cold 5% acetic acid for at least 30 min to extract the acid-soluble materials and were centrifuged at 11 500 × g for 10 min. The supernatants were analyzed b y thin-layer chromatography on poly(ethyleneimine)-impregnated cellulose on plastic sheets [19].

Ultraviolet ligh t-induced DNA degradation The cells were grown for at least three generations (2 • 108--3 • 10 s cells/ml) in the synthetic medium supplemented with requirements containing 20 nmol/ ml [2-14C]thymine (0.1 pCi/ml, specific activity 11 090 cpm/nmol). These prelabelled cells were collected by centrifugation, washed, resuspended in synthetic medium supplemented with non-radioactive requirements at the same concentration, and grown for a further 30 min to exhaust any endogenous radioactivity, and then irradiated for 60 s (29 J / m 2) with ultraviolet light at room temperature. These irradiated cells were collected by centrifugation, washed, resusp e n d e d in the same grown medium and at the same cell concentration described above and then aerated at 37°C in the dark. At the times indicated samples were withdrawn and DNA was precipitated in ice-cold 5% trichloroacetic acid. Materials insoluble in the acid were collected in sartorious membrane filters, washed twice with 5% trichloroacetic acid and with distilled water. The filters were dried and their radioactivity determined in a Nuclear Chicago scintillation spectrophotometer (Unilex II). DNA synthesis after ultraviolet radiation Irradiated cells (29 J/m 2) were suspended in synthetic medium supplemented with requirements containing 20 nmol/ml [2-14C]thymine (0.1 pCi/ml, specific activity 11 090 cpm/nmol) and aerated at 37°C in the dark. At the indicated time intervals, samples were withdrawn and DNA was precipitated in ice-cold 5% trichloroacetic acid for at least 30 min. Materials insoluble in the acid were collected on membrane filters, washed with 5% trichloroacetic acid and with distilled water. The filters were dried and their radioactivity was determined in a Nuclear Chicago scintillation spectrometer described above. R N A synthesis after ultraviolet irradiation Irradiated cells (29 J/m 2) were suspended in synthetic medium supplemented with requirements containing 10 pg/ml [2-14C]uracil (0.290 pCi/ml, specific activity 5.089 • 103 cpm/nmol) and aerated at 37°C in the dark. Other methods for the determination of radioactivity were as described in the section on DNA synthesis. Measurement o f pyrimidine dimers The cells (2--3 • 108 cells/ml) labelled with [6-3H]thymine (15 pCi/ml, specific activity 1 9 2 . 1 0 3 cpm/nmol) were irradiated with ultraviolet light (29 J/ m2), washed and incubated routinely in synthetic growth medium containing 20 nm non-radioactive thymine. At the times indicated, the samples were withdrawn, harvested and treated with cold 5% trichloroacetic acid for at least 30 min. By centrifugation of these samples, acid-soluble and acid-insoluble fractions were separated. The acid-soluble fractions were treated three times with

194 ether to remove the trichloroacetic acid. Thereafter, these acid-soluble and acid-insoluble fractions were dried and hydrolyzed with 98% formic acid for 30 min at 176°C according to the method of Carrier and Setlow [20], and analyzed by two-dimensional thin-layer chromatography as described by Goldman and Friedberg [21]. The photoproducts analyzed were presumed to constitute dimers of thymine-thymine and thymine-cytosine. Results

Survivors after ultraviolet light irradiation The number of colonies formed on agar plates after ultraviolet irradiation of mutant strains (Ter-15 and Ter-21) is compared with the number of colonies on agar plates after ultraviolet irradiation of wild-type strains (parents). The percent survival is plotted in Fig. 1 as a function of irradiation time. The mutant strains are about three times as sensitive as their parents to ultraviolet irradiation as judged by the final slopes of the curves. Fig. 2 shows that irradiated cells have a slower growth rate than unirradiated cells, and that in the mutants there is lysis beginning at around 90 rain, with considerably more in Ter-21. Fig. 3 shows the cell numbers as a function of post-irradiation incubation. In

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Fig. 3. T o t a l cell n u m b e r s i n s y n t h e t i c m e d i u m a f t e r u l t r a v i o l e t irradiation. C o n d i t i o n s w e r e t h e s a m e as t h o s e d e s c r i b e d in Fig. 2. T o t a l cell n u m b e r s at t h e t i m e s i n d i c a t e d w e r e m e a s u r e d as d e s c r i b e d i n Materials and Methods. ¢ -', w i l d t y p e I; X . . . . . . X, Ter-15; ~ ..... -~, w i l d t y p e H ; o . . . . . . o, Ter-21.

the wild-type strains the cell numbers are constant for the first 120 min, and then increase slowly, and filaments are observed. The number of Ter-15 cells is approximately constant for 60 min, after which it decreases to about 65% of the initial value. The number of Ter-21 cells during the first 60 min decreases slowly to about 90% of its initial value, after which it decreases rapidly and reaches about 40% of the initial value by 120 min. In the observation of the cell shape after ultraviolet irradiation under the phase-contrast microscope, p a r e n t strains of E. coli K12 used here form a long filament in cell culture after ultraviolet irradiation, but Ter-15 cells form a long filament as a rate faster than that of parents and show lysis after 90 min. Also, Ter-21 cells show a spherical or elliptical form without filament during the first 15 min after ultraviolet irradiation and exhibit lysis gradually over a 120 min period of cell culture.

Thymidine nucleotide concentrations after ultraviolet irradiation In the wild type strains, the thymidine triphosphate (dTTP) concentration increases within the first 30 min to 8--9 times that of unirradiated controls, and is constant for the next 20 min, after which time dTTP concentration decreases to that of the control cells (Figs. 4 and 6). The m u t a n t strains have 3--4 times higher concentrations of dTTP than do the parent strains in normal cell growth [19]. In Ter-15 (Fig. 5) the dTTP concentration after ultraviolet irradiation increases within the first 30 min to 7--8 times that of unirradiated controls, and then rapidly decreases over the next 75 min to the dTTP concentration of the control. In contrast, dTTP in irradiated Ter-21 cells increases from the control concentration only in the first 5 min and then decreases to the control value (Fig. 7). The thymidine diphosphate (dTDP) in wild-type I after ultraviolet irradia-

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tion increases for the first 30 min to 7--8 times that of unirradiated controls, after which time it decreases over a 50 min period to the dTDP concentration of the control. In wild-type II, dTDP increases to about 7 times that of unirradiated controls w i t h o u t any decreasing pattern during the period of cell culture. Also, dTDP concentration in Ter-15 cells exhibits the same p h e n o m e n o n as that of wild-type I, showing a 7--8 higher concentration of dTDP within the first 30 min compared to that of unirradiated controls, and decreases to the control concentration within 75 min. Ter-21 cells s h o w the same dTDP con-

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centration after ultraviolet irradiation as that of the unirradiated control culture. The dTMP concentration in wild-type I or II increases to 5--8 times higher values and dTDP-sugar increases to 5--6 times higher values than those of unirradiated controls with constant values sustained for 50 min during the period of cell culture. But in Ter-15 or Ter-21 cells dTMP and dTDP-sugar concentrations do not show any remarkable variation after ultraviolet irradiation when compared to the unirradiated control culture. R N A synthesis as a function o f ultraviolet irradiation The rate of RNA synthesis in irradiated wild-type I is only slightly decreased

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degradation after ultraviolet irradiation and non-irradiation. The radioactivities w e r e m e a s u r e d as d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s . A : w i l d t y p e I a n d T e r o ) ; w i l d t y p e I, u l t r a v i o l e t i r r a d i a t i o n (X . . . . . X ); T e r - 1 5 u l t r a v i o l e t i r r a d i a t y p e II a n d Ter-21, n o n - i r r a d i a t i o n (e I ) ; w i l d t y p e II, u l t r a v i o l e t i r r a d i a u l t r a v i o l e t i r r a d i a t i o n ((~. . . . . . o).

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and in wild-type II is about half that seen in the control (Fig. 8). Irradiated Ter15 synthesizes RNA at about one third the rate of the control, and Ter-21 behaves similarly to its parent strain.

DNA degradation and post-irradiation synthesis Fig. 9 shows degradation of D N A after irradiation. Degradation in the mutants is only about 1/4 that in the wild types and thus is not correlated with ultraviolet-sensitivity. In the mutants, but not in the wild-type strains degradation ceases at about 30 min. Fig. 10 shows that D N A synthesis after irradiation is somewhat more inhibited in the mutants than in their respective parent strains. The excision of pyrimidine dimers We determined whether ultraviolet-induced pyrimidine dimers remained

200 TABLE I D I M E R E X C I S I O N IN E. C O L I K 1 2 W 2 2 5 2 - I I U - A N D T E R M U T A N T S E X P O S E D T O 29 J / m 2

Specific activity o f [ 6 - 3 H ] t h y m i n e was 1 9 2 • 103 c p m / n m o l . C o n d i t i o n s w e r e t h e s a m e as t h o s e d e s c r i b e d in Fig. 4. D i m e r radioactivities of acid-insoluble a n d acid-soluble m a t e r i a l s w e r e d e t e r m i n e d as d e s c r i b e d in Materials a n d M e t h o d s . X - T / T d e n o t e s t h e p e r c e n t a g e o f t h y m i n e ( T ) c o n v e r t e d to t h y m i n e - c o n t a i n ing d i m e r s (X • T). I n c o n t r o l s ( n o n - i r r a d i a t e d cells) v a r i e d f r o m 0 . 0 0 1 2 t o 0 . 0 0 2 1 % . Bacterial strains

Incubation (min)

Acid insoluble

X - T/T (%)

Thy mine ( c p m X 10 - 4 )

Dimers (cpm)

Acid soluble Thymine ( c p m × 10 - 4 )

Dimers (cpm)

W2252-11U( w i l d - t y p e I)

0 15 30 45 60

20.00 20.08 17.36 17.03 16.70

405.0 340.9 298.0 226.0 178.0

0,202 0,169 0.172 0.132 0,107

2.88 2.44 1.96 1.88 1.75

28.7 68.0 98.6 151.0 198.0

Ter-15

0 15 30 45 60

39.16 39.00 35.83 34.697 33.08

570 611 558 563 510

0.145 0.157 0.155 0.162 0.154

0.33 0.26 0,21 0.25 0,22

31.6 22.0 20.5 32.0 58.0

W 2 2 5 2 - i 1 U(wild-type II)

0 15 30 45 60

22.7 20.9 20.3 19.3 19.6

429 360 285 280 270

0.188 0.172 0.140 0.145 0.137

3.59 3.38 2.25 2.00 1.88

28.4 79.0 93.0 118.0 145.0

Ter-21

0 15 30 45 60

30.76 32.48 28.74 30.3 30.4

478 482 445 472 445

0.155 0.148 0.154 0.155 0.146

0.26 0.21 0.27 0.22 0.30

26.7 32.5 45.5 42.0 49.0

acid-insoluble following exposure of cells to ultraviolet irradiation (29 J/m2). In parent strains dimers are excised and such excision occurs most efficiently when the cells are ~incubated in complete medium, because dimers appear immediately in the acid-soluble fraction. The ratio of dimers/thymine decreases on incubation of the wild type cells, but not the mutants, in which the ratio remains approximately constant (Table I).

Discussion Although Ter-15 and Ter-21 produce mucoid colonies in minimal agar [19] and are ultraviolet-sensitive like Escherichia coli K12 known to carry a Ion m u t a t i o n [11,14,15,18,22--25], Ter-21, u n l i k e the lon mutants [18], assumes a sPherical shape after ultraviolet irradiation, rather than forming filaments. Thus Ter-21 may be m u t a n t at a different locus. The fact that Ter-15 and Ter-21 lyse after a period of incubation following irradiation suggest t h a t t h e y contain an inducible prophage [26]. To test this possibility, the following procedure was applied. When the cells lysed in the cell culture after ultraviolet irradiation, the cell debris in the lysate was removed by centrifugation at 10 000 rev./min for 10 rain and then the precipitate was collected by ultracentrifugation at 40 000 rev./min for 90 min. Although this pre-

201 cipitate can be directly observed with the e l e c t r o n microscope (×20 000), no phage-like particles were seen with this instrument. Since bacterial rigidity is based on the structure of the cell wall according to WeibuU [27], it seems probable that there would be a class of mutants with defects in part of the cell wall, which can grow in liquid medium without ,the stabilization of sucrose. Mangiarotti et al. [28] isolated a class o f mutants with apparent envelope defects, which is dependent on high concentration of sucrose for growth and which often exhibits growth of filaments or lysis in the absence of sucrose. But Ter-15 and Ter-21 can not only grow in liquid medium without the stabilization of sucrose, b u t can grow at a rather faster rate than that of parent strains, lysing within 90--120 min after ultraviolet irradiation, while the parent strains form long filaments in the cell culture after ultraviolet irradiation. This shows a class of mutants with apparent fragile cell walls synthesized by deficient concentration in dTDP-sugar. The CET (Colicin E-two) mutants isolated b y Holland et al. [29] show increased breakdown over the parental type after large doses of ultraviolet light. However, they could still be distinguished from typical r e c A mutants which characteristically show extensive spontaneous DNA breakdown, have large intracellular nucleotide pools and rapidly degrade their DNA after small doses (5.0 J / m 2) of ultraviolet light [30]. Howard-Flanders reported the two systems for the "dark repair" of ultraviolet radiation-damaged DNA, that is, excision repair and post-replicational repair [31]. In general, E. c o i l K12 can recover from ultraviolet irradiation produced damage through the excision repair mechanisms which consist of the excision of dimers [5,6], filling of the resulting gaps by resynthesis [7], and rejoining of single polynhcleotide strands [32]. The m u t a n t strains of E. coli K12 designated uvr A, uvr B, and uvr C by Howard-Flanders et al. [33,22] and van de Putt et al. [34] are more ultraviolet sensitive than uvr ÷ strain and are unable to excise thymine-containing dimers from their DNA after ultraviolet irradiation. In particular, the uvr A mutant is approximately 60 times more sensitive than the uvr ÷ strain [31]. Also, Boyce and Howard-Flanders found that E. coli K12 degrade their DNA during incubation following ultraviolet irradiatiom However, degradation occurs to a lesser extent in excision defective mutants than in wild type cells [5]. From Fig. 9 and Table I, the DNA degradation in Ter mutants after ultraviolet irradiation is a b o u t 10% of the original radioactivity for 90 min of incubation, and most of the dimers in the DNA of Ter mutants remain for 60 min Of incubation in complete medium. The results show that Ter mutants cannot excise thymine-containing dimers in their DNA after ultraviolet irradiation. Although these data of Ter mutants show similar results to the uvr mutants after ultraviolet irradiation, the uvr A mutant is approximately 20 times more sensitive than the Ter mutants. From this, we consider that the DNA synthesis after ultraviolet irradiation in the Ter mutants would be different from that of the post replicational repair process that is the only known dark repair system remaining in the uvr A strain. The incorporation of [3H]thymidine in the uvr A6 after ultraviolet irradiation is reduced more and more as the ultraviolet dose is increased, and is almost blocked for 90 min of incubation after ultraviolet irradiation of 9 J / m 2 doses

202

[35]. In the Ter mutants, the incorporation of the ['4C]thymine after ultraviolet irradiation (29 J/m 2 doses) is completely blocked for 45 min, after which time the thymine incorporation is recovered from the inhibition, and this is very different from the incorporation of thymidine in uvr A strain. As the Ter mutants have a three to four times larger dTTP pool and synthesize the D N A at faster rate than those of wild-type cells in normal cell growth [19], the D N A synthesis by the measurement of the incorporation of ['4C]thymine in the Ter mutants is different from that of the incorporation of [3H]thymidine in thymine-prototrophic uvr A strain. Also, the D N A synthesis after ultraviolet irradiation in the Ter mutants is now under investigation. References 1 Wacker, A., Dellweg, H. a n d W e i n b l u m , D. ( 1 9 6 0 ) N a t u r w i s s e n s c h a f t e n 47, 4 7 7 2 Wacker, A. ( 1 9 6 3 ) Progress in Nucleic Acid R e s e a r c h , Vol. I, pp. 2 6 9 - - 3 9 9 , A c a d e m i c Press, New York 3 BoUum, F.J. a n d Setlow, R.B. ( 1 9 6 3 ) Bioehim. B i o p h y s . A c t a 68, 599---607 4 Setlow, R.B., S w e n s o n , P.A. a n d Carrier, W.L. ( 1 9 6 3 ) Science 142, 1 4 6 4 - - 1 4 6 6 5 Boyee, R.P. a n d H o w a r d - F l a n d e r s , P. ( 1 9 6 4 ) Proe. Nail. Acad. Sei. U.S. 51, 2 9 3 - - 3 0 0 6 Setlow, R.B. a n d Carrier, W.L. ( 1 9 6 4 ) Proc. Natl. Acad. Sei. U.S. 51, 2 2 6 - - 2 3 1 7 P e t t i j o h n , D. a n d H a n a w a l t , P.C. ( 1 9 6 4 ) J. Mol. Biol. 9 , 3 9 5 - - 4 1 0 8 S w e n s o n , P.A. a n d Setlow, R.B. ( 1 9 6 6 ) J. Mol. Biol. 15, 2 0 1 - - 2 1 9 9 Suzuki, K., M o r i g u c h i , E. a n d Horii, Z. ( 1 9 6 6 ) N a t u r e 212, 1 2 6 5 - - 1 2 6 7 10 Ma~kovitz, A. ( 1 9 6 4 ) Proc. Natl. A c a d . Sci. U.S. 5 1 , 2 3 9 - - 2 4 6 11 Markovitz, A. a n d R o s e n b a u m , N. ( 1 9 6 5 ) Proc. Natl. Aead. Sci. U.S. 54, 1 0 8 4 - - 1 0 9 1 12 Markovitz, A., L i b e r m a n , M.M. a n d R o s e n b a u m , N. ( 1 9 6 7 ) J. Bacteriol. 94, 1 4 9 7 - - 1 5 0 1 13 Adler, H.I. a n d H a r d i g r e e , A.A. ( 1 9 6 4 ) J. Bacteriol. 87, 7 2 0 - - 7 2 6 14 H o w a r d - F l a n d e r s , P., S i m o n , E. a n d T h e r i o t , L. ( 1 9 6 4 ) Genetics, 49, 2 3 7 - - 2 4 6 15 B u c h a n a n , C.E. a n d Markovitz, A. ( 1 9 7 3 ) J. Bacteriol. 115, 1 0 1 1 - - 1 0 2 0 16 H a m e l i n , C. a n d Chang, Y.S. ( 1 9 7 5 ) J. Bacteriol. 122, 1 9 - - 2 4 17 Markovitz, A. a n d Baker, B. ( 1 9 6 7 ) J. Bacteriol. 9 4 , 3 8 8 - - 3 9 5 18 Bush, J.W. a n d Markovitz, A. ( 1 9 7 3 ) Genetics 74, 2 1 5 - - 2 2 5 19 O h k a w a , T. ( 1 9 7 6 ) Eur. J. Biochern. 61, 8 1 - - 9 1 20 Carrier, W.L. a n d Setlow, R.B. ( 1 9 7 1 ) in M e t h o d s in E n z y m o l o g y (L. G r o s s m a n a n d K. Moldave, eds.), Vol. XXI, pp. 2 3 0 - - 2 3 7 . A c a d e m i c Press, New Y o r k 21 G o l d m a n , K. a n d F r i e d b e r g , E.C. ( 1 9 7 3 ) Anal. B i o c h e m . 5 3 , 1 2 4 - - 1 3 1 22 H o w a r d - F l a n d e r s , P., B o y c e , R.P. a n d T h e r i o t , L. ( 1 9 6 6 ) Genetics 53, 1 1 1 9 - - 1 1 3 6 23 D o n c h , J. a n d G r e e n b e r g , J. ( 1 9 6 8 ) Mol. Gen. Genet. 103, 1 0 5 - 1 1 5 24 D o n c h , J. a n d G r e e n b e r g , J. ( 1 9 7 0 ) M u t a t . Res. 10, 1 5 3 - - 1 5 5 25 L i e b e r m a n , M.M., B u c h a n a n , C.E. a n d Markovitz, A. ( 1 9 7 0 ) Proc. Natl. Acad. Sci. U.S. 65, 6 2 5 - 6 3 2 26 Brooks, K. a n d Clark, A.J. ( 1 9 6 7 ) J. Virol. 1, 2 8 3 - - 2 9 3 27 Weibull, C. ( 1 9 5 3 ) J. Bacteriol. 6 6 , 6 8 8 - - 6 9 5 28 M a n g i a r o t t i , G., A p i r i o n , D. a n d Schlessinger, D. ( 1 9 6 6 ) Science 153, 8 9 2 - - 8 9 4 29 H o l l a n d , I.B., Threlfali, E.J., H o l l a n d , E.M., D a r b y , V. a n d S a m s o n , A.C.R. ( 1 9 7 0 ) J. Gen. MicrobioL 62, 3 7 1 - - 3 8 2 3 0 H o w a r d - F l a n d e r s , P. a n d T h e r i o t , L. ( 1 9 6 6 ) Genetics 53, 1 1 3 7 - - 1 1 5 0 31 H o w a r d - F l a n d e r s , O. ( 1 9 6 8 ) A n n u . Rev. Biochem. 37, 1 7 5 - 2 0 0 32 Olivera, B.M. a n d L e h m a n , I.R. ( 1 9 6 7 ) Proc. Natl. A c a d . Sci. U.S. 57, 1 4 2 6 - - 1 4 3 3 33 H o w a r d - F l a n d e r s , P., B o y c e , R..P., Simson, E. a n d T h e r i o t , L. ( 1 9 6 2 ) Proc. Natl. Acad. Sci. U.S. 4 8 , 2109--2115 34 V a n de Putts, P. a n d v a n Sluis, C.A. ( 1 9 6 5 ) M u t a t . Res. 2, 9 7 - - 1 1 0 3 5 R u p p , W.P. a n d H o w a r d - F l a n d e r s , P. ( 1 9 6 8 ) J. Mol. Biol. 3 1 , 2 9 1 - - 3 0 4