Effect of monochromatic ultraviolet light on macromolecular synthesis in Escherichia coli

283

BIOCHIMICA ET BIOPHYSICA ACTA

E F F E C T O F M O N O C H R O M A T I C U L T R A V I O L E T L I G H T ON MACROMOLECULAR SYNTHESIS IN ESCHERICHIA

COLI

P H I L I P HANAWALT* AND RICHARD SETLOW

Biophysics Department, Yale University, New Haven, Conn. (U.S.A.) (Received October 29th, 1959)

SUMMARY

Tracer techniques were used to follow the synthesis of protein and nucleic acids in Escherichia coli, strains B and I5T-, after the irradiation of exponentially growing cultures with monochromatic ultraviolet light (u.v.). DNA synthesis is most sensitive to u.v., the observed effect being a delay in the initiation of 3,p incorporation into DNA. Doses above 15o ergs/mm * at 2652 A result in cessation of DNA synthesis for a period greater than one normal division time. The incorporation of ~5S into protein and of 32p into RNA is linear with time after u.v. doses which stop DNA synthesis. Protein synthesis is inhibited more than RNA synthesis, and the dose-effect curves indicate a higher multiplicity for the effect of u.v. on RNA synthesis than on protein synthesis. Action spectra for the effect of u.v. on protein synthesis and RNA synthesis are similar and follow a nucleic acid absorption spectrum. The implications of these results, especially of linear synthesis, are discussed.

INTRODUCTION

The studies of m a n y workers have shown that DNA synthesis is the cellular function most sensitive to the action of u.v. light 1-4. However, there has been little quantitative evaluation of the effects on DNA synthesis as compared to the effects on synthesis of other macromolecular species in the bacterial cell. Such a comparison might be expected to provide clues to mechanisms of synthesis in the cell and should therefore be worthwhile. SWENSON AND GIESE5 have shown that much higher doses of u.v. are required to inhibit induced enzyme synthesis in yeast than are required to stop DNA synthesis. The action spectrum for the inhibition of enzyme induction implicated nucleic acid. HALVORSON AND JACKSONe studied the effect of u.v. on induced synthesis of flgalactosidase and on incorporation of 32p into the nucleotides of RNA in yeast. The loss of capacity to utilize free amino acids in proteins paralleled the loss of the The following abbreviations will be used: Tris, tris(hydroxymethyl)aminomethane; DNA, deoxyribonucleic acid; RNA, ribonucleic acid; TCA, trichloroacetic acid. " Abstracted in part from a dissertation for the degree of Doctor of Philosophy in Yale University. Present address: University Institute of Microbiology, Oster Farimagsgade 2a, Copenhagen, Denmark.

Biochim. Biophys. Acta, 41 (196o) 283-294

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PH. C. HANAWALT, R. SETLOW

ability to synthesize the enzyme. Doses of u.v. which inhibited RNA metabolism by 22 % were found to suppress enzyme formation by 95 %. DEERING AND SETLOW7 in a quantitative study of the effect of u.v. on division in E. coli B, found no detectable effect on RNA and DNA synthesis after doses which inhibited division in most of the cells. IVERSON AND GIESE¢ looked specifically at RNA and DNA synthesis in cultures of E. coli B after heavy incident u.v. irradiation. However, synthesis was followed for over a 4-h period, within which time the less affected cells may have had a chance to multiply several times (and repair mechanisms could operate), thus obscuring the initial effects on synthesis in the severely damaged cells. The incident doses used in their studies were much larger than those to be reported here because they made no correction for the absorption of light in the highly absorbing sample. It seemed to us that the most interesting time to take a detailed look at the effects of u.v. on synthesis in E. coli (with a division period of 5° min) should be in the early period of growth, immediately subsequent to the irradiation. COHEN AND BARNER 8, in their studies on unbalanced growth in bacteria, found that the permanent effects of inhibition of DNA synthesis were already manifest within one division period after this inhibition. In the present studies exponentially growing bacteria were irradiated with monochromatic u.v. light. Special attention was paid to dosimetry, and macromolecular synthesis was examined closely in the critical period of "growth" immediately following the irradiation.

MATERIALS AND GENERAL PROCEDURES Bacterial strains and growth conditions The studies were carried out primarily on Escherichia coli I5T_8, although several parallel experiments were performed on Escherichia coli B for comparisons as to generality. The bacteria were cultured aerobically at 37 ° and at pH 7.4 in a synthetic medium containing in I 1 of water: 12 g Tris, 2.25 g KC1, 1.5 g NH4C1, 0.04 g MgC12"6H~O, O.Ol4 g Na~HPO4, 0.026 g Na~SO,, and 0.5 % glucose as carbon source. For balanced growth of strain I5T_ , thymidine was added to the culture tube to a concentration of 0.8 t~g/ml. A Io-ml culture was inoculated from a nutrient agar slant and allowed to grow up overnight on a growth-limiting glucose concentration of 0.02%. When glucose concentration was increased to 0.5 % the next morning, exponential growth resumed with little lag. Growth rate and cell concentration were estimated from the optical density of the culture at 625 m/z in a Coleman model 6A spectrophotometer. When the culture attained 8.8. IO~ cells/ml concentration, the bacteria were harvested by centrifugation for 2 min at 6700 x g. After resuspension in Tris buffer and a second centrifugation the cells were suspended in Tris to the desired concentration for the u.v. irradiations. Two centrifugations were found sufficient to remove essentially all thymidine from cultures of strain I5T . Upon addition to the glucose-salt medium within a period of half an hour, the control cultures would resume exponential growth immediately. Biochim. Biophys. Acia, 41 (196o) 283 294

SYNTHESIS IN E.

coli

AFTER ULTRAVIOLET LIGHT

285

Isotope incorporation and radioactivity assay ~S incorporation into the "TCA-insoluble" fraction was used as a measure of protein synthesis 1°. Cells prepared as described above were pipetted into the isotopically labeled medium (containing o.I t,c/t,g ~5SO~) at time zero. 0.2 ml aliquots were taken from the culture in duplicate at short intervals thereafter and pipetted into small test tubes. 5 ml ice-cold 50/jo TCA was added to each tube and the contents were then emptied into collodion filters (Schleicher & Schuell, 27 m m diam., coarse porosity) to collect the "TCA-insoluble" fraction 9, lO. The filters on a suction filtration apparatus were then rinsed with 5 ml distilled water before they were allowed to dry in air. The radioactivity on the dried filters was determined by conventional GMcounter methods employing an Amperex 1. 4 mg/cm 2 thin mica window counting tube, placed in a ½-inch thick lead shield, at a distance of 3 m m from the filter surface. Counting times were set so as to allow roughly 5 % variation from statistical fluctuations in counting rate. Background counting rate and the radioactivity adsorbed to the filters from the medium, as determined prior to incorporation experiment, were subtracted from the observed rate to give the "TCA-insoluble" rate. 32p incorporation was used to follow nucleic acid synthesis. An adaptation of the SCHMIDT-THANNHAUSERseparation procedure ix to the collodion filter technique of ROBERTS et al. 1° permitted the simultaneous observation of 32p incorporation into the RNA and the DNA fractions of the same sample from the culture. The details of this procedure and an estimate of reliability are reported elsewhere 9,12. Bacteria were grown in IO ml Tris medium containing 0.04 t~c/t~g 82PO4 and, as for the 35S assay, 0.2 ml aliquots were withdrawn in duplicate for the determination of incorporation into the RNA, DNA, and phospholipid fractions.

U.v. irradiations A large water-prism monochromator utilizing a 250 W low-pressure mercury arc lamp was used for the irradiations 13. The intensity was controlled, within limits, by the exit-slit width. The resolution of the instrument allowed an 8 m m separation between the 2805 • and 2652 A lines at the position of the exit-slit. Scattered radiation at wave-lengths above 2400 A amounted to less than 1 % of the intensity. The intensity at the wave-length to be used was determined at the position of the sample tube prior to the irradiation, by means of a calibrated GI-955 phototube with a I cm * mask. The phototube output was measured by a d.c. amplifier and read on a calibrated meter scale. The phototube was calibrated over the u.v. spectral range against a thermopile whose output had been calibrated by standard lamp. Irradiations were performed on bacteria from an exponentially growing culture, suspended in Tris buffer to a concentration of about 2.5"1o s cells/ml. The percent transmission of the sample to be irradiated was determined prior to irradiation, in a Beckman model DU spectrophotometer, and the average effective intensity was then calculated b y the method of MOROWlTZ14. An additional correction factor of 0.9 accounted for the light scattered from the front window of the irradiation cell. 2.3 ml of the bacterial suspension was placed in a i cm path-length quartz cell, and the sample was stirred manually by means of a 2 m m twisted glass stirring rod during the irradiation. Irradiation times were of the order of I to 5 min. The irradiated samples and the unirradiated control sample were kept at 37 ° before, during, and after the irradiation. No effect on isotope incorporation was observed as due to keeping Biochim. Biophys. Acta, 41 (196o) 283-294

286

P H . C. H A N A W A L T , R. S E T L O W

the E. coli cells in buffer at 37 ° for up to half an hour before or after the irradiation. Samples were kept in subdued light until at least 20 min of growth in the Tris medium after the irradiation, to minimize photoreactivation effects. All irradiations reported here were performed at 2652 A unless otherwise specified. CONTROL EXPERIMENTS

Preliminary incorporation studies

Before undertaking a study of macromolecular synthesis by the incorporation of radioisotopes into bacteria which have been treated in various ways, it is important to have a thorough knowledge of the kinetics of isotope incorporation by normal bacterial cells. This has been examined in detail by ROBERTS et al. 1° for both 32P0~ and for 85S04 labeling. However, it was necessary to verify their kinetics for the conditions and media to be used in the present studies. A simple model for the incorporation of **P (from n'P04) into the TCA-insoluble fraction of exponentially growing cells can be represented by the following expression 1°: Z = [Poear/(b - - a)] [b(I --

e - a t ) - - a ( I - - e-&t)]

in which Z represents the total incorporated activity per unit volume of culture at time, t, after the addition of isotope, a is the growth constant, and b is the pool "equilibrium" constant, which is dependent on the rate of formation of small molecular weight precursors as well as on the rate of exchange between the pool and the external environment. P0 is a constant dependent on the initial concentration of cells, and it represents the extrapolation of the logarithmic incorporation rate obtained at large values of t, to the ordinate at t ~ o. 50

10

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curve

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0.5

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Time in min Fig. I. The incorporation of 32p into the TCA-insoluble fraction of normal exponentially growing E . coli B i n T r i s m e d i u m

a t 3 7 °.

B i o c h i m . B i o p h y s . A c t a , 41 ( t 9 6 o ) 2 8 3 - 2 9 4

SYNTHESIS IN

E. coli

AFTER ULTRAVIOLET LIGHT

287

The model was verified experimentally as shown in Fig. I, where data on the incorporation of a*P into the TCA-insoluble fraction of exponentially growing E. coli B is presented. The growth constant was determined from a semilog plot of colony count vs. time. The constants Po and b were then evaluated b y trial and error to achieve the best fit to the theoretical expression. The value obtained for b corresponded to a "half-life" of 4.5 min for the attainment of the pool steady-state condition. Effectively this means that a logarithmic rate of incorporation into the TCA-insoluble fraction is not approached until about 30 min from the time at which the isotope is added to the growth medium. A similar picture but with a much shorter "half-life", was obtained for the incorporation of 35S (from 35S04) into the TCA-insoluble fraction of exponentially growing E. coli B. This effect must be borne in mind in studies of synthesis rates in the cell as measured by the incorporation of radioisotopes. Turnover E. coli I5T- were grown in a~PO4-containing medium until highly labeled. Cells were then separated from the labeled medium by centrifugation and were resuspended in Tris buffer. A dose of 400 ergs/mm 2 u.v. at 2652 A was administered. Then equal aliquots of irradiated and control samples were added to non-labeled Tris medium. The radioactivity per unit volume of culture (TCA-insoluble fraction) was determined at intervals by the filter method. The procedure was repeated using 3~S04 as label. 3~p loss amounted to less than 0.5 To per hour in the first hour for both control and irradiated cultures, although after an hour of growth activity was lost rapidly from the irradiated cells. Roughly 1 % loss of 85S was obtained in the first hour b y both control and irradiated cultures, and a similar drop in 35S content in the irradiated cultures occurred after this period. It was concluded that turnover and autolysis effects are negligible in the experiments to be described, since incorporation studies were carried out within the first hour after irradiation. Reciprocity In studies on the effects of different wave-lengths it is necessary either to use the same intensity for irradiation at each wave length, or to demonstrate that reciprocity holds over the range of intensities to be used. Within experimental error the same incorporation curve (for either 32p or 3sS) was obtained with E. coli I5Tafter a n.v. dose of 200 ergs/mm 2 at 2652 A as intensity was varied over a factor of four. A further check on reciprocity was obtained at 23Ol A and at 24o0 A where the data obtained using the low intensity lamp were in agreement with those obtained using a General Electric H-6 high pressure mercury arc which gave a ten times higher intensity. EXPERIMENTAL

Colony-forming ability in E. coli I5T_ For purpose of comparison with the effects on molecular synthesis, data were obtained on the effect of u.v. on colony-forming ability in E. coli IST_. Immediately after irradiation at 2652 A, dilution series (to give about 200 colonies per plate) were carried out in Tris buffer at room temperature and aliquots were spread on Difco (23 g/l) nutrient agar plates. Incubation at 35 ° was begun immediately after plating. The resulting dose-survival curve for colony-forming ability is shown in Fig. 2, Biochim. Biophys. Acta, 41 (196o) 283-294

288

PH. C. HANAWALT, R. SETLOW

and indicates 37 To survival after a dose of 65 ergs/mm ~. Using a different plating procedure (delay after irradiation and lower incubation temperature) SETLOW A~D CUMMINGS (unpublished) obtained a 37 % survival dose of 50 ergs/mm *. However, it is well known that post-irradiation treatment affects the viability of irradiated bacteria 15. DEERING16 obtained 37 % survival of colony-forming ability in E. calM B after doses of 30 ergs/mm 2 or IO ergs/mm ~, depending on plating procedure used.

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Fig. 2. A d o s e - s u r v i v a l c u r v e for colonyf o r m i n g a b i l i t y of E. calM I5T_ a f t e r u.v. irr a d i a t i o n a t 2652 ~.

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Fig. 3. S a m p l e d a t a on t h e i n c o r p o r a t i o n of 35S into t h e TCA-insoluble fraction of E. coli I5T_ a f t e r different doses of 2652 -~ radiation.

Protein synthesis

Sample data from a 3~S-incorporation run with E. calM I5T- after different doses of u.v. at 2652 A are shown in Fig. 3. The control culture and the five different irradiated cultures were started in labeled medium simultaneously, after the irradiation. The filtering apparatus, holding six filters at once, allowed sampling from alternate groups of three cultures (in duplicate) at the same times, providing control on growth conditions for purposes of comparing effects of different doses. All points in the incorporation data represent a numerical average of two samples taken from the culture at the same time. Note that incorporation into the TCA-insoluble fraction appears to be linear after doses above about 15o ergs/mm *. The linear synthesis of protein after high doses of u.v. was verified in a more detailed check, using E. calMB. This strain does not lyse after high doses of u.v., so growth can be followed for a longer period after the irradiation. In Fig. 4 are shown the data for the incorporation of 35S into the TCA-insoluble fraction of E. calMB after a dose of 15o ergs/mm 2. In this experiment, in contradistinction to the others described, the bacteria were grown for several generations in the radioactive medium before irradiation. Thus, the problem of t h e attainment of pool equilibrium could be disregarded, even at short times after the irradiation. The data in Fig. 4 are plotted both on a linear scale to show the linear Biochim. Biophys. Acta, 41 (196o) 283-294

SYNTHESIS IN E. coli AFTER ULTRAVIOLET LIGHT

28 9

incorporation after u.v. and on a logarithmic scale to show the exponential incorporation in the unirradiated control culture. KELNER17 has interpreted data for total cell mass similar to those shown in Fig. 4b, to indicate that irradiated cultures show an abrupt change in exponential rate of mass increase. We would rather interpret our data as indicating linear rather than two exponential rates. I t is important to note that the 35S-incorporation rate for a dose of 15o ergs/mm ~ is the same as that in the control culture immediately after the irradiation. As time goes on, the incorporation rate in the control culture increases exponentially, but the rate in the irradiated culture remains the same. Larger doses than 15o ergs/mm 2 yield linear rates of incorporation which are less than the initial control value. Smaller doses give rise to incorporation curves which are between linear and exponential. 30O

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Fig. 4. S a m p l e d a t a on t h e i n c o r p o r a t i o n of asS into t h e TCA-insoluble fraction of E. coli B a f t e r 15o e r g s / m m 2 a t 2652 A. a. T h e d a t a on a linear scale, b. T h e s a m e d a t a on a l o g a r i t h m i c scale.

For a check on the use of 35S incorporation as a measure of protein synthesis in E. coli I5T- after irradiation, the effect of u.v. (two different doses) was determined

on the incorporation of [3sSJmethionine and of [14Clproline. The results obtained were consistent with those obtained by using 35SO4 as label after the same doses of U.V.

The linear incorporation curves of Fig. 3 allow the effects of different doses to be compared b y comparing the slopes of these incorporation curves. Taking into account the pool saturation effect mentioned earlier, it seemed best to measure these slopes at a time 35 min after addition of cells to labeled medium. The slopes of the exponential control and the near-exponential curves obtained after low doses were approximated by the tangents to the curves at the 35-min point. It should be noted that any method of comparison at low doses is somewhat arbitrary since the shapes of the incorporation curves change with dose in this region. I t is easier experimentally to determine the slopes at 35 min than at zero time, even if the cells were fully labeled to eliminate the problem of pool equilibrium. A dose-effect curve for protein synthesis was constructed b y plotting the relative incorporation rates at 35 min (compared to the unirradiated control) after different doses. A simple exponential function resulted from this analysis, yielding a 37 % Biochim. Biophys. Acta, 41 (196o) 283-294

290

PH. C. HANAWALT, R. SETLOW

survival dose of 16o ergs/mm 2 for E. coli I5T- E. coli B also gave a first-order dosesurvival curve for protein synthesis, but with a 37% survival dose of 21o ergs/mm *. The shape of the dose-effect curve is arbitrary for low doses. If rates had been compared at zero time, no effect would have been observed until doses greater than IOO ergs/mm 2 for strain I5T- and 15o ergs/mm 2 for strain B were given. However, the slope of the straight line portions would still be the same as shown in Fig. 5. Within experimental error the same survival for protein synthesis in E. coli I5Twas found in cultures growing in the presence and in the absence of thymidine subsequent to the irradiation.

Phospholipid synthesis The incorporation of 3zp into the phospholipid fraction showed too much

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Fig. 5" Dose-effect curves for 35S and 32p incorporation rates, measured 35 min after 2652 A irradiation. Each point represents the data from one curve of the type shown in Fig. 3.

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Time in min Fig. 6. Sample data on the incorporation of 32p into the RNA fraction of E. coli I5r_ after different doses of 2652 -~ radiation.

fluctuation to be used as a valid measure of synthesis in this fraction after u.v. treatment of E. coli I5T-. Qualitatively the incorporation appeared to be exponential, even after doses of u.v. above 15o ergs/mm z. A 37 % survival dose for phospholipid synthesis was estimated at between 400 and 600 ergs/mmL based on a comparison of rates of incorporation of s2p into the phospholipid fraction, measured as tangents to incorporation curves at the 35-min point.

R N A synthesis Sample data from an experiment on s2p incorporation into the RNA fraction of E. coli I5T- after different doses of u.v. are shown in Fig. 6. The incorporation is linear after doses above 15o ergs/mm 2. The slopes of the incorporation curves were measured, as with the ssS studies, at the 35-min point. The resulting dose-effect Biochim. Biophys. 3cta, 41 (196o) 283-294

SYNTHESIS IN

E. coli AFTER ULTRAVIOLET LIGHT

291

curve (Fig. 5) for RNA synthesis does not give the simple exponential relationship found for protein synthesis, nor does it follow multiple target kinetics in the low dose range. (In a comparison of eight irradiations with doses below 6o ergs/mm 2, the rates of incorporation at 35 min by the irradiated samples were always within experimental error of that of the controls.) Even though the dose-effect curves are again somewhat arbitrary in shape for low doses, it is evident that RNA synthesis is considerably less sensitive to u.v. than is protein synthesis. Dose-effect curves at different wave-lengths showed the same multiplicity found at 2652 A, the exponential part of the curves extrapolating back to an intercept of roughly 1.5. Similiar kinetics were found for E. coli B. After irradiation at 2652 the 37 % survival dose was 240 ergs/mm 2 for E. coli IST_ and 280 ergs/mm 2 for E. coli B. The exponential parts of the dose-survival curves for RNA synthesis were parallel to those obtained for protein synthesis, but displaced from them. RNA synthesis was found equally sensitive to u.v. in cultures growing in the presence or absence of thymidine after the irradiation TM. D N A synthesis

A sample experiment on the incorporation of 32p into the DNA fraction of E. coli I5T_ cells after u.v. irradiation is shown in Fig. 7. The effect appears to be a temporary inhibition of DNA synthesis, although doses below about 20 ergs/mm z seemed to have no effect and doses above 15o ergs/mm 2 destroyed DNA-synthesizing ability permanently. It is difficult to obtain more than a qualitative idea of the effect on DNA synthesis from the data obtained, although it is evident that the effect does not involve merely a reduction in the rate of synthesis. These data are in agreement with the finding of KELNER1 that DNA synthesis is inhibited by u.v. for a period roughly proportional to dose. l

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Fig. 7. S a m p l e d a t a on t h e i n c o r p o r a t i o n of asp into t h e D N A fraction of E. coli 15T- a f t e r different doses of 2652 A radiation.

Action spectra

The shapes of the dose-effect curves for protein synthesis and for RNA synthesis Biochim. Biophys. Acta, 4~ (196o) 283-294

292

PH. C. HANAWALT, R. SETLOW

were shown to be independent of the wave-length, but the exponential slopes of these curves were wave-length dependent. The relative sensitivity to different wave-lengths m a y thus be expressed as the reciprocal of the 37 % dose obtained from the exponential portion of the dose-effect curves at these wave-lengths. The sensitivity at each wave-length can be expressed in terms of cm ~per incident quantum. Fig. 8 shows the u.v. action spectra obtained in this fashion. An absorption spectrum for DNA is given for comparison. xlO ~i

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Fig. 8. Action spectra for the inhibition of protein and RNA synthesis. The absorption spectrum of nucleic acid is shown for comparison. Each point represents a dose-effect curve constructed from 3 or more irradiated samples. SUMMARIZING DISCUSSION

The action spectra for the effects of u.v. on RNA synthesis and on protein synthesis have been shown, within experimental error, to be the same and to follow a nucleic acid absorption spectrum. The nucleic acid-type action spectrum has been found by others workers for induced enzyme synthesis 5, and the similarity of protein and RNA action spectra is not surprising in the light of current evidence for the association of nucleic acid in cellular synthesis. The effect of u.v. on DNA synthesis supports the findings of other workers. It is evident that recovery mechanisms can eventually restore the DNA synthesis rate to nearly that of the unirradiated control, except after very high doses. Also, it appears that recovery is a step-process, in that when DNA synthesis resumes, it does so at a rate similar to that of the control initially. However, it is very possible that much of the DNA synthesized after recovery is biologically non-functional, as suggested by the high sensitivity of colony-forming ability to u.v. It is significant that the synthesis of protein and RNA are linear after u.v. doses Biochim. Biophys. Acta, 41 (196o) 283-294

SYNTHESIS IN E.

coli

AFTER ULTRAVIOLET LIGHT

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which inhibit DNA synthesis for appreciable periods of time. Linear synthesis of RNA has also been observed in E. coli I5T- when DNA synthesis is inhibited by thymine deficiency 9, although protein synthesis continues for a period at an increasing rate under this condition TM. R. B. ROBERTS (personal communication) has evidence from studies with this strain that particulate RNA synthesis is constant in the absence of DNA synthesis, but that both soluble RNA and protein synthesis rates increase. Such results could be explained by supposing that the rate of (particulate) RNA synthesis is determined in part b y the number of intact DNA "units" in the cell. This idea is supported by the fact that when DNA synthesis resumes in a culture after a low dose of u.v., the RNA synthesis rate increases. One might ask if the linear rates observed after u.v. are not merely a fortuitous combination of exponential rates in some cells, counteracted b y decreasing rates of synthesis in other, more seriously damaged, cells? However, then one might expect to see decreasing rather than linear rates of synthesis after still higher doses of u.v. Our results show that this is not the case, linear rates being found in all cases where DNA synthesis is stopped. The output in synthesis of RNA and protein per unit DNA could be controlled partially b y the supply of precursors. Thus, linear synthesis would be expected in cases in which precursors were supplied at a constant rate. A case of this type m a y have been observed by COHENTM who observed linear growth in E. coli K - I 2 in the presence of high valine concentrations. The rate of precursor synthesis could have been limited by the concentration of enzymes existing before the cells began to produce non-functional enzymes containing valine as an amino acid analogue. A striking difference in the effect of inhibition of DNA synthesis by u.v. and by thymine deficiency is the fact that a linear rate of protein synthesis is observed in the former and an increasing rate is found in the latter, RNA synthesis rates being constant in both cases. W h y does protein synthesis appear to be less sensitive than RNA synthesis to thymine deficiency, but more sensitive than RNA synthesis to u.v. action? An increasing protein synthesis rate would be expected from the increase in RNA, assuming the current picture for the involvement of ribosomes in protein synthesis. It m a y be that damaged DNA specifies much "nonsense" RNA which cannot sustain protein synthesis. Or at the next lower level it could be the protein which is non-functional and which then limits the rate of precursor synthesis. MCFALL et al. 2° have provided evidence that the integrity rather than the synthesis of bacterial DNA is a precondition for protein synthesis. The fact that the effect of u.v. on protein and RNA synthesis is the same in the presence as in the absence of thymine in E. coli I5T- would support this picture also. Now, consider the dose-survival curves. The effect on protein synthesis can be described by single-hit kinetics in which the surviving fraction is equal to N / N o e a1~, where N o is the protein synthesis level for zero dose, D is the u.v. dose, and is the inactivation cross section, which m a y be expressed in terms of area per incident quantum at a given wave-length. The 37 % survival of protein synthesis after 16o ergs/mm 2 at 2652 A leads to an inactivation cross section of 5 .~2 by this analysis. The effect on RNA synthesis can be approximated b y a multiple-target analysis, although as we have pointed out, the shape of the dose-effect curve depends somewhat on the time at which the a2p incorporation rates are compared. But, if we assume two-target kinetics for the effect on RNA (the actual intercept was 1.5), the relationship N / N o ~- i - - ( i - - e - a D ) 2 leads to two targets of cross section 5 A2, Biochim. Biophys. Acta, 41 (196o) 283-294

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PH. C. HANAWALT, R. SETLOW

each of which must be inactivated. If the suggested conclusion that the two strands of a DNA molecule are involved, is true, then it would appear that damage to one strand is sufficient for unit effect on protein synthesis, but that both strands must be damaged for unit effect on RNA synthesis. The implications of such a model are perplexing at best. Assuming that DNA is the structure involved in u.v. action and taking the quantum yield of Io -2 for damage to DNA ~1, the absorption cross section for the sensitive unit at 2652 A should be 500/k2/photon. (Quantum yield ---- inactivation cross section/absorption cross section.) Since the u.v. absorption cross section for nucleic acid is about 20 times that for protein and since there is about 6 times as much RNA as DNA in the exponentially growing E. coli I5T- cell 12, the absorption at 2652/k is due predominantly to the ribosomes which contain most of the RNA in the cell. The u.v. absorption (corrected for scattering) at 2652 2k by 2.5" lOs cells/ml gives an optical density of 0.45 (ref. 12). Assuming 50,000 ribosomes per cell leads to a concentration of 12. lO12 ribosomes/ml (2. IO-s moles/l). As an approximation say that these ribosomes accounted for 0.4 units of the observed optical density. Then the molecular extinction coefficient for the ribosomes would be 0.2. lOs, which leads to an absorption cross section of about lO -13 cm*/photon. The quantum yield for protein synthesis (assuming ribosomes to be the active centers for this synthesis) is then 5" lO-16 cm2/P hoton (Inact. cross sect.) i o-la cm2/photon (Absorption) or 5" lO-3, which is within a factor of 2 of the quantum yield for damage to DNA. I t is suggested that the primary sites of action of u.v. are the ribosomes, but that the primary damage is to the DNA which m a y be associated with the ribosomes. ACKNOWLEDGEMENTS

This work was assisted by Grant E-I285 from the U.S. Public Health Service. We are grateful to JOHN BUEHLER and NED LYLE for excellent technical assistance. REFERENCES 1 A. KELNER, J. Bacteriol., 65 (1953) 252. 2 D. KANAZIR AND M. ERRERA, Biochim. Biophys. Acta, 14 (1954) 62. 3 D. KANAZlR AND M. ERRERA, Cold Spring Harbor Symposia Quant. Biol., 21 (1956) 19. 4 R. M. IVERSON AND A. C. GIESE, Biochim. Biophys. ,4cta, 25 (1957) 62. 5 B. A. SWENSON AND A. C. GIESE, J. Cellular Comp. Physiol., 36 (195 o) 369 . e H. O. HALVORSON AND L. JACKSON, Bacteriol. Proc. (Soc. Am. Bacteriologists), 117 (1954). 7 R. A. DEERING AND R. B. SETLOW, Science, 126 (1957) 397. 8 S. S. COHEN AND H. n . BARNER, Proc. Natl. Acad. Sci. U.S., 4 ° (1954) 885. 9 p. C. HANAWALT, Science, 13o (1959) 386. 10 R. ]3. ROBERTS, P. H. ABELSON, D. ]3. COWlE, E. T. BOLTON AND R. J. BRITTEN, Studies o[ Biosynthesis in Escherichia Coli, Carnegie Inst., W a s h i n g t o n D.C., 1955, r e v i s e d 1957. 11 G. SCHMIDT AND S. J. THANNHAUSER, jr. Biol. Chem., 161 (1945) 293. 12 p. C. HANAWALT, Ph.D. Thesis, Y a l e U n i v e r s i t y , 1958. 13 D. FLUKE AND R. B. SETLOW, J. opt. Soc. Am., 44 (1954) 327. 14 H. MOROWlTZ, Science, i i i (195o) 229. 15 V. G. BRUCE AND O. MAALOE, Biochim. Biophys. Acta, 21 (1956) 227. is R. A. DEERING, J. Bacteriol., 76 (1958) 123. 17 A. KELNER AND L. L. JACOBS, J. Bacteriol., 77 (1959) 281. 18 p. C. HANAWALT AND J. BUEHLER, Bioehim. Biophys. Acta, 37 (196o) 141. 19 G. N. COHEN, ~Inn. inst. Pasteur, 94 (1958) 152o E. MCFALL, A. B. PARDEE AND G. S. STENT, Biochim. Biophys. Acta, 27 (1958) 282. 21 R. ]3. SETLOW AND ]3. DOYLE, Biochim. Biophys. Acta, 15 (1954) 117.

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