The effect of amino acid deprivation on subsequent deoxyribonucleic acid replication

BIOCI-IIMICA ET BIOPHYSICA ACTA

9

O F A M I N O ACID D E P R I V A T I O N

ON S U B S E Q U E N T

BBA 8302

THE

EFFECT

DEOXYRIBONUCLEIC

ACID R E P L I C A T I O N

KARL G. LARK*, TRENT REPKO AND EDWARD J. HOFFMAN** Department o] Microbiology, Saint Louis University, St. Louis, Mo. (U.S.A.) (Received March 5th, I963)

SUMMARY The pattern of replication of DNA was studied using a quadruple auxotroph of Escherichia coli requiring thymine, methionine, arginine, and tryptophan. Cells were labelled with a pulse of tritiated thymine and then grown in bromouracil medium either immediately or after an intervening period of amino acid starvation. The patterns of conversion of light-radioactive into hybrid-radioactive DNA under these different conditions provide evidence that a sequential pattern of replication for the bacterial chromosome exists. Prolonged amino acid deprivation was shown to align the DNA replication cycles of individual cells. However, that DNA which is replicated in the absence of amino acids is abnormal. It is subsequently replicated at a slower rate than the DNA normally made in the presence of amino acids. INTRODUCTION Amino acids are required for the continued synthesis of nucleic acids. Bacterial auxotrophs requiring one or more amino acids cease to synthesize RNA and, eventually, DNA when deprived of the required amino acid 1-5. Similarly, animal cells cease to synthesize DNA upon refiaoval of a required amino acidd, 7 or upon addition of an amino acid ai~alogue such as ethionine 8& ~¢[AALOE AND HANAWALT5 hypothesized that upon the removal of amino acids, cells which had embarked upon the synthesis of their new DNA complement (i.e., upon a cycle of DNA synthesis ) could complete that cycle of synthesis but not initiate another. Studies of DNA synthesis in individual cells during and following amino acid starvation 1° were consistent with this hypothesis but did not serve to establish it. The present paper reports on a series of experiments designed to test their hypothesis that: (a) The intracellular synthesis of DNA in bacteria occurs via some sequence such that each single portion of the chromosome once replicated cannot be replicated again until all other portions of the chromosome have also been replicated and that this sequence remains the same in successive replications, i.e., there exists a cycle of DNA synthesis with a defined beginning and end. (b) During amino acid starvation cells complete the synthetic cycles upon which they are embarked but do not initiate new ones. Abbreviation: 5-BU, 5-bromouracil. " Present address, Department of Physics, Kansas StateUniversity, Manhattan, 1(an. (U.S.A.). " ~,~resent address. Deoartment of Chemistry, Stanford University, Calif. (U.S.A.).

I0

K.G.

L A R K , T. R E P K O ,

E.

J. HOFFMAN

The experimental procedure used consists of incorporating a radioactive label into a portion of the bacterial chromosome and then studying the replication of this radioactive portion by means of subsequent density labelling. If the hypothesis stated is correct, one would predict that when a randomly dividing culture is subjected to a "pulse" of radioactive label and subsequently grown in the presence of a density label, the radioactive material should retain its original density until all other portions of the chromosome have replicated, i.e., a cycle of DNA synthesis has passed. At this time, the radioactively labelled DNA should change its density. On the other hand, if the radioactive pulse to the random culture is followed by a period of amino acid starvation to align the individual DNA cycles before addition of the density label, the density of the radioactive material should change immediately and continue to do so in a manner consistent with the random distribution of cell cycles and consequently of the position of the radioactive pulse in different cells. MATERIALS AND

METHODS

Bacterial strain and growth conditions The quadruple auxotroph of Escherichia coli, strain I5T- (557) requiring thymine, methionine, tryptophan, and arginine used in these experiments was obtained from Dr. D. ]3ILLEN. The organism was grown in M9 synthetic medium (Na~HPQ, 0.7 %; KH2P04, 0.3 %; NaCI, 0.05 %; NH4C1, o.I %; glucose, 0.4 %; CaCI~, 0.002 %; MgSO 4, o.o2 % (pH 7-7.2)) supplemented with 2 #g thylnine/ml, 34 ttg L-arginine/ml, 3o#g L-methionine/ml and 14#g L-tryptophan/ml. The organism grows exponentially in this medium at 37 ° with a generation time of 45 rain. The concentration of thymine is ten-fold greater than that required for exponential growth to a cell density of lO9 per millilitre. When [3H]thymine was used, the concentration of radioactive thymine was usually reduced to o.2 #g/ml or, on one occasion, less. 5-Bromouracil, when used, was substituted at a concentration of 2 ttg/ml for thymine. I5T- (557) is partially "relaxed ''11 with respect to RNA synthesis since it will synthesize some RNA in the absence of amino acids supplement. The incorporation of [3H]uridine into the cold-acid or boiling water insoluble cell fraction is shown in Table I. TABLE

I

INCORPORATION OF [3I~]URIDINE BY THE QUADRUPLE AUXOTROPH I 5 T - 5 5 7 IN DIFFJ~RENT MEDIA C e l l s g r o w i n g i n c o m p l e t e m e d i u m ( M 9 + a r g i n i n e , m e t h i o n i n e , t r y p t o p h a n , a n d t h y m i n e ) plus added uridine (2o/*g/ml) were collected and washed by filtration and then resuspended at a titer o f 1 . 6 . lO s c e l l s p e r m i l l i l i t r e in t h e m e d i a s h o w n p l u s [ 3 H ] u r i d i n e ( 2 0 / t g / m l ) . I n c o r p o r a t i o n of u r i d i n e w a s m e a s u r e d b y c o l l e c t i o n o f i - m l s a m p l e s a t t h e t i m e s i n d i c a t e d (t ~ m i n u t e s a f t e r resuspension) on Schleicher and Schuell membrane filters and washing with boiling water. ( S i m i l a r r e s u l t s w e r e o b t a i n e d if s a m p l e s w e r e w a s h e d w i t h c o l d t r i c h l o r o a c e t i c a c i d . )

t

Io 22 62

Complete counts/rain

15 5 6 3 5 ° 987 181 7Ol

Complete without arginin¢ t counts/rain

12 24 66

3 492 13 0 2 4 19 5 1 2

Complete without methionine t counts~rain

14 26 68

20 5 9 2 24 6 9 9 41 8 8 8

Complete without tryptophan t counts/rain

6. 5 28 56

7 5°8 41 9 3 7 61 3 6 I

M9 t

3° 32 60

M9 + T

counts]rain

t

counts]rain

5 229 io 5o8 17 35 °

8 3° 58

2 81o 9 699 I4 207

Number o[ bacteria per millilitre 9 ° win a#er resuspension

6 ' IO s

2.1 • 10 s

2.1 - l O s

2 . 2 3 - IO s

1.6" 108

2.O" lO s

AMINO ACID DEPRIVATION AND SEQUENTIAL

DNA

REPLICATION

II

Changes of media w~.re accomplished by collecting the cells on a Schleicher and Schuell membrane filter (coarse, A, 15 cm diameter), washing on the filter, and resuspension in the new medium. This procedure yields a 75-80 % cell recovery and lasts a total of 1-2 min depending upon the extent of washing.

Measurement o] cell number, DNA content, and absorbancy Cells were counted in a Coulter Counter Model B (using a 3o-/z orifice, a current setting of 3, maximum gain and lower threshold of 8). The total number of cells measured by this method was compared with colony counts of exponentially growing cells and of starved cells. Cells grown under either condition 3delded identical plating efficiencies of 80 %. 4-ml samples were frozen at intervals and analyzed at the end of the experiment for DNA. This was done by thawing the samples, precipitating with cold trichloroacetic acid at a concentration of 5 %, washing with cold trichloroacetic acid once, and extracting the washed pellet at 7 °o twice into a total of 0.25 ml of 0.5 N perchloric acid. o.I ml of this extract was assayed for deoxyribose by the method of BURTON12 suitably reduced in scale. In some experiments DNA was measured as incorporation of radioactive thymine into cells. When such cells were washed with cold trichloroacetic acid. by centrifugation, the results obtained were the same as those obtained with the diphenylamine reaction. However, cold trichloroacetic acid washing led to large errors when cells were collected and washed on membrane filters. This could be circumvented by collecting cells on membrane filters and washing with boiling water instead of cold trichloroacetic acid. Such results were consistent with those obtained by centrifugation from cold trichloroacetic acid. Incorporation of uridine could be measured reproducibly by collecting cells on membrane filters and washing either with boiling water or cold 5 ~o trichloroacetic acid. The absorbancy of cultures was measured at 595 m# in a Bausch and. Lomb spectronic 2o spectrophotometer using a tube with a 1-in light path.

Counting o/radioisotopes Low specific activity (145 mC/mmole) [3Hlthymine was obtained from Volk Radiochemical Co. High specific activity (IO C/mmole) [Me-3H]thymine was obtained from New England Nuclear Corp. Counting of tritium-labelled radioactive samples was carried out in a Packard Tricarb Scintillation Counter. Samples on membrane filters were dried and counted in an organic scintillation fluid: 300 mg 1,4-bis-2-(4-methyl-5-phenyl°xazole)-benzene, 3 g 2,5-diphenyloxazole, dissolved in I 1 toluene. Samples from CsCl-gradient centrifugation were counted in a scintillation fluid for aqueous samples: 80 g naphthalene, 5 g 2,5-diphenyloxazole dissolved in a mixture of 360 ml dioxane, 360 ml toluene and 216 ml ethanol. Despite precipitation of the CsC1 no interference of CsC1 with the counting of tritium samples was observed.

Analysis o/DNA samples by gradient eentri/ugation 2o-ml samples of cells were pipetted quickly onto 6 g of crushed M 9 medium ice at --50 °, centrifuged, and the resulting pellet frozen. The frozen pellets were subsequently thawed and 0.55 ml of a 60 ° solution of I °/o Dupanol, o.oi M Tris, and o.oi M EDTA at pH 7.5 added. The samples were stirred at 60 ° for 30 min. This crude lysate was then divided into two parts. Biochim. Biophys. Acta, 76 (1963) 9-24

12

K.G.

LARK, T. REPKO, E. J. HOFFMAN

I. 80/~1 of crude lysate were added to 800/zl of a 61.2 % solution of CsC1 and the mixture centrifuged in the Model E analytical centrifuge 13 at 42 040 rev./min for 20 h. Pictures were analyzed using a modified analytrol densitometer (in which the coated layer photocell and recorder unit were replaced by a Macbeth Quanta log photo multiplier tube and amplifier and by a Sargent recorder)*. Thi~ analysis yielded the total material present as light (i.e., thymine-thymine), half-heavy hybrid (i.e., thymine-bromouracil), and all-heavy (bromouracil-bromouracil) DNA's from the areas under the densitometer curves. A densitometer tracing of a mixture of light and half-heavy material to which a standard Pseudomonas aeruginosa DNA (supplied by Dr. SUEOKA) was added is shown in Fig. Ia. The density of the allheavy DNA (not shown) corresponded to 1.824. LIGHT

STANDARD

HYBRID A

L. 1.714

1.730

1. 770

1300 t 1100

,'~

00

E

=~ 700 500

1.78

l

~-

1.76

~

1.72-

~P 1,70300 ACKGROUHD

100

J 4

] 8

J I I I 12 I'6 210 21 18 32 16 40 44 ..~

I

l

SAMPLE HUMBER

Fig. I. (A) D e n s i t o m e t e r tracing of an ultraviolet p h o t o g r a p h obtained after 24 h centrifugation of a CsCI-DNA m i x t u r e at 42 040 rev./min in the analytical ultracentrifuge. A crude lysate was p r e p a r e d (see MATERIALS AND METHODS) f r o m bacteria g r o w n in n o r m a l m e d i u m and s u b s e q u e n t l y in 5-BU medium. This m a t e r i a l mixed w i t h a Ps. aeruginosa s t a n d a r d o b t a i n e d ' f r o m Dr. S U E O K A , WaS added to a CsC1 solution and analyzed. (B) Swinging b u c k e t profile of radioactive light and h y b r i d DNA. Bacteria were g r o w n in n o r m a l m e d i u m plus jail]thymine and s u b s e q u e n t l y in 5-BU medium. A crude lysate was p r e p a r e d and centrifuged. 18oo countsJmin were obtained f r o m the s c u m layer at the t o p of the tube. * This modification was suggested b y Drs. CAVALIERr AND ROSENBERG.

Biochim. Biophys. Acta, 76 (1963) 9--24

AMINO ACID DEPRIVATION AND SEQUENTIAL D ~ A

REPLICATION

13

2. 0. 4 ml of crude lysate were added to 4.1 ml ot 59.5 % CsC1 and centrifuged at 29 ooo rev./min in the SW 39 L rotor of a preparative Spinco ultracentrifuge for three days. The tubes were punctured and drops collected 14 directly into bottles of aqueous scintillation fluid. Fifty fractions were collected from each tube and the radioactivity of each determined. Fig. Ib gives a typical result. The amount of material in each peak was directly calculated by subtracting background. The results to be presented are given as the per cent of material in either the light or the (hybrid+heavy) peaks. Difficulty in placing counts between two peaks such as in Fig. Ib or in establishing the baseline in the densitometer tracings such as in Fig. Ia may give rise to errors as high as 20 % in establishing the amount of material in the smallest peaks. This has not been too important since the conclusions to be drawn do not rest on differences between viz. IO % and IO~ 2 % material. A more serious objection is the finding that 50 % of the radioactivity in the crude lysate is trapped in the dupanol-protein scum which floats to the top during centrifugation. The possibility thus arises that the results obtained may be in large part due to selective removal of a given type of DNA (light, hybrid, or heavy). However, we have found no selective trend in the per cent of material trapped in this scum. Thus, the same amount of material is floated in lysates in which the DNA bands in the light form and in lysates in which it is partially or all in the hybrid or h y b r i d + h e a v y form. (Further purification of the lysates was not attempted since yields of less than 50 % were consistently obtained following either chloroform or phenol extraction.) RESULTS

(A) Replication o[ DNA in the presence o[ amino acids MESELSON AND STAHL13 demonstrated that bacteria will not begin a second round of replication of any portion of their chromosome unless all other portions have already completed at least one round of replication. Thus, upon transfer of cells from 15N to 14N medium, a point was reached (at the end of one generation) when only a hybrid material was observed. A similar experiment was done with I5T- (557). Cells growing exponentially were transferred into complete medium plus [SH]thymine for 5 min and then further transferred to medium in which 5-bromouracil was substituted for thymine. The plan of the experiment and the observed growth of the cells are shown in Fig. 2. Samples were taken at intervals during growth in 5-BU and analyzed for the distribution of radioactivity between the light and hybrid bands. The results are shown in Fig. 3. (It should be recalled that upon extraction the bacterial chromosome fragments into smaller DNA subunits 18.) As might be expected, the transfer of radioactivity from the light to the hybrid band only begins after a period of growth and DNA synthesis has elapsed. This is more clearly seen in Fig. 3b where the percent of radioactivity in the hybrid band is presented as a function of the proportion of material in the h y b r i d + h e a v y bands. (The percent hybrid DNA is a measure of the amount of newly synthesized DNA. Since the hybrid DNA contains one strand of parental material, only half of it is % 5-BU(DNA) newly synthesized, thus the fraction DNA replicated ---- 200--% 5-BU(DNA) )" Biochim. Biophys. Aria, 76 (1963) 9--24

14

I<. G. LARK, T. REPKO, E. J. HOFFMAN

j

DNA

< + T + AA-~-~ 4

+ BU+ AA

E

K

cO <

-I

U

I -60

I

I -20

!1 0

t

t

a0

t

I

80

I

I

120

I

I

160

TIME ( r a i n )

Fig. 2. B e h a v i o r of a c u l t u r e pulse-labelled w i t h [ 3 H ] t h y m i n e a n d t r a n s f e r r e d to 5 - B U m e d i u m . I5 T - 557 w a s g r o w n in c o m p l e t e m e d i u m . A t m i n u s 5 m i n it w a s t r a n s f e r r e d to E3H]thymine (o.2/~g/ml) m e d i u m (stippled area) a n d a t o m i n into 5 - B U m e d i u m . A A s t a n d s for a m i n o acid.

No transfer of radioactive material from the light to the hybrid band can be detected until about 70 ~o of the DNA is in the hybrid form or when about 55 ~o of the cell's DNA content has replicated. Although this result is consistent with some sequential pattern controlling DNA synthesis, the amount of DNA already synthesized when this commences would indicate that some cells have proceeded to replicate DNA at a faster rate than others. Thus a labelling period of 5 rain, possibly 7 if we include the period of washing and transfer to 5-BU medium, represents at most only an eighth of the generation period, i.e., the replication of at most 12 O/,oof the DNA content. One might therefore expect that more than 85 ~o of the initial DNA content of the culture would have replicated before a transfer of label took place. This deviation cannot be accounted for as an increase in the actual duration of the radioactive pulse due to a large residual intracellular pool of radioactive thymine since transfer of cells to 5-BU medium instantly halts incorporation of radioactive thymine (see Table II). Despite the early onset of the transfer of label into the hybrid band, very little transfer of DNA from the hybrid into the heavy band has been observed prior to completion of the replication of light material. Thus, no heavy material was detected until 50 rain when approx. 3 °/o each of the light and heavy bands were observed. This would indicate that although variations in the rate of DNA synthesis may occur during replication, they are not of long duration and have usually been compensated when replication is completed.

(B) Replication o~ DNA /ollowing amino acid starvation The pattern of DNA replication is markedly changed following amino acid starvation. This is shown in Fig. 5 which summarizes the results of an experiment in which the pulse of radioactive label is followed by a 9o-min period during which the auxotroph is starved of all three amino acids in the presence of thymine and then Biochim. Biophys. Acla, 76 (1963) 9 - 2 4

AMINO ACID DEPRIVATION AND SEQUENTIAL I ) N A

REPLICATION

15

RA~DIOACTIVE

8°t TOTAL + ci >~

4O

20 A v

v

20

40

I

I

TIME (mln)

I

60

I

80

J /

_z

6o

?

.<

o Q

40

7'/

20

20

40

60

110

100

% (HYBRID + HEAVY)

Fig. 3. (A) T r a n s f e r of r a d i o a c t i v e m a t e r i a l f r o m t h e light to t h e h y b r i d b a n d . S a m p l e s f r o m t h e c u l t u r e in Fig. 2 were a n a l y z e d for t h e p e r c e n t a g e of b a n d e d m a t e r i a l w h i c h a p p e a r e d in t h e light a n d t h e h y b r i d b a n d s . F o r t h e r a d i o a c t i v e c u r v e e a c h s a m p l e a n a l y z e d c o n t a i n e d a t o t a l of a p p r o x . 5ooo c o u n t s / m i n of w h i c h a p p r o x . 5 ° % w a s lost as s c u m (see MATERIALS AND METHODS). T h e d a s h e d line i n d i c a t e s t h e e n d of one g e n e r a t i o n period. (B) P a t t e r n of replication of t h e r a d i o a c t i v e pulse. T h e d a t a in (A) were r e p l o t t e d to p r e s e n t t h e r a d i o a c t i v e m a t e r i a l as a fraction of t h e t o t a l 5 - B U m a t e r i a l . F r a c t i o n D N A replicated = % 5 - B U ( D N A ) 2 o 0 - - °/oS-BU(DNA )' see t e x t . T h e n u m b e r s n e x t to t h e p o i n t s indicate t h e t i m e of s a m p l i n g a f t e r t r a n s f e r to 5 - B U m e d i u m . A mount o/ Light material

A t 50 m i n A t 60 rain

4 (?) i

Hybrid material

93 90

Heavy materiaZ

3 9

T h e d a s h e d line r e p r e s e n t s t h e e x p e c t e d course of s y n t h e s i s if t h e r a t e of s y n t h e s i s of D N A in all labelled cells is t h e s a m e a n d is also c o n s t a n t t h r o u g h o u t t h e replication cycle.

Biochim. Biophys. Acta, 76 (1963) 9 - 2 4

16

K. G. LARK, T. REPKO, E. J. HOFFMAN TABLE n

INCREASE IN RADIOACTIVITY IN 5 - B E MEDIUM FOLLOWING INCORPORATION OF ESH]THYMINE IN COMPLETE MEDIUM

To test the actual duration of a 5-rain pulse of [SH]thymine (see text), [SH]thymine was added to an exponentially growing culture (A) at o min and samples taken at intervals, collected on membrane filters, and washed with boiling water. An aliquot (B) of this culture was transferred at 5 rain (see MATERIALSAND METHODS) to medium in which 5-BU was substituted for [SH]thymine and samples taken at intervals. At 8 min aliquot (A) contained 2.1 • lO8 cells per millilitre. At io-min aliquot (B) contained 1.9" lO8 cells per millilitre. Time (rain) Culture

A B

[SH~thymine added at t = o mid Culture A filtered into 5-BU medium at t =

5

zr

I8

36

Counts/min

2030

5545

IO 686

25 444

Counts/miD

2o4o

1663

196o

2490

54

4 ° 072

5 rain

J

< Z t%

)

.6

3

:~

I

--C~.~ '~

.3

>" U z < rn

.15 ~ < U

1

20

~

60

,

100

140

TIME

(mfn)

180

,- ~ 260

220

Fig. 4. Behavior oi a culture pulse-labelled with [SH]thymine, starved of amino acids and then transferred to 5-BU medium. I5T- 557 was labelled for 5 min (stippled area) in complete medium + o.2 #g [SH]thymine/ml. The culture was then transferred with washing to starvation medium minus amino acids (M9+2 ~ug thymine/ml). After 9o rain it was further transferred, to 5-BU medium ~ontaining amino acids (AA) and samples were taken for analysis. t r a n s f e r r e d to a m e d i u m c o n t a i n i n g a m i n o acids a n d 5 - b r o m o u r a c i l . T h e p l a n of t h e e x p e r i m e n t a n d t h e g r o w t h of t h e c u l t u r e is s h o w n in Fig. 4. T h e b e h a v i o r of t h e c u l t u r e is i d e n t i c a l to t h a t d e s c r i b e d b y MAALOE AND I-IANAWALT5 for I 5 T - A - U - . I n g e n e r a l a lag of 2 0 - 3 0 m i d p r e c e d e d t h e r e s u m p t i o n of D N A s y n t h e s i s f o l l o w i n g s t a r v a t i o n . H o w e v e r , if cells w e r e k e p t for a l o n g e r t i m e o n t h e filter d u r i n g collect i o n a n d t r a n s f e r ( 3 - 4 m i n i n s t e a d of 1-2) t h e d u r a t i o n of this~lag m i g h t be i n c r e a s e d to as l o n g as 45 rain. As m a y be s e e n f r o m Fig. 5, t h e t r a n s f e r of r a d i o a c t i v i t y f r o m t h e l i g h t t o t h e h e a v y b a n d follows a p a t t e r n c o n s i s t e n t w i t h t h a t e x p e c t e d of cells w h o s e s e q u e n t i a l p a t t e r n of D N A r e p l i c a t i o n is t h e s a m e , w h i c h h a v e b e e n l a b e l l e d d u r i n g r a n d o m Blockish. Biophys. Acta, 76 (1963) 9--24

DNA

AMINO ACID DEPRIVATION AND SEQUENTIAL

1oo

5 MINPRELABEL

~,

17

1~2o}O~

j!

THEN 90MINSTARVE

>"~'"'7"r80

REPLICATION

/

fT°,

60

E

v,u o_ 40 °

~ 0cooi //

,¢v SO

2O

~/t~

l 20

l

l

l

40

l

l

60

l

l

80

l !00

% (HYBRID, HEAVY)

Fig. 5 P a t t e r n of replication of the radioactive pulse from cells exposed to a 9o-min period of amino acid starvation. Two e x p e r i m e n t s are shbwn. © - © , d a t a from the experiment shown in Fig. 4; O - O , from a n o t h e r experiment with the same protocol in which the lag in D N A synthesis and cell n u m b e r following s t a r v a t i o n was 2o m i n longer t h a n in Fig. 4. The total radioactivity per sample analyzed was a b o u t 6ooo eounts/min. D a t a are presented as in Fig. 3B. A m o u ~ o~

At 80 m i n At IOO m i n At 12o m i n

Light ma*erial

Hybrid material

Heavy material

4° 13 15

54 71 65

6 i6 2o

MAALOE AND HANAWALT m a d e the simplifying a s s u m p t i o n t h a t D N A was synthesized linearly t h r o u g h o u t the replication cycle i.e., a 5-min pulse would label all cells equally no m a t t e r w h a t p a r t of the chromosome was being replicated. If this is true, we can represent the transfer of label to the hybrid b a n d b y the equation: SH Hybrid =

f

x ln2 ( I - - f (5-BU)) % SH in 5-BU e d(f (5-BU)) where f(5-BU) = 0 200 -- % SH in 5-BU

The dashed curve represents these predicted values. Considering the deviation of the d a t a in Fig. 3 B the fit of t h i s curve to the d a t a is good.

growth at different stages of DNA replication, and subsequently all brought to the same stage in the replication cycle. Moreover, this does not appear to depend upon the physiological state of the cells since cells which showed an abnormally extended lag following collect.ion show the same pattern. A theoretical curve constructed on the assumptions of MAALOEand HANAWALT5 is also shown. The shift from the pattern of replication observed in Fig. 3b to that in Fig. 5 is completed with the termination of that DNA synthesis observed during starvation. Thus Fig. 6a and b present the pattern of DNA synthesis observed following 15 and 40 min starvation. As may be seen, by 40 rain the pattern is essentially that obBiochim. Biophys. Acta, 76 (I963) 9-24

18

K.C.

LARK, T. REPKO, E. J. HOFFMAN

served after 9 ° min. Table III indicates the period required for I)NA synthesis to be completed.

100

-

,..//

5 MIN PRELABEL THEN

(sl)y"~Olss}

80--

ca

>,-z >,I.-

V-U

60

40

0

I

7

12o~ ~ I 20

I 40

A

I

t 60

I

I 80

I

I

100

%(HYBRID * HEAVY)

100

.,.., 8O 1701 >-r

/

Z 60 >,I,>. I,U o<

40

1/)

(6010/

e~ 401 20

Z I

B

I 20

t

I I I I I 40 60 80 % (HYBRID + HEAVY)

I

I

100

Fig. 6. P a t t e r n of r e p l i c a t i o n of a r a d i o a c t i v e p u l s e in cells e x p o s e d to d i f f e r e n t p e r i o d s of a m i n o acid d e p r i v a t i o n A. P r o t o c o l as in Fig. 4 e x c e p t t h a t t h e p e r i o d in M 9 + t h y m i n e (starvation p e r i o d ) lasted 15 m i n ; ]3, p r o t o c o l as in Fig. 4 e x c e p t t h a t t h e p e r i o d in M9 + t h y m i n e lasted 4 ° rain. T h e t o t a l n u m b e r of c o u n t s p e r s a m p l e w a s a b o u t 5ooo c o u n t s / m i n .

Biochim. Biophys. Acta, 76 (1963) 9--24

AMINO ACID DEPRIVATION AND SEQUENTIAL

DNA

REPLICATION

19

TABLE III INCORPORATION OF [SH]THYMINE DURING AMINO ACID STARVATION Cells growing in complete m e d i u m were collected and suspended in s t a r v a t i o n media (lacking added a m i n o acids) a t a t i t e r of 1. 7 • lO 8 cells per millilitre. T h y m i n e c o n c e n t r a t i o n was o.2/~g/ml. j a i l ] T h y m i n e w a s added to one aliquot i m m e d i a t e l y and to a n o t h e r after 20 min. The incorpor a t i o n into i - m l s a m p l e s was m e a s u r e d as in the e x p e r i m e n t s in Table I or II. W h e n a l i q u o t (A) was diluted four-fold a t 90 min into a m e d i u m containing 2 o / , g / m l non-radioactive t h y m i n e no change in radioactivity was observed over an incubation period of 2 h or a four-fold increase in cell n u m b e r . The label incorporated during s t a r v a t i o n was therefore metabolically stable. Minutes after resuspension in starvation media IsMopeadded

A B

at o rain (counts/min) at 20 rain (counts/min)

zo

20

30

40

50

1274 --

274 ° --

3472 lO55

4283 1956

5143 3188

60

4957 3148

7°

5195 3338

80

5279 3179

(C) The replication o/that DNA made during amino acid starvation Unlike the experiments in Fig. 3, the late samples from the experiments in Figs. 5 and 6 all revealed the presence of relatively large (15-2o %) quantities of both light and heavy material present simultaneously. Since all of the radioactive label was being transferred to the hybrid band, this observation suggested that the DNA made in the absence of amino acids was replicating more slowly than that formed either prior to starvation or that made subsequently. This proved to be the case. Fig. 7 presents the pattern of replication of DNA made during amino acid starvation. Cells were labelled with [~Hjthymine during a 9o-min period of amino acid starvation and then transferred to complete 5-BU medium. The patterns of growth were identical to those observed in Fig. 4. It will be observed that the transfer of label from the light to the hybrid band is delayed until about 25 % (40/200-4 o) of the cell's DNA has replicated. Subsequent replication did not occur at a markedly rapid rate. Indeed, by the end of the experiment more than 30 % of the label had not replicated despite the fact that a large quantity (24 %) of all heavy (5-BU-5-BU) DNA had been made (representing the further replication of 50 % of the hybrid I)NA). Moreover, at this time, all of the prelabelled DNA in the experiment in section (B) had replicated viz. Fig. 5.

(D) The effect o/amino acid starvation on the DNA replication cycle The data obtained in Figs. 5 and 6b may result from cells completing their DNA complement in the absence of amino acids and then failing to initiate new cycles of synthesis. On the other hand, it is possible that starvation results in a rearrangement of the sequence of synthesis, which is subsequently initiated at some point chosen at random on the chromosome. To differentiate between these two possibilities, an experiment was carried out in which a culture was starved of amino acids for 60 min (to allow the cells to achieve their full DNA complement (see Table III)) and then transferred to complete media containing thymine. After 27 rain further incubation the cells, which were resuming DNA synthesis, were transferred to a medium containing [3H]thymine (0.02/,g/ml; IO C/mmole) for c~min and then again transferred back to normal medium conBiochim.

Biophys.

Acta,

76 (1963) 9-24

20

K . G . LARK~ T, REPKO, E. J. HOFFMAN

10o

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g (HYBRID +HEAVY) Fig. 7. Pattern of replication of DNA synthesized during amino acid starvation. The protocol for the experiment was the same as that in Fig. 4 except that cells were incubated with [SH]thymine during the 9o-min amino acid starvation. Different experiments are represented by different symbols. In one experiment ( D - D ) an unusually long lag was encountered between the termination of starvation and the onset of DNA synthesis. In the experiments represented by ( Q - O ) and ([~-N) samples were starved in M9+2/~g [SH]thymine/ml. The total radioactivity per sample analyzed was about 400o counts]rain, In the other, ( O - O ) the cells were starved in M9+o.2/~g thymine]ml in which the specific activity of the [SH]thyrfline had been increased five-fold. The total radioactivity per sample was 22 ooo counts/rain. The data are presented as in Fig. 3B. Amount o/ Light material

At 72 rain At 79 rain At 86 rain At 93 rain At xoo rain At 12o min

45 4° 34 31 2o ii

Hybrid material

51 53 57 59 62 65

Heavy material

4 7 9 io 18 24

t a i n i n g non-radioactive t h y m i n e . T h e y were then grown for two generations in one experiment, four in another, a n d again s t a r v e d of amino acids for 0o min. Finally, t h e y were resuspended in complete m e d i u m c o n t a i n i n g 5 - B U a n d samples t a k e n at intervals. D u r i n g the period in which [SH]thymine was incorporated, 7 % of the t o t a l D N A c o m p l e m e n t was replicated as measured b y t h y m i n e incorporation in a control aliquot. I n the period between the end of s t a r v a t i o n a n d the a d d i t i o n of the isotope, only 7 ~/o replication h a d a l r e a d y occurred. Thus, the pulse essentially labelled a small portion of the D N A c o m p l e m e n t near the onset of replication. D u r i n g b o t h the first a n d second s t a r v a t i o n periods a n a m o u n t of D N A equal to a b o u t 45 ~/o of the original c o m p l e m e n t was made a n d b o t h s t a r v a t i o n periods were followed b y a lag of 2o-30 rain before the onset of D N A synthesis; the lag following Biochim. Biophys. Acta, 76 (1963) 9--24

AMINO ACID DEPRIVATION AND SEQUENTIAL

DNA REPLICATION

21

the second starvation was somewhat longer for Expt. b (4 generations interval between successive starvation) than for Expt. a (2 generations interval between successive starvation). Fig. 8a schematically summarizes the treatment and behavior of the cultures. Fig. 8b presents the pattern of DNA synthesis following the second round of amino acid starvation. It is apparent that the replication of DNA (tl:ansfer of radioactivity to the hybrid band) does not follow the random course seen in Fig. 5, but occurs in Part as a pulse. However, 2o % remains in the light band for a much longer time. This may be most easily explained on the basis of the finding, discussed in the previous section above, that DNA replicated in the absence of amino acids is subsequently replicated much more slowly than other DNA. A small proportion of the DNA labelled with [SH]thymine will be replicated during the second starvation period. Thus, if one assumes that (a) DNA is synthesized linearly throughout the cell cycle, (b) cells entering the second starvation period are thoroughly randomized with respect to their replication cycles (see DISCUSSION below), (c) that the 14 % replication which had taken place in the period between the first starvation and the termination of labelling with [3H]thymine takes place at the beginning of the replication sequence; then the fraction of cells whose label will be replicated during the second starvation may be approximated as: 0.14 ln2

f

e(ln2_kt )

d(kt) = 0.2 or 20 ~o

O

This would not be expected to affect the experiments in section (B) since in those experiments the radioactive label was inserted immediately prior to starvation and would not be replicated during starvation. It may be concluded that amino acid starvation does not randomize the intracellular pattern of DNA synthesis or the point at which DNA synthesis is re-initiated. Instead, amino acid starvation wonld appear to align DNA synthetic cycles whose sequential pattern has been maintained for up to four generations.

DISCUSSION

With the reservations concerning the yield of material in the gradient centrifugations already discussed in MATERIALS AND METHODS, the data presented support the hypothesis advanced by MAALOE AND HANAWALT5. The argument may be recapitulated as follows. A pulse of [3H]thymine has been used to label a fraction of the DNA within individuals of a randomly dividing cell population. Presumably, this fraction is different, i.e., near the beginning, middle, or end of the sequence, in different cells depending upon which portion of the replication cycle they are in. Upon continued growth, it is observed that the labelled portions of all of the cells tend to be replicated at the same time (one generation later). This indicates some sequential pattern of replication. If cells are starved of amino acids, it is observed that the labelled portions of their DNA do not tend to replicate simultaneously. This may be due to either an alignment of the replication sequence in the previously randomized population thus making the random insertion Biochim. Biophys. Acta, 76 (1963) 9-24

22

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% (HYBRID +HEAVY) Fig. 8. P a t t e r n of replication of a radioactive pulse introduced after amino ~cid s t a r v a t i o n and replicated a f t e r a second period of a m i n o acid s t a r v a t i o n . (A) Schematic r e p r e s e n t a t i o n of the experiment: I 5 T - 557 was starved of amino acids in M 9 + 2 / ~ g t h y m i n e / m l for 60 rain. Amino acids (AA) were then added and after 27 rain the culture was transff,rc'd to M 9 + a m i n o acids + o . o 2 / ~ g / m l of io C/mmole radioactive t h y m i n e . T h e y were t h e n labelled for 9 rain (stippled area) and r e t u r n e d to complete m e d i u m containing 2/~g t h y m i n e / m ] . The labelled culture was diluted at the time of t r a n s f e r either four-fold (A) or sixteen-fold (B). This was done because of a limitation on the n u m b e r of cells which can be filtered rapidly (400 ml of cells, density lO s per millilitre). Aliquots A and B were then g r o w n for an additional a p p r o x . 8o or 16o rain, respectively, to alk)w for 2 or 4 generations of f u r t h e r growth. The cultures were then filtered and starved of a m i n o acids for 60 rain in M 9 + 2 # g t h y m i n e / m l . Finally, t h e y were t r a n s f e r r e d into 5-BU media and the replication p a t t e r n studied. (B) The replication p a t t e r n of the radioactive pulse f r o m the e x p e r i m e n t s described in Fig. 8A: O - O , two generations g r o w t h before the second s t a r v a t i o n period each sample contained a total of a b o u t 20 ooo counts/rain; [] [7, four generations g r o w t h before the second s t a r v a t i o n , each sample contained a total of a b o u t 600o counts/rain. D a t a are presented as in Fig. 3 B. A mount o~ Light

At 73 rain At 81 rain At 88 min

material

31 15 II

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55 76 78

Heavy material

4 9 II

Biochim. Biophys. Acta, 76 (1963) 9-24

AMINO ACID DEPRIVATION AND SEQUENTIAL

DNA

REPLICATION

23

of the label apparent or to a random distortion of the replication sequence within individual cells. However, the finding that cells in which DNA is labelled immediately after amino acid starvation will, after several generations' growth, tend to replicate a portion of their I)NA at the same time, argues that starvation aligns the replication cycles ~f the population. The fact that not all of the I)NA is replicated in this manner detracts, somewhat, from the strength of the experimental evidence but is to be expected since DNA replicated during amino acid starvation subsequently replicates more slowly than normal DNA. These experiments, while supporting the existence of some pattern of sequential DNA replication do nothing to relate this to the structure of I)NA or the replication of genetic material. However, the recent studies of YOSHIKAWA AND SUEOKA 15 and of N AGATA16,17have demonstrated the sequential replication of genetic markers on the bacterial chromosome. In addition, the radioautography experiments of CAIRNSTM have indicated a single point of replication in the chromosome of E . coll. The present studies are consistent with their findings. Although the pattern of DNA synthesis in individual cells may be aligned by amino acid starvation we have no direct evidence that this occurs as a result of the completion of those cycles in progress at the onset of amino acid deprivation. However, the fact that transfer to the hybrid band after 15 min starvation (Fig. 6a) is completed prior to total DNA replication 7 ° vs. IOO % (hybrid+heavy) supports this idea. The existing stochiometric evidence has already been evaluated pro (ref. 5) and contra (ref. 19). From the data of SCHAECHTER19 it would appear that some bacteria may undergo a second cycle of replication before DNA synthesis ceases. Our results explain the failure of HANAWALT et al. 1° to observe synchronization of I~NA synthesis and cell division following amino acid deprivation. The I)NA made during starvation appears to replicate more slowly than either the I)NA synthesized prior to, or after, starvation. Moreover, recent radioautographic studies in our laboratory have shown that the total amount of DNA synthesized during starvation differs from cell to cell, a result consistent with findings on single cells which had been pulse-labelledX°, ~9. If individual cells contain different amounts of slowly replicating DNA, one would expect their pattern of DNA synthesis to go rapidly out of phase, provided that this material was not bypassed. This would lead to the observed result TM that shortly after the resumption of DNA synthesis some cells are making DNA at a much faster rate than others. Since replication of this I)NA lags behind that of normal DNA, we could thus predict that cells with a large amount of normal DNA would proceed rapidly to arrive at the end of their cycles at a time when others may still be struggling with the initial phases. An alternative explanation would be that some cells are incapable of initiating any DNA synthesis until after a long lag. This seems unlikely since prelabelled cells always replicate all of their DNA (Figs. 5 and 6b) and the pattern of replication both in time and quantity is inconsistent with such a lag*. At the moment, it is not clear why the DNA replicated during starvation should subsequently fail to duplicate normally. The data in Fig. 8b strongly suggest that this " L o n g e x p o s u r e a u t o r a d i o g r a p h s have s h o w n t h a t following s t a r v a t i o n all of the cells in the p o p u l a t i o n are synthesizing D N A at the earliest time at which D N A synthesis can be detected (i.e., 3 ° min after starvation).

Biochim. Biophys. Acta, 76 (1963) 9-24

24

K. G. LARK, T. REPKO, E. J. HOFFMAN

failure is characteristic of both strands, that synthesized in the absence of amino acids and that acting as a template. The production of this material may be related to the absence of protein synthesis rather than to a low rate of RNA synthesis, since we have also found this to be characteristic for I)NA made in the absence of only tryptophan or only methionine, a condition in which this "relaxed" strain appears to synthesize RNA readily for at least 30 rain (see Table I). Further studies that are underway to characterize this material may clarify this problem. However, it seems clear that if protein or RNA synthesis is necessary for the initiation of a new cycle of DNA synthesis 5, protein synthesis is also necessary during DNA synthesis if the I)NA made is subsequently to replicate normally. ACKNOWLEDGEMENTS This work was initiated by one of the authors (K. G. L.) with a hurried experiment conducted with Dr. MAALOE in Copenhagen in the summer of 1961. This experiment ended with a bang when the Spinco Model L centrifuge exploded. Although the experiments above have followed a different course, K. G. L. wishes to thank Dr. MAALOE for his explosive send-off. The authors are also indebted to Dr. SUEOKA for his comments on the manuscript. This work was supported by Grant AI-oi391 from the National Institutes of Health and by Grant GI9577 from the National Science Foundation. REFERENCES PARDEE AND L. S. PRESTIDGE, J . Bacteriol., 71 (1956) 677. F. GROS AND F. GROS, Exptl. Cell Res., 14 (1958) lO 4. ~:{. OKAZAKI AND T. OKAZAKI, Biochim. Biophys. Acta, 28 (1958) 47 o. A. G6LDSTEIN, D. G. C-OLDSTEIN, ]3. J. BROWN AND S. CHOU, Biochim. Biophys. Acta, 36 (1959) 163. O. MAALOE AND P. C. HANAWALT, J. Mol. Biol., 3 (1961) 144. j. PAUL, p e r s o n a l c o m m u n i c a t i o n . K. G. LARK, Progress in Molecular Genetics, Vol. i, A c a d e m i c P r e s s , N e w Y o r k , 1962, p. 177. j. H . I-IOLZNER, T. BARKER AND H. J. POPPER, Natl. Cancerlnst., 23 (r959) 1215. j . H. SCHNEIDER, ~ . CASSIR AND F. CHORDIKION, J. Biol. Chem., 235 (196o) 1437. 19. C. HANAWALT, O. IV[AALOE,D. I. CUMMINGS AND IV[. SCHAECHTER, J . Mol. Biol., 3 (1961) 156. G. S. STENT AND S. BRENNER, Proc. Natl. Acad. Sci. U.S., 47 (1961) 2bo5. K. BURTON, Biochem. J., 62 (1956) 315. IV[. ]V[ESELSON AND F. \¥. STAHL, Proc. Natl. Acad. Sci. U.S., 44 (1958) 672j . WEIGLE, M. I~ESELSON AND K. PAIGEN, J. Mol. Biol., i (1959) 379. H. YOSHIKAWA AND i . SUEOKA, Proc. Natl. Acad. Sei. U.S., 49 (1963) 559T. NAGATA, Biochem. Biophys. Res. Commun., 8 (1962) 348. T. NAGATA, Proc. Natl. Acad. Sci. U.S., 49 (1963) 551j. CAIRNS, J. Mol. Biol., 6 (1963) 208. M. SCHAECHTER, Cold Spring Harbor Syrup. Quant. Biol., 26 (1961) 53.

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Biochim. Biophys. Acta, 76 (1963) 9-24