Replication of a specific terminal chromosome segment in Escherichia coli which is required for cell division

J. Mol. Biol. (1973) 78, 211-228

Replication of a Specific Terminal Chromosome Segment in Escherichia cob which is Required for Cell Division TOHRU MmuNoucHIt

AND WALTER MESSER

Max- Plan&-Institut fiir molekulare Genetik Berlin, West Germany (Received 26 June 1972, and in revised form 26 Murch IY73) In the absence of protein synthesis, DNA replication does not proceed to the very end of the chromosome. A terminal segment remains unreplicated. This segment can be selectively labelled. Its size is about O+F& of the chromosome and

its replication is required for subsequent cell division.

1. Introduction Inhibition of protein synthesis in Ederichia wli blocks initiation of DNA replication but allows chromosomes that are in the process of replication to replicate up to, or close to, the end (Ma&e & Hanawalt, 1961; Lark et al., 1963; Abe & Tomizawa, 1967; Caro & Berg, 1968; Cerda-Olmedo et al., 1968; Wolf et al., 1968; Bird & Lark, 1970). The amount of DNA synthesized during inhibition of protein synthesis corresponds to the amount required for the completion of already initiated replication cycles. The same is true if mutants, temperature sensitive for the initiation of replication, are shifted to a non-permissive temperature (see Gross (1972) for a review of the relevant literature). Replication of the chromosome to the very end is a necessary requirement for subsequent normal cell division. But cell division becomes insensitive to the inhibition of DNA synthesis after completion of the chromosome replication cycle (Clark, 1968; Helmstetter t Pierucci, 1968; Marunouchi & Messer, 1972). A signal is given at the end of the replication cycle that initiates a chain of events leading to cell division (Hoffman et al., 1972). Cell division may thus be used as a marker for successful termination of the replication cycle. We report here experiments designed to determine whether the “ends” reached during amino acid starvation and at the non-permissive temperature in initiation mutants are identical. The effects of these treatments on cell division have been investigated. t Present address: Mitsubishi-Kasei Institute of Life Sciences, Tokyo, Japan. 211

212

T. MARUNOUCHI

AND

W. MESSER

2. Materials and Methods (a) Strain8

Eecherichiu coli B/r301, F-,

leu-, pro-,

and media

luc-, gal-, try-,

his-, arg-, thy-, SmR, met-, dra- or drm-, tipKla, is a derivtative of strain HB50 from H. Boyer. E. coli B/r301 dna A was obtained from R. Schindler. The dnaA marker w&s transferred from E. c&i K12 W1485 dnaA PC5 (Carl, 1970) to E. wli B/r301 by Pl transduction. Glucose-minimal medium (Helmstetter, 1967) contained 2 g NH&l, 6 g NazHPO,. 2H 2O, 3 g KH ,PO ,, 3 g N&l, 0.175 g MgSO *.7 H 2O and 2 g glucose in 11 of demineralized water. Required amino acids (Cal Biochem, Los Angeles, U.S.A.) and thymine (Sigma, St. Louis, U.S.A.) were added at 20 pg/ml each. I%-, 15N-labelled medium contained 2g lSNH,C1 (99 “/b 15N, Bio-rad, Richmond, Calif., & Dohme, Montreal, Canada) per U.S.A.) and 2 g [13C]glucose (55 74 13C, Merck-Sharp 11 of minimal medium instead -of the light isotopes. Changes of media were accomplished by rapid filtration through 0.45-pm Millipore filters, prerinsed with hot distilled water, and by washing with 50ml of warmed medium. The procedure was completed within 2 min. For amino acid starvation all required amino acids except methionine were omitted from the medium. DNa.se S1 was a gift from H. Hirokawa. (b) Synchronization In most experiments a modification of the membrane selection technique of Helmstetter & Cummings (1964) w&9 used (Messer, 1972). For one experiment (Table 1) the technique of Mitchison & Vincent (1965) was used with the following modifications: 80 ml of an exponential culture with 10” cells/ml were centrifuged at 4”C, resuspended in 0.6 ml of cold minimal medium and layered on a g-ml sucrose gradient (5 y0 to 30 y0 sucrose in conditioned minimal medium).The gradient was centrifuged for 4.5 min at 2000 g. The top of the band containing bacteria was selected and diluted into warmed minimd medium. Synchronous growth of the cultures wa8 followed with a Coulter counter combined with a Nuclear Data multichannel analyser.

(c) Rate of DNA syntheaia Rate of DNA synthesis was determined with 4-min pulses of [ 3H]thymidine (Amersham, England) (20 to 30 Ci/mmol, 5 @i/ml). Incorporation was stopped with 50/, trichloroacetic acid, containing 100 pg thymidine/ml. The precipitate waz~ collected on 0.45-c” membrane filters (Sartorius, Gattingen, Germany) soaked with 5% trichloroacotic acid containing 100 /Ig thymidine/ml, washed thoroughly with 5% trichloroacetic acid, and counted in a Packard Tri-Carb liquid scintillation spectrometer. (d) End of a round

of DNA

replication

A 0.3-ml fraction of the culture was added to 0.3 ml of warmed medium containing all amino acids, thymine and 20 pg of nalidixic acid/ml (Winthrop Lab., Newcastle, Great Britain). The cell number reached after 60 min was plotted at the time of addition of nalidixic acid, giving the potential to divide in the presence of the drug (Clmk, 1968; Marunouchi & Messer, 1972). (e) 18opycmic ce&&gation Incorporation wae stopped by adding fractions to frozen medium containing KCN (10d3 M). Cells were harvested by centrifugation, resuspended in O-2 M TrisaHCl buffer, pH 7.5, and mixed with cells grown for more than 9 generations in medium containing or 15NH,C1/[13C]glucose, respectively, as density [‘%]thymine and 1*NH,C1/[12C]glucose markers.

TERMINAL

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Cells were lysed with 0.1 mg lysozyme/ml (Worthington Biochemical Co., N.J., U.S.A.), 20 mM-EDTA, 1% sodium dodecyl sulphate (Bonhoeffer & Gierer, 1963). DNA W&B subjected to controlled hydrodynamic shear (Creeger & Spatz, 1969) to give fragments with a molecular weight of 25 x lo8 or 5 x 106, as determined by the calibration curves given by these authors. 3.80 g of CsCl were dissolved in 3.0 g of lysate. The samples were centrifuged at 30,000 revs/min for 60 h at 20°C in a Spinco R50 Ti rotor, or (for the experiment in Fig. 9) in a Spinco SW50 rotor. Fractions were collected on glass filter paper (Sohleicher and Schiill, Damsel, Germany, no. 8), washed twice with 50/, trichloroacetic acid and twice with ethanol.

3. Results (a) Cell divisio~z us u nmker

for the termination

of DNA

rep&x&ion

Initiation of DNA replication can be inhibited by a block in protein synthesis or by shifting a temperature-sensitive initiation mutant to the non-permissive temperature. In both cases, DNA replication proceeds to the end of the chromosome, thenreplication stops. Termination of the replication cycle is a necessary requirement for subsequent cell division (see Introduction). If it were also a sufficient requirement we should expect all cells to divide after both treatments. Extensive residual cell division occurs in initiation mutants on incubation at the non-permissive temperature (Hirota et al., 1970; Beyersmann et al., 1971). After inhibition of protein synthesis, on the other hand, only limited cell division can be observed (Pierucci & Helmstetter, 1969). The following experiments were designed to look at this difference in more detail. A culture of E. co.%B/r301 dnu A growing exponentially at 33°C was dividedin two. One part was incubated at 42°C in the presence of amino acids, the other part was starved for amino acids at 33°C. At 42°C the cell number increased 2*4-fold, then stopped (Fig. l(a)). This is about the increase expected if those cells that had initiated at the time of the temperature shift but had not yet divided proceed to divide, and then all of the cells divide again after finishing the round of replication in progress (calculated in the same way as the example below). Cells growing under the conditions used (glucose-minimal medium) terminate their replication cycle and initiate a new cycle in the middle of the division cycle (Helmstetter, 1967). In the absence of amino acids the increase in cell number was I*4-fold at 33°C (Fig. l(a)). At 119 min the amino acid-starved culture was shifted to 42”C, and 1 minute later amino acids were added. Cell division started about 40 minutes later, and the mid-point of the curve when half of the population had divided occurred 67 minutes after the addition of amino acids. The plateau in cell number reached eventually was the same as that reached at 42°C without preceding amino acid starvation. In the culture that had first been starved for amino acids the rate of DNA replication was determined with pulses of [3H]thymidine as described in Materiels and Methods (Fig. l(b)) and the amount of DNA was determined by continuous incorporation of [’ 4C]thymine (Fig. l(c)). During the amino acid starvation period the rate of DNA replication dropped continually, as was expected since more and more cells reach the end of the replication cycle and stop synthesizing DNA. The amount of DNA increased 1.48-fold, consistent with the expectation that all chromosomes replicate up to, or close to, the terminus.

214

T.

MARUNOUCHI

AND

W.

MESSER

After the shift to 42°C and addition of amino acids there was a sudden burst in the rate of DNA synthesis (Fig. l(b)), after which the rate rapidly dropped again. This burst in the rate of synthesis was not accompanied by a similar increase in the amount of DNA replicated (Fig. l(c)). This indicates that the burst in the rate of replication reflects the synthesis of a small portion of DNA, too small to become evident in the measurements of the amount of DNA. 20 0

r (b)

3 0

60

120

180

11

’ 0

240

I

60

--

I

120

0

60

I

I

IS0

240

120

180

240

Tme(mml

FIG. 1. Cell division (a), rate of DNA replication (b), and amount of DNA synthesized (c) in E. coli B/r301 dna A. From 0 to 119 min incubation was at 33”C, -amino acids. At 119 min the culture was shifted to 42°C and at 120 min amino acids were added. (a) (0) Cell division; (0) cell division from 0 to 120 min at 42°C in the presence of amino acids.

This experiment shows that all cells divide if chromosome replication is terminated in the presence of ongoing protein synthesis, i.e. by raising the temperature of a mutant that is temperature sensitive for initiation. On the other hand only part of the cells divide when termination is by inhibition of protein synthesis; those which did not divide can do ao later, if protein synthesis is restored while the initiation of new replication rounds is inhibited at 42°C. Pierucci & Helmstetter (1969) have demonstrated that cell division requires protein synthesis but only approximately up to the time of the termination of DNA replication, i.e. until about 20minutes beforecell division. Theterminalstepsof celldivisioncantherefore occur without protein synthesis. This agrees with our finding that the cell number increased l.Cfold after removal of amino acids from an exponentially growing culture

TERMINAL

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216

(Fig. l(a)). Using the cell age distribution function derived by Powell (1956), we can deduce from this increase that cell division escaped the inhibition, by amino acid starvation about 20 minutes before cell division, i.e. at about the same time as the chromosomes terminate. i There are, therefore, two possible explanations for the results shown in Figure l(a). (1) Protein 8yn&&8 i.9required 8?wrtly after the termination of replication. The sequence of events would be termination-protein synthesis-cell division. (2) Protein synthesis is rsquir& 8h5rL?y before ternhuth, i.e. it is a prerequisite for termination. With this hypothesis, replication would stop shortly before the end of the chromosome in the absence of amino acids, a small terminal segment remaining unreplicated. Only when protein synthesis is allowed can this segment be replicated. The sequence of events would be protein synthesis-termination-cell division. The fact that we find a burst in the rate of DNA synthesis if we add amino acids to a starved culture (and shift at the same time to 42”C, Fig. l(b)) supports hypothesis (2) and this burst in the rate of replication could reflect the replication of the terminal segment. The following experiment was designed to decide between the two hypotheses. We starved a culture for amino acids. After different times of starvation, amino acids were added and at the same time DNA synthesis was inhibited with nalidixic acid. If hypothesis (1) is correct, all the cells that had terminated during the period of starvation should proceed to divide. If hypothesis (2) is correct, we should not observe any cell division after such a treatment, because the required replication of the terminal DNA segment is inhibited. In order to make the experiments easier to interpret and to get additional data on the time when cell division escapes the inhibition due to amino acid starvation, we used synchronized cells for this experiment. E. coli B/r301 was synchronized with the modified membrane selection technique (Messer, 1972), and the synchronized culture was divided into two parts (Fig. 2(a)). One part was immediately starved of amino acids and one part was not starved and served as a control. At various times, samples were removed from each culture and either used for a direct measurement of cell number, time of cell division, or given nalidixic acid, and amino acids in case of the starved culture, and then measured for cell number 60 minutes later, i.e. time of the end of a round of replication. The whole experiment was then repeated with the sole difference that starvation did not start until 35 minutes (Fig. 2(b)). The average time of cell division in the controls was determined from the mid-point of the stepwise increase and occurred at 42 and 84 minutes, giving the same generation time as for cultures growing exponentially in this medium. There is a time interval of about 20 minutes between the curves for the end of a round of replication and for cell division, and the average time for termination of replication was 20 and 60 minutes, again determined from the mid-point of the increase. No cell division occurred if protein synthesis was blocked early in the cell cycle t If the total cycle is 1 Bnd the cell age (expressed as fraction escapes the inhibition is 2, we can write ln2ji

21-zdx+21n2j:

of the tots1 cycle) at which a cell

21-zdx=1.4,

then 2=0-51. When t,he generation

time ia 42 min,

x correspondsto the time 21 min.

216

T.

MARUNOUCHI

AND

W.

MESSER

(Fig. 2(a)), as predicted by hypothesis (2). If amino acid starvation began at 35 minute+ (Fig. 2(b)), when most cells in the population had finished the DNA replication cycle (and about 25% of them had already divided), the level of the cell number reached was the same as the number of cells that had terminated DNA replication at the time of removal of amino acids, and about 90% of the plateau level which was reached after the first division in the control (Fig. 2(b)). This agrees with the previous observation that cell division ceases to be sensitive to the inhibition of protein synthesis at about the time that termination of replication occurs. Identical results were obtained when a single amino acid (leucine or proline) was omitted instead of five (see Materials and Methods).

IO - -Amino f 2

oclds

-

$52 E 2 = 2u" I 0

I

20 -

I

I 40

- Amino

I 80

1

acids

I 120

I

I 160

I

(b)

1 T 3 b 2 Y d

IO1

E 52 _ % u 3+

IL---J--J40 O

80 "

120 160 200

Time (min)

FIG. 2. Cell division in synchronized E. coli B/r301 after amino acid starvation and nalidixio acid treatment. (a) Starvation starting at 0 min; (b) starvation starting at 35 min. Control. Cell division (0) and end of a round of DNA replication (A) were determined as described in Materials and Methods. End of a round data were corrected for the twofold dilution mentioned in Materials and Methods. Arrows are set at the mid-points between 2 plateaux (arithmetic mean) to indicate the times when half of the population had divided or terminated DNA replication. Amino acid-starved cultures. Cell division (0) and end of a round of DNA replication ( A) were determined as in the control cultures. Amino acids were added back at the same time as nalidixic acid was added.

The most interesting curves are those showing the end of a round of replication in the amino acid-starved cultures. Samples were removed from the starved cultures and nalidixic acid and amino acids were added. Cell number in these samples was determined 60 and 120 minutes later (and was identical at both times) and plotted at the

TERMINAL

CHROMOSOME

SEGMENT

OF

217

E. COLI

time of addition of amino acids and nalidixic acid. If amino acid starvation started early in the life cycle, i.e. at time 0 minutes (Fig. 2(a)), no cell division was observed in these samples. Similarly, cells starved for amino acids starting at time 35 minutes and treated the same way showed no division in the second cycle (Fig. 2(b)) . Although as demonstrated in the previous experiment, release of amino acid starvation does eventually result in cell division, this is not true if DNA replication is inhibited, indicating that hypothesis (2) is oorrect. The increase in the amount of DNA during amino acid starvation was 1*48-fold in Figure I(c). The failure of the cells to divide under conditions in which protein synthesis is again allowed but DNA replication is blocked can hardly be due to a stop of the replication at a point a long way from the end of the chromosome, as has been postulated for Boxillus subtilis (Copeland, 1971). In order to confirm this conclusion, we determined the amount of DNA synthesized in a synchronized population during amino acid starvation. In one type of experiment a culture synchronized with the membrane selection technique (see Materials and Methods) was transferred to amino acid-free medium at 35 minutes and the amount of DNA synthesized was determined chemically by the method of Ceriotti (1952). The results are given in Table 1 (expt 1). The time TABLE

1

DNA synthesis in synchronized E. coli B/r301 starved for amino acids from time 35 minutes

Time (min)

Experiment Amount of DNA (pug/20 ml)

38 40 70 100 120

5.2hO.4 7.4kO.4

1 Relative increase

1 1.4

Experiment 2 [ sH]thymine incorporated (ots/min/ml)

Relative increase

3390 3750 4660 4910 4680

1 1.09 1.37 l-44 l-38

In experiment 1, a culture synchronized with the modified membrane selection technique was transferred to amino acid-free medium at 35 min and the amount of DNA was determined by the method of Ceriotti (1952). In experiment 2, cells prelabelled with [3H]thymine for more than 8 generations were synchronized in a sucrose gradient (Mitohison & Vincent, 1966). They were resuspended in medium containing the same specific activity of [3H]thymine and the incorporation of radioact,ivity was determined after removing amino aoids at time 35 min.

35 minutes was chosen because at this time all of the cells have finished the previous replication cycle and initiated a new one (Helmstetter, 1967; see also Fig. 3). In another type of experiment, cells labelled with [3H]thymine for more than eight generations were synchronized in a sucrose gradient as described by Mitchison & Vincent (1965) (see Materials and Methods). They were resuspended in medium containing the same specific activity thymine, and the amount of [3H]thymine incorporated into DNA was determined after removing amino acids at 35 minutes (Table 1, ext. 2). At 35 minutes the average cell has replicated about three-eighths of the length of the chromosome. Thus, if DNA replication proceeds up to or close to the end of the chromosome, an increase in the amount of DNA by a factor of I.45 is expect,ed. which is in good agreement with the experiments.

T.

218

MARUNOUCHI

AND

W.

MESSER

Enough DNA is thus synthesized during inhibition of protein synthesis to replicate the chromosomes close to the end, but cell division occurs thereafter only if both protein and DNA synthesis are allowed. The sequence of events which trigger cell division must therefore be protein synthesis-termination of replication-signs1 for t Amino acids I-

2 5

.L T

-Amino

acids

.I.

+Amino

acids

4

F50-

: 0 -7 20.!? 5 f $ IO2 c1 6 al 6 n--

5-

20 IO = 3 3

5-

‘0 x

2 E z 500”

DIV. I

Div.2

-ib P

IO 5 20 1 40

80

1

120

160

200

1-ic

240

Time (mln)

Fro. 3. Rate of DNA synthesis (a), end of a round of replication (b), and cell division (c) in synchronized E. coli B/r301. Amino acids were removed at t = 35 min and added back at 119 min. From 120 to 124 min a pulse with [8H]thymidine was given, at 126 min the culture was transferred to IaN., 1%labelled medium (see protocol Table 2). Closed symbols, controls before starvation or to which amino acids were added baok immediately. Arrows at the mid-points of the curves indicate when half of the population had initiated (In.), arrived at the end of a round of replication (Eor) or divided (Div.). The numbers refer to events belonging to the same cycle. For the determination of the end of a round of replication during the starvation period (36 to 119 min), amino acids were added together with nalidixio acid. End of a round data are not corrected for the t,wofold dilution (see Materials and Methods).

TERMlNAL

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219

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cell division. In other words during amino acid starvation replication apparently does not proceed to the very end of the chromosome, a small terminal segment remaining unreplicated. The inhibition of initiation in a temperature-sensitive mutant, on the other hand, does not interfere with the completion of the replication cycle, nor therefore with the release of the signal for cell division. Of course, the experiments do not rule out hypothesis (1) completely, because they do not tell us whether any additional steps requiring protein synthesis occur shortly after termination. (b) Selective labelling of the terminal chromosome segment and time of its replication It should be possible to label this postulated terminal DNA segment selectively with a pulse of [3H]thymidine given immediately after the release from amino acid starvation. If our interpretation is correct for the experiments in which cell division was used as a marker for termination, we can predict that this DNA segment should replicate in the next cycle at the time when the ends of the chromosomes replicate, not the beginnings. This would be a direct proof for the existence of such a segment. In synchronized cells we can choose the conditions such that a new cycle of replication is initiated before the cycle, in which the labelled segment replicates again, is terminated. This allows a clear discrimination whether the labelled DNA is at the end or at the beginning of the chromosome. Synchronized cells of E. coli B/r301 were starved for amino acids beginning at TABLE

2

Protocol to the experiment in Figures 3, 4 and 5 Time (min)

Experimenlal pocedure

o-35

t Amino acids

35-119 119 120 - 124

-Amino

Chromosome configwation (as Inferred from Fig31 -T\.----J/-

~.

_

acids

t Amino acids Pulse wifh[3H]thymidine (IOp(;i/ml) Incubated in heavy mednxn 05NH.,Cl and [‘3C]glucose) + Amino acids

(-) uniabelled

126 - 162

Sample (a 1

126 - 175

Sample (b)

126- 182

Sample (c)

126- 192 126-202

x--. Sampie (d) s Sample (e 1 s

126-212

Sample (f 1

U~abelled light (lrN, IV) heevy (lBN, 13C) DNA.

DNA;

~~

-L ----J/----

(.

. . . .) 3H-labelled

light (14N, I’%) DNA;

(-)

2’0

T.

MRRUNOUCHI

AND

W.

BIESSER

time 35 minutes, i.e. when all cells had initiated replication (Fig. 3, 11~1). After 85 minutes of starvation amino acids were added and a pulse of [3H]thymidine was given. Cells were then transferred to complete medium containing the heavy isotopes, 15NH,C1 and [13C]glucose. The detailed protocol of the experiment is given in Table 2. Rate of DNA replication, end of a round of replication and cell division are shown in Figure 3. The time when half of the population of cells had initiated or finished repliI 6

(a) t

6

Cd)

(cl t

-1.. 6-

A-

(e)

Frac:ion

no

Fro. 4. Density profiles of DNA samples taken at different times during the experiment shown in Fig. 3 and in Table 2. Incubation in 15N-, ‘W-labelled medium was from (a) 126 to 162 min, (b) 126 to 175 min, (c) 126 to 182 min, (d) 126 to 192 min, (P) 126 to 202 min, (f) 126 to 212 min. (See protocol Table 2.) ( . . . -) ‘*C-labelled reference DNA (HH and LL). Total cts/min in the gradients were (a) 54,000, (b) 59,000, (c) 51,000, (d) 54,000, (e) 57,000, (f) 50,000.

TERMINAL

CHROMOSOME

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OF

E. CO.51

22 I

cation was determined from the mid-points of the corresponding curves. Numbers have been assigned to corresponding events, i.e. Eorl is the end of that cycle of replication which was initiated at In.1, and so on. The times for initiation 2 and 3 were assigned on the basis of the following reasoning. For each observed end of a round of replication a corresponding initiation event is required, and the time interval between the two must not be less than 40 minutes. The small dip at 160 minutes, corresponding to the level reached in initiation 2 in the control, and the plateau at around 200 minutes in the measurements of the rate of DNA synthesis were observed in all experiments and were used to assign a defined time for initiation 3 at the mid-point of the curve between the two levels. The time at which the pulse-labelled material in replicated again was determined by analysing the DNA by isopycnic centrifugation at different times after the transfer to heavy medium (Fig. 4). There was no significant increase in the amount of [3H]thymidine incorporat’ed after t,he shift to heavy medium, indicating that the termination of the pulse and the shift were clean. However, there is a small amount of fully heavy material in Figure 4(e) and (f) which may be due to pool effects, i.e. the availability of some[3H]thymidine after t,he density shift.

12

FK. 5. The pattern of the density profiles in Fig. Increase in the number replication (-A-A-)

Time of replication of the 3H-labelled chromosome segment. second cycle of replication of the 3H-labelled segment is taken from the 4, expressed as percentage of totalradioactivity at hybriddensity (-e-a-). of cells which initiated (-m-m-), or arrived at the end of a round of DNA is taken from Fig. 3.

Due to amino acid starvat,ion, initiation and the end of a round of DNA replication, occurring at the same time in normal cultures (see In.1 and EorO), are displaced in time by about 14 minutes in the second cycle after starvat.ion (Figs 3 and 5). In Figure 5 the measurements of rate of DNA replication and end of a round given in Figure 3 are normalized to 1 at time 160 minutes. An increase by a factor of two reflects in this plot the increase in the number of cells that have initiated or arrived at the end of a round after 160 minutes. The replication of the labelled segment, measured as per cent 3H in DNA of hybrid density, coincides with the end of a round of replication, not with initiation. The DNA segment synthesized immediately after release of amino acid starvation is thus replicated again at a time when the terminus

T.

222

MARUNOUCHI

AND

W.

MESSER

of the chromosome replicates, assuming that the labelled DNA reflects the whole population (see Discussion). In another experiment of similar design (the protocol of which is given in Table 3), amino acids were again removed at 160 or 180 minutes, i.e. 40 or 60 minutes after the re-addition of amino acids and transfer to “heavy” medium. Incubation was continued in heavy medium and the DNA analysed in CsCl gradients. Control samples that had not been starved for amino acids a second time were taken at 180 minutes and at 250 minutes. These show the same pattern as in the previous experiment (Fig. 6). Again, a small amount of fully heavy material in Figure 6(b) possibly reflects incorporation into DNA synthesized after the density shift due to pool effects. TABLE 3

Protoeot to the experiment in Figure 6 Tlme(min)

Q-35

Experimental procedure

t Amino acids

35- 119

- Amino acids

119

-t Amino acids

120- 124

Chromosome conflguratlon (see Fags 3 and 61 --.-..I

z..-3’

\ i/ -~

Pulse with[3H] thymldme ---(IO~Ci/ml) A-

1-r ~‘2 ..----

lncuboted in heavy medum (15NH4 CL and [13C] glucose)

k----

unlabeled

124- 180

t Amino oclds w sample (a) ------J/

124 - 250

+ Ammo oclds sample (b)

124- 160

t Ammo suds

160 - 250

-Ammo acids sample (c)

124- I80

+ Amino acids

) U&belled light (14N, lzC) DNA; heavy (16N, 13C) DNA.

1. ..+ -zrz,.. --.. y/-

~.

-I---

____i?-m-

(. . . . . . .) 3H-labellod

light (‘*N,

I’%) DNA;

(-)

When amino acids were removed again at 160 minutes only a small fraction of the radioactive label was replicated upon further incubation. When amino acid starvation began at 180 minutes most of the labelled DNA was replicated, showing hybrid density on incubation in heavy medium free of amino acids. This occurred despite the fact that at the beginning of the starvation only a fraction of the 3H-labelled segments was replicated (see for comparison Fig. 4(b) and (c)). Thus if amino acids are removed shortly before the terminal segment is replicated, its replication is no longer inhibited.

TERMINAL

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223

This could indicate that the protein required for replication of the terminus was synthesized some time before its use, or that effective inhibition of the synthesis of this protein took longer than inhibition of over-all protein synthesis. The experiments thus agree with the predictions made. Protein synthesis is required for the replication of a specific DNA segment, and this segment is at the the terminus of the chromosome. __ (0)

_

id)

HH

i

Fraction

FIG. 6. Density profiles of DNA samples in the HH and LL density markers is indicated.

no

experiment given in Table 3. The position of

(c) Structure of the terminal chromosome segment; selective labelling in dna A mutants During amino acid starvation, replication stops some distance before the end of the chromosome; at 42°C in an initiation mutant replication proceeds to the very end. In an experiment of similar design to the one described in Figure 1, we determined whether the segment between these two sites is replicated again in a second cycle of amino acid starvation and whether the “amino acid stop” and the “42°C stop” are reached again in a successive cycle with the same treatments. It turned out that this experiment gives also an estimate for the size of the segment.

T.

224

MARUNOUCHI

AND

W.

MESSER

The terminal segment of the chromosome was labelled in a temperature-sensitive mutant E. c& B/r301 dna A. An exponential culture was starved for amino acids for 120 minutes at 33°C (see Fig. 1 and Table 4). A pulse of [ 3H]thymidine was given during the replication of the terminal segment after the re-addition of amino acids at 42°C. After all chromosomes finished replication, reinitiation was allowed at 33°C in the presence of amino acids, and the culture was t,ransferred to medium containing heavy isotopes (l”NH,Cl and [13C]glucose) and further incubated with or without amino acids at 33 or 42°C. DNA was prepared and the density determined as described in Materials and Methods. A protocol to the experiment is given in Table 4. TABLE 4 Protocol to the experiment

in Figures

1, 7 and 8

___~ ~.-----.-

-.

O-119

129- 240 240 - 260

~~-..-

-7L

33OC-Amino acids Shifl to 42°C

119 120- 125 I 25 - 129

Expected chromosome configuratlon

Expenmentol procedure

T~rnekn~n)

r-

‘~

42’C + Amino acids Pulse with13H] thymldine (IOpWml) Further lncubotion at 42’C -I- Amino oclds Reinltotlon: 33°C+Am~no acids

-i

--~~ -T}

--7 ~~-/ / I ~~ ---

Culture dwided, incubated in heavy medium ( 15NH4Cl and

260 - 360

[‘JClglucose)at (a) 42Y+Amino

acids

-1,

(b) 33’C -I- Amino acids --=5-

(c) 42°C-Aminoacids

______.._~ k----

unlabelled

) Unlabelled light (l*N, IV) heavy (IEN, 13C) DNA.

(d) 33’C -Amino acids _ .._.. ~~~-.

DNA;

(.........)

Y --

\ :,, ~. ._~~~---

3H-labelled

1

light (14N, IV)

DNA;

(--)

As shown in Table 4 we would expect the labelled segment to be replicated and therefore to become hybrid in density in the presence of amino acids at 33°C and at. 42°C. Without amino acids at 33°C and at 42°C the labelled material should remain light in density. Labelled DNA replicated in the presence of amino acids at 42°C showed the same buoyant density of I.720 g/cm3 as the control replicating uninhibitedly at 33°C with amino acids (Fig. 7(a) and (b)). After incubation in heavy medium in the absence of amino acids the density of the 3H-labelled segment was 1.713 g/cm3, both at 42°C (Fig. 7(c)) and at 33°C (Fig. 7(d)). This density is intermediat’e between hybrid DNA and the 14C-labelIed, light reference DNA (2.707 g/cm3).

TERMINAL

CHROMOSOME

SEGMENT

OF

E. COLI

226

There was a small amount of 3H-label at the position of light DNA in all gradients, which may be due to repair synthesis or could be explained by a failure to replicate again for very few of the labelled segments. The intermediate density of the terminal chromosome segment isolated after density labelling in the absence of amino acids is not due to the presence of a small amount of single-stranded DNA attached to the segment. Single-strand specific DNase S, (Ando, 1966) did not alter the density of the segment.

- (a) 42Of

Amino acids I

12 -

(b) 33OC+ Amno acids

I

8-

(dl 33’C

G

20

- Amlna acids

40 Fraction no.

Fm. 7. Density profiles of the terminal chromosome segment after a second replication cycle in heavy medium (see Table 4). Incubation during the second cycle in IeN-, WXabelled medium was at (a) 42% + amino acids, (b) 33% + amino acids, (c) 42”C-amino acids, (d) 33’C-amino acids. Molecular weight of the DNA analysed was 25 x 10s. (. * . .) 14C-labelled reference DXA (HH and LL).

About 50% of the 3H label, however, was converted to light density when the molecular weight of the DNA fragments analysed in the gradients was reduced from 25 x lo6 to 5 x lo6 (Fig. 8(b)). Denaturation converted all 3H-labelled DNA to the density of light reference DNA (Fig. g(c)). Thus the intermediate density of the terminal chromosome segment results from its size being similar to the size of the analysed DNA fragments (for details see Discussion). 16

T.

MARUNOUCHI

AND

W.

MESSER I

0

20

40

60

0

20

0

20 Frocfm

40

60

40 no.

Fra. 8. Density profiles of the terminal chromosome segment. The same sample as in Fig. 7(a) was centrifuged in an SW60 rotor at 30,000 revs/min for 60 h. (a) 33”C-amino aoids, mol. wt of the fragments 26 x 10s; (b) 33°C-amino acids, mol. wt of the fragments 5 x log; (c) 33°C -amino acids, denatured DNA. ( * * . a) 14C-labelled reference DNA (HH and LL).

4.

Discussion

Three lines of evidence indicate that a specific segment at the end of the chromosome of E. wli fails to be replicated in the absence of protein synthesis. (1) No signal for division is given if replication proceeds towards the end of the chromosome in the absence of protein synthesis. The signal can occur later when protein synthesis is again allowed, but only if DNA replication is not inhibited. Since cell division can occur after the re-addition of amino acids without a substantial increase in the amount of DNA (Fig. l), the signal for division cannot be given by a newly initiated replication cycle (see also Fig. 3). Cell division occurring after recovery from amino acid starvation must, therefore, be due to the completion of a replication cycle that could not be finished in the absence of protein synthesis. (2) The DNA segment which is replicated in initiation mutants (dna A) after release from amino acid starvation at the non-permissive temperature does not replicate in the next replication cycle if amino acids have again been removed.

TERMINAL

CHROMOSOME

SEGMENT

OF

E. COLI

227

(3) The DNA segment which replicates immediately after the re-addition of amino acids to a starved culture replicates again at a time when the terminus of the chromosome is replicated, not the beginning (Fig. 5).

FIG. 9. Possible model for the creation of a fragment of intermediate density due to hydrodynamic shear. ) U&belled light (14N, l W) DNA; (. . . . . ...) 3H-labelled light (14N, laC) DNA; (-1 (---u&belled heavy (15N, 13C) DNA.

The terminal chromosome segment which was 3H-labelled in amino acid-starved E. cola’B/r dna A after the re-addition of amino acids at the non-permissive temperature had a density intermediate between hybrid and light DNA, if in the second replication cycle DNA synthesis occurred with heavy isotopes in the absence of protein synthesis. This indicates that the size of the DNA segment with heavy label associated with 3Hlabelled DNA is smaller than the size of the DNA fragments analysed in the gradients, and this fact allows an estimate of the size of the terminal segment (Bonhoeffer & Gierer, 1963). Figure 9 shows one way in which such a fragment could originate during the hydrodynamic shear treatment of the DNA. Since the average molecular weight of the fragments showing intermediate density was 25 x 10s, the terminal chromosome segment must be of the order of 10 x 106, i.e. about O*5o/oof the E. coli chromosome. No extensive turnover or repair synthesis has been observed. Repair synthesis in DNA which has replicated during amino acid starvation would give labelled material at hybrid density in an ensuing cycle of amino acid starvation in 13C-,15N-labelled medium (see Table 3). An upper estimate for possible repair synthesis can be obtained from Figure 6(c). About 20% of the label is found at hybrid density. This, however, includes chromosomes that at the time of removal of amino acids have already replicated their terminal segments. We conclude from this that there is very little, if any, repair synthesis. Turnover of DNA synthesized after release from amino acid starvation and an inaccurate termination of the radioactive pulse due to pool effects would result in the availability of labelled precursors during the incubation in heavy medium and, thus, would lead to fully heavy labeled material. As seen in Figure 4(e) and (f) and in Figure 6(b) there is very little such material. Incorporation into DNA did not increase significantly after termination of the pulse (Fig. 4). There are several indications that the replication of the labelled segment is a true reflection of the population, i.e. that we have not labelled the DNA in an unrepresentative minority of cells. With respect to cell division and end of a round of replication, all cells behave alike in the different treatments. Increases in cell number and end of a round of replication are by factors of two and there are no unexpected discontinuities

228

T.

MARUNOUCHI

AND

W.

MESSER

in the curves (Figs 3 and 5). The time spread during which the labelled segments replicate, i.e. become hybrid, is the same as the time spread during which the total population arrives at the end of a round of replication (Fig. 5). We do not know the accuracy of the determination of the site at which replication stops in the absence of protein synthesis. The homogeneous density distribution in Figure 7(c) and (d) and Figure S(a) suggests, however, that the variation with which this site is reached again in another cycle of amino acid starvation must be small. Replication of the chromosomes to the very end is a prerequisite for normal cell division. The morphogenetic events with which the cell prepares for division require a signal given at the end of replication (Hoffman et uZ., 1972). We have shown that the production of this signal requires protein synthesis and is connected with the replication of a small terminal portion of the chromosome. Whether other processes like the release of the replication machinery from the DNA, the release of t,hc DNA from the membrane, or the formation of closed circular chromosomes are also correlated with the replication of the terminal segment remains to be determined and may lead to an understanding of the biological significance of our finding. We are grateful to R. Schindler for making strain E. coli B/r301 dna A available. We thank D. Beyersmann, H. Hirokawa and T. A. Trautner for many stimulating discussions. The able technical assistance of T. Hantke and H. Papendorf is gratefully acknowledged.

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Ceriotti, G. (1952). J. Biol. Chem. 198, 297-303. Clark, D. J. (1968). Cold Spring Harbor Symp. Quant. Biol. 33, 823-838. Copeland, J. C. (1971), J. Bacterial. 105, 595-603. Creeger, E. S. & Spatz, H. C. (1969). 1woZ. Gen. Genet. 106, 25-31. Gross, J. D. (1972). Current Topics in Microbiology and Immunology, 57, 39974. Helmstetter, C. E. (1967). J. Mol. Biol. 24, 417-427. Helmstetter, C. E. & Cummings, D. J. (1964). Biochem. Biophys. Acta, 82, 608-610. Helmstctter, C. E. & Pierucci, 0. (1968). J. Bacterial. 95, 1627-1633. Hirota, Y., Mordoh, J. & Jacob, F. (1970). J. MoZ. BioZ. 53, 369-387. Hoffmann, B., Messer, W. & Schwartz, U. (1972). J. Supramol. Structure, 1, 29-37. Lark, K. G., Repko, R. & Hoffmann, E. J. (1963). Biochim. Biophys. Acta, 76, 9-24. Ms.&e, 0. & Hanawalt, P. C. (1961). J. Mol. Biol. 3, 144-155. Marunouchi, T. & Messer, W. (1972). J. Bacterial. 112, 1431-1432. Messer, W. (1972). J. Bacterial. 112, 7-12. Mitchison, J. M. & Vincent, W. S. (1965). Nature, 205, 9877989. Pierucci, 0. & Helmstetter, C. E. (1969). Fed. Proc. Fed. Amer. Sot. Exp. BioZ. 28, 17551760.

Powell, E. 0. (1956). J. Gen. Microbial. 15, 492-511. Wolf, B., Newman, A. & Glazer, D. A. (1968). J. Mol.

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