The ‘DNA-Membrane Complex’ of Escherichia coli B/r : Its Composition and Properties and the Fate...

Eur. J. Biochem. 94, 65 - 75 (1979)

The ‘DNA-Membrane Complex’ of Escherichia coli B/r Its Composition and Properties and the Fate of Nascent and Genome DNA during DNA Synthesis

William ROBERTSON and David WATKINS

Medical Research Council Experimental Radiopathology Unit, Hammersmith Hospital, London

(Received February 15/0ctober 25, 1978)

The composition and properties of the ‘DNA-membrane complex’ of Escherichia coli Bjr have been investigated. The ‘complexes’ contain most of the DNA and membrane of the cells, and about SO % and 2.5 % of the RNA and protein respectively. The properties of DNA synthesized by the ‘complexes’ are described and the process is concluded to be largely mediated through polymerase I.

Nascent DNA synthesized by the ‘DNA-membrane complexes’ was of two main classes, one of molecular weight around 600000- 800000 and the other of higher molecular weight. Polynucleo- tide ligase activity was not detectable. The onset of synthesis coincided with the dissociation of at least 70 % of the genome DNA and all of the nascent DNA from the ‘complexes’ and was concomitant with the action of a nuclease on parental DNA. This nuclease activity was not ATP-dependent.

Isolation of ‘DNA-membrane complexes’ has been achieved by gentle lysis of bacterial cells, with the subsequent purification of a membrane fraction either by high-speed [ l ] or low-speed [2] centrifugation. Most work on these systems utilized ‘complexes’ obtained from polA - mutant bacteria (lacking polymerase I) derived from Escherichia coli K12 strains. However, both Knippers and Stratling [ l ] and Oka- zaki et al. [2] used, as a control, the equivalent wild- type strain of the bacteria containing polymerase I. Both groups of workers concluded that most of the polymerase I activity was lost during the lysis procedure, did not co-sediment with the membrane, and played little part in the DNA-synthesizing activity of the ‘complexes’ subsequently isolated.

In studies into radiation effects on DNA synthesis [3-51 we examined ‘complexes’ isolated from the radiation-resistant and radiation-sensitive strains of E. coli, Bir and Bs-1 respectively. Both these strains of E. coli have the full complement of polymerase I, and in the present work an attempt was made to investigate whether the strain variation (E . coli B as opposed to K12) influenced the DNA-synthetic cha- racteristics of the isolated ‘complexes’.

Abbreviation. Brij 58, poly(oxyethy1ene cetyl ether). Enzymes. DNA polymerase (EC 2.7.7.7); polynucleotide ligase

(EC 6.5.1.2); endodeoxyribonuclease I (EC 3.1.4.30); lysozyme (EC 3.2.1.17); deoxyribonuclease I (EC 3.1.4.5); ribonuclease I (EC 3.1.4.22) ; 5’-3’-exodeoxyribonuclease (EC 3.1.4.26).

MATERIALS AND METHODS

Growth Conditions

E. coli Bjr was grown to late-log phase in glucose- supplemented minimal medium as described by Cramp et al. [4]. At the end of the incubation the culture was chilled and the bacteria were harvested by centrifugation. The bacterial pellet, containing approximately 10” cells, was washed with sterile ice-cold 0.02 M Tris (pH 7.6), re-centrifuged and finally re-suspended in 2 ml of ice-cold 2.5 % (wiw) sucrose in 0.02 M Tris (pH 7.6).

Lysis of E. coli Bjr

The lysis procedure was based upon the method of Godson and Sinsheimer [6] and was performed at 4 “C.

After 10 min in the hypertonic sucrose solution, a mixture of lysozyme (0.2 ml, 5 mgiml) and EDTA (0.2 ml, 20 mgiml) was added to the bacterial suspension and mixed gently for 60 s. Both the lysozyme and the EDTA solutions were made up in ice-cold 0.02 M Tris (pH 8.0). The suspension was incubated for a further 5 min. To the spheroplasts produced by this treatment, a solution containing 0.3 ml of 0.14 M MgS04 and 0.3 ml of 5 % Brij 58 (both solutions made up in ice-cold 0.02 M Tris, pH 7.6) was added. The suspension was mixed by repeated gentle pipetting through a wide-mouthed 5-ml pipette for

66 The 'DNA-Membrane Complex' of E. coli B/r

45-60 s. The mixture was then incubated for 10 min at 4 "C. Lysis was completed by the addition of 6 ml hypotonic 5 mM MgSO4 in 0.02 M Tris (pH 7.6) to give 9 ml of lysate.

Isolation of the 'DNA-Membrane Complexes'

The method of isolation of the 'DNA-membrane complexes' was based upon the work of Knippers and Stratling [l]. The lysate was gently layered onto a discontinous sucrose gradient, consisting of 12 ml 20% (wjw) sucrose on 5 ml60% (w/w) sucrose, both in 0.02 M Tris/5 mM Mg2'. (pH 7.6). The gradient was formed .in 30-ml polyallomer centrifuge tubes (Beckman) and spun at 100000 x g and 4 "C for 30 min in the Beckman L2/65B high-speed centrifuge, using the SW27 rotor head. The 'DNA-membrane complex' sedimented on to, but did not penetrate, the 60% sucrose layer. This material was gently drawn up through a wide-bore capillary tube attached via Teflon tubing (2-mm internal diameter) to a 2-ml syringe. Between 1.2 ml and 1.5 ml of material were collected in this way. The 'DNA-membrane complexes' (in 20% sucrose) were then diluted to 5 ml with 0.02 M Tris/5 mM Mg2+ (pH 7.6). A homogeneous suspension was obtained by gently inverting the tube 8 - 10 times.

(50 p1 of [14C]thymidine, 62 pCi/ml, 20 pCi/mmol) and membrane (50 pl of [2-3H]glycerol, 0.2 pCi/ ml, 0.5 Ci/mmol, see [S]) were each labelled in a similar manner.

Precipitation, Filtration and Quantification of Radioactive Labelled Macromolecules

'DNA-membrane complex' suspensions were precipitated in tubes with excess ice-cold 5 % trichloroacetic acid. The acid precipitates, after at least 1 h at 4 "C, were filtered and washed on Whatman GFjC glass fibre filters using a Millipore filtration mani- fold. Filters were pre-soaked in 1 mM ATP to prevent non-specific binding of unincorporated [3H]dCTP present in the precipitate suspension. Dried filters were placed in 12 ml of scintillation fluid composed of 5 parts toluene, containing 0.3 % 2,5-diphenyl- oxazole (PPO) and 0.01 % 1,4-bis(5-phenyloxazolyl-2)- benzene (POPOP) to one part of ethanol. The scintillation bottles were stored overnight in the dark at 4 "C before counting on a Beckman LS/250 liquid scintillation spectrometer. When both 3H and 14C isotopes were present, tritium was totally excluded from the I4C channel; the counts were recorded on punch tape and analysed on the Medical Research Council's Hewlitt-Packard 21 16 B computer. The counting efficiencies were 25.6 % for 3H and 50 % for l4C.

Pulse-Labelling of DNA Synthesized by 'DNA-Membrane Complexes'

Conditions of Sonication Radioactive labelling of the new DNA synthesized

by the 'DNA-membrane complexes' was performed with equal volumes of the 'DNA-membrane complexes' and a nucleoside triphosphate mixture containing [3H]deoxycytidine triphosphate ([3H]dCTP). The nucleotide mixture was basically that described by Smith et al. [7] containing 0.02 mM deoxynucleoside triphosphates and supplemented with 1 mM tRNA and [3H]dCTP (11.6 Ci/mmol, 200 pCi/ml) to give a solution containing 4 pCi/ml.

The reaction mixture was mixed on a whirlymixer (Fisons Scientific Instruments Ltd) for 1-2 s and, unless stated otherwise, incubated for 2 min at 37 "C. The reaction was stopped by the addition of 2vol. of ice-cold 5 % trichloroacetic acid.

Radioactive Labelling of Protein, RNA, DNA and Membrane in Growing Bacteria

Cellular protein was labelled by including 100 pl [3H]leucine (50 pCi/ml, 29.8 Ci/mmol) in the bacterial culture (100 ml) throughout growth. RNA (50 p1 of [3H]uracil, 25 pCi/ml, 20 Cijmmol), genome DNA

Sonication was performed by inserting the 5-mm- diameter probe of the Mullard sonicator (19 kHz 50 W) into 0.5-ml aliquots of the 'DNA-membrane complexes'. The tubes were cooled with ice during the treatment which never exceeded 60 s.

Neutral Sucrose Gradients

Neutral sucrose gradients (4.5 ml) of linear den- sitysucrose(5-20%, w/w)in0.02 MTrisj5 mM Mg2' (pH 7.6) were prepared in Beckman polyallomer tubes (5.1 x 1.3 cm) by the freeze-thaw method of Baxter-Gabbard [9]. This involved the freezing of 4.5 ml of 12.5% sucrose in the tubes at - 30 "C, and the subsequent slow thawing in an icelwater slurry. After the gradient had been formed, an 0.2-ml 60% sucrose cushion (in 0.02 M Trisj5 mM Mg2+) was introduced below the gradients. The radioactive sample to be centrifuged (0.3 ml) was then gently layered on the gradient and centrifuged for 2 h at 100000 x g and 4 "C in the Beckman 50.1 rotor on the Beckman L2/65B high-speed centrifuge. After

W. Robertson and D. Watkins 61

centrifugation, fractions were collected by upward displacement and precipitated with excess ice-cold trichloroacetic acid in the presence of 20 pg calf thymus DNA. The precipitates were left for at least 1 h in the cold before filtration and counting.

Alkaline Sucrose Gradients

The alkaline sucrose gradients were 4.5-m15 - 20 % (w/w) linear sucrose in 0.1 M NaOH/0.9 M NaCl/ 0.001 M EDTA (pH 12.0). The preparation of the gradients was identical to that described for the neutral gradients, The gradients were again cushioned with 0.2 ml 60% sucrose made up in the alkaline solution described above. Samples (0.2 ml) to be centrifuged were denatured with 0.1 ml NaOH (0.1 M) before being layered onto the gradient. Centrifugation was for 5 h at 100000 x g and 20 "C. All other conditions were as described for the neutral gradients.

The Determination of Sedimentation Coefficients and Molecular Weights from Sucrose Gradients

The values for sedimentation coefficients of DNA centrifuged in linear sucrose gradients were computed using the formulae and data tables of Barber [lo] and McEwen [l 11. The sedimentation coefficients obtained in this way were converted into molecular weights by the equation described by Studier [12].

CsCl Density Gradient Studies

E. coli Bjr was grown in the modified medium (100 ml) of Gillies [13] supplemented with 1 pCi ['4C]thymidine and 0.2 % sulphanilamide and containing 5-bromouracil (75 pg/ml) as density marker. In control cultures thymine (75 pgjml) replaced the 5-bromouracil. After 3.5 h (three doubling times), the bacteria were harvested, washed and re-grown for 1 h in the absence of 5-bromouracil. Lysis was then performed as described above. 'Complexes' were pulse-labelled for 2 min at 37 "C and the reaction stopped with 0.2 M EDTA. The suspensions were then treated with solid sodium dodecyl sulphate (to give a final concentration of 1 %) for 1 h at 37 "C. Pancreatic RNase I, pre-treated for 10 min at 80 "C, was then added to a final concentration of 25 pg/ml and the solution was incubated for 1 h at 37 "C. Finally pronase, self-digested for 2 h at 37 "C, was added at a concentration of 100 pgjml. The solution was then left for 1 h at 37 "C [14].

Samples in either 20 mM Tris, pH 7.6, or 20 mM Tris/O.l M NaOH (3.3 ml) were added to 4.241 g CsCl, overlaid with liquid paraffin and centrifuged in polyallomer tubes in the Beckman SW5O.la swing-out rotor (6 x 5 ml) for 48 h at 100000 xg," and 25 "C.

After centrifugation, 6-drop fractions were collected from the bottom of the tubes directly onto GFjC filters (pre-treated with 1 mM ATP). The filters were dried, soaked for 15 min in ice-cold trichloroacetic acid (5 %) and then washed, dried and counted. Control experiments were run to establish the caesium chloride density gradient, by measuring the refractive index of each fraction (collected into tubes).

Source of Chemicals

The radiochemicals were obtained from the Radio- chemical Company, Amersham. All substrates and enzymes were obtained from the Sigma (London) Chemical Company Ltd, with the exception of the 4-deoxynucleoside triphosphates which were purchased from the Boehringer Corporation (London) Ltd.

Blood and nutrient agar bases were obtained from the Difco Laboratories Ltd.

The chemicals used in scintillation counting (PPO and POPOP) were obtained from Koch-Light Chem- icals. Scintillation-grade CsCl (purity at least 99.9 %) was purchased from the Harshaw Chemical Co. (Ohio, U.S.A.).

General chemicals used in this study were of Analar grade (Hopkins and Williams) or the purest grade commercially available.

RESULTS

Composition of the 'DNA-Membrane Complexes'

Lysates were prepared from cultures with labelled DNA, membrane, protein and RNA respectively. Samples (1 ml) were then centrifuged as for the preparation of 'complexes' but in this case the discontinuous gradients, composed of 3 ml of 20% sucrose layered on 1 ml of 60 % sucrose, were spun in the Beck- man SW 50.1 rotor. Fractions (0.2 ml) were collected, precipitated, and filtered, and the radioactivity was measured. The results (Table 1) are expressed as the percentage of total radioactivity in pooled fractions 1-8, 9-16, 17-21 and 22-25. The 'complexes' (found in fractions 17-21) contained 86% of the DNA, 89 % of the membrane, 24 % of the protein and 54% of the RNA. The material which did not sediment onto the 60 % shelf was found in fractions 1 - 8.

DNA Synthesis by 'Complexes'

In pulse-labelled 'complexes' incorporation of [3H]dCTP into DNA was linear to 2min and then gradually slowed until at 20 min it had ceased. When the 'complexes' were centrifuged out after synthesis had stopped, the supernatant nucleotide mixture was able to support synthesis by a second sample of un-

68 The 'DNA-Membrane Complex' of E. co/i B/r

Table 1. The distribution of labelled constituents from lysed E. coli 3lr on discontinuous neutral sucrose gradients Lysates were prepared from bacteria labelled with ['4C]thymid~ne (for DNA), [3H]glycerol (for membrane), [3H]leucine (for protein) and ['Hluracil (for RNA) respectively. Aliquots (1 ml) were layered onto discontinuous neutral sucrose gradients composed of 3 ml of 20 % (w/w) sucrose on 1 ml of 60 % (w/w) sucrose. Centrifugation was at 100000 x g for 30 min at 4 "C in the SW 50.1 rotor; 15-drop fractions were collected from the top of the tube and the radioactivity in each fraction was assayed. Recoveries from the gradients were between 85 and 100 % in all cases

Fraction Radioactively labelled material numbers

[14C]DNA 3H-labelled 3H-labelled [3H]RNA membrane protein

% of total

1- 8 9 9 68 44 9- 16 2 1 8 2

17-21 86 89 24 54 22 - 25 3 1 - -

reacted 'complex' (held in ice) for a further 20 min. This indicated that the substrates were not rate- limiting. Moreover, when a similar unincubated 'complex' suspension was added to a complete reaction mixture which had been already incubated for 20 min, DNA synthesis was reinitiated. Thus, the cessation of activity was not due to the appearance of inhibitory substances produced during synthesis.

Synthesis of DNA was found to be temperature- dependent. At 25 "C it was only 40% of that at 37 "C; there was no observable synthesis at 4 "C. Storage for 24 h at 4 "C had no effect on this uptake; a 13 loss of activity was observed after 7-days storage at this temperature. Freezing and thawing the 'complex' abolished all activity.

Properties of the DNA Synthesis

The properties of DNA synthesis mediated by the 'DNA-membrane complex' are shown in Table 2.

Synthesis was totally dependent on the presence of all four deoxynucleoside triphosphates and M$ i. ions. Activity was reduced by about 30 %in the absence of ATP, and was totally depressed by pre-digestion of the 'complex' for 2 h at 37 "C with deoxyribonuclease (1 5 pg/ml). The genome DNA was totally degraded by this treatment. A similar experiment with ribonuclease (25 pg/ml) had no effect. The presence of the sulphydryl protector, dithiothreitol (0.1 M), sti- mulated synthesis by about 20%. The sulphydryl- blocking agents, p-chloromercuribenzoate (0.3 mM) and N-ethylmaleimide (10 mM), had differing effects. N-Ethylmaleimide inhibited the activity to a small degree (about 14 %), whereas p-chloromercuribenzoate had no effect. Finally, increasing the concentra-

Table 2. DNA synthesis in 'DNA-membrane complexes' isolated from E. coli Blr Aliquots (0.2 ml) of the 'complexes' were reacted for 2 min at 37 "C under the conditions indicated. The complete system is de- fined in Materials and Methods. The reaction was terminated by the addition of 2 ml ice-cold trichloroacetic acid (5 %) and the radioactivity was assayed. The results represent the mean of four experiments and the range was

Experimental conditions Uptake of [3H]dCTP

4 % of the mean

% control

Complete system (control) 100

- magnesium 2

+ dithiothreitol (0.1 M) 121

+ 15 pg/ml pancreatic DNase"

- ATP 72 - dTTP 2

+ p-chloromercuribenzoate (0.3 mM) 99 + N-ethylmaleimide (10 mM) 86

+ potassium chloride (0.2 M) 96 2

+ 25 pg/ml pancreatic RNase" 92

a For 2 h prior to the reaction.

Table 3. The effect of various shearing forces on the integrity and DNA-synthetic activity of 'DNA-membrane complexes' Two suspensions of 'DNA-membrane complexes', labelled for DNA (with ['4C]thymidine) or membrane (with [3H]glycerol), were prepared. Aliquots (1 ml) were then treated as indicated. In the [14C]DNA preparation the DNA-synthetic activity was then assayed for 2 min. Finally, duplicate 10O-pl aliquots from both preparations were precipitated with trichloroacetic acid and assayed for radioactivity

Shearing force ['4C]DNA 3H-labelled Uptake recovered membrane of 13H]dCTP

recovered into acid-insoluble material

counts/min ("/, control)

Control 553 (100) 2031 (100) 603 (100) 2-min vortexing 525 (95) 1848 (91) 596 (99) 2-min pipetting 508 (92) 1827 (90) 560 (93) 10-s sonication 442 (80) 1442 (71) 850(141) 30-s sonication 420 (76) 1053 (52) 1091 (181)

tion of KCI to 0.2 M was found to have no effect. It has been shown [15] that purified polymerase 111 has no activity at this concentration of KCI.

When lysates were subjected to mild shear (vortexing or pipetting), DNA synthesis was only mini- mally depressed (Table 3) and 90% or more of pre- labelled membrane or DNA was still acid-precipitable. After sonication, however, marked effects were observed; treatment for 10 s and 30 s reduced the acid- insoluble [I4C]DNA by approximately 20 % and membrane losses were 30 % and 50 % respectively (Table 3). This breakdown of both DNA and lipid was coupled with an overall increase of DNA synthetic activity


Table 4. The release of genome D N A from ‘DNA-membrane complexes’ of E. coli Bjr during incubation under various conditions ‘DNA-membrane complexes’, in which the genome DNA was labelled with [I4C]thymidine, were prepared. Aliquots (0.5 ml) of the ‘complexes‘ were treated as described and centrifuged at 3000 x g for 15 min at 4 “C. Duplicate 100-p1 aliquots were removed from the supernatant and assayed for radioactivity

Expt Conditions Genome [I4C]DNA found in supernatant

%

no incubation 10

incubation for 10 min at 37 “C 12

4. As (2) + 1 mM ATP 24 5.

1. ‘ DNA-membrane complex’ ;

2. ‘DNA-membrane complex’;

3. As (2) + 0.2 M EDTA 12

As (2) + 5 mM Mgz+, 80 mM KCI, 50 mM Tris, 1 mM ATP 25

6. As (2) + 5 mM Mgz+, 50 mM Tris, 1 mM ATP 22

7. As (2) + 5 mM Mg2+, 50 mM Tris, 80 mM KC1 26

8. As (2) + full nucleotide mixture 61 9. As (2) + nucleotide mixture

lacking ATP 65 10. As (2) + nucleotide mixture

lacking dTTP 51

of 40% (after 10 s of sonication) to 80% (after 30 s of sonication).

The Distribution of New and Parental DNA after DNA Synthesis

Suspensions of ‘complexes’, pre-labelled for DNA, were incubated under various conditions for 10 min at 37 “C. The incubated samples were then centrifuged at 3000 x g, the supernatants poured off and the pellets resuspended. The two fractions were then precipitated and the radioactivity measured.

The results, summarized in Table 4, are expressed as the percentage of the total radioactivity found in the supernatant. It can be seen (expt 1) that over 90 % of the genome DNA was membrane-bound after the ‘complexes’ had been isolated. Incubation of the ‘complexes’ at 37 “C for 10 min (expt 2) and in the presence of EDTA (expt 3) had little effect upon the amount of rapidly sedimenting genome DNA. The effect of various mixtures of the non-substrate com- ponents of the nucleotide solution on the ‘complexes’ (expu 4- 7) led to no more than 20 - 25 % of the parental DNA appearing in the supernatant. However, incubation of the ‘complexes’ with either the full nucleotide mixture or the nucleotide solution lacking either ATP or dTTP (expts 8 - 10) resulted in solu- bilization of at least 60% of the genome DNA. In the case of expts 8 and 9 the reaction was terminated by adding 0.2 M EDTA. In the two samples where

new DNA was synthesized (expts 8 and 9), over 90 % of this DNA was not attached to the rapidly sedimenting structures.

Parallel experiments to those described above, but in which the membrane was radioactively labelled with [l-’4C]glycerol, did not show any release of the membrane into the supernatant.

Analysis of Nascent D N A on Alkaline Sucrose Gradients

‘Complexes’ were prepared from bacteria pre- labelled with [‘4C]thymidine and aliquots pulse-labelled for 1 and 10 min; samples of the pulsed material were then analysed on alkaline sucrose gradients. For both incubation times tested, the gradient profiles of newly synthesized DNA were biphasic. For example, after a 1 rnin pulse (Fig. 1 A) 70 % of the nascent material was recovered in fractions 1 - 10 with maximum tritium content in fraction 6, consistent with a molecular weight of 650000. The remaining 30 % of the new DNA had sedimented to the bottom of the tube in a position coincident with the parental material, and corresponded to an M , of greater than 10’. The distribution of activity did not vary with pulsing times, and it can be seen that even after a 10-min pulse (Fig. 1 B) the tritium profile was similar to that obtained after 1 min. Once again the nascent DNA sedimented sharply at fraction 6, and the parental DNA at fraction 20. After the 10-min pulse a slight degradation of the parental DNA to smaller molecular weights was apparent. The co-factor of the polynucleotide ligase enzyme of E. cofi [16] was not present during the incubation described above. However, further experiments were performed with added NAD’ in order to establish the contribution, if any, of polynucleotide ligase in this membrane system.

‘Complexes’ were prepared and pulse-labelled with the nucleotide mixture supplemented with NAD +

at a concentration of 0.4 mM [17] for 1 min, or 1 rnin followed by the addition of 1000-fold concentration of unlabelled dCTP for a further 20-min incubation. The pulsed samples were centrifuged on alkaline sucrose gradients.

The gradient profile for the 1 -min pulse (Fig. 2 A) was similar to that described in the previous experiment (Fig. 1 A). The main features were the biphasic distribution of nascent tritium-labelled DNA, with 64% of the new DNA appearing in the first 10 fractions, and with the peak at fraction 7 (13 S). This was consistent with a molecular weight of about 800000. The remaining tritiated DNA was in a broad peak and contained about 30 %of the nascent material. After a 20-min chase (Fig.2B) the second broader peak was virtually absent, although there was an almost identical amount of DNA synthesized (1413 counts/min compared with 1448 counts/min for the

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Fig. 1. The distribution of nascent QH]DNA andgenome [‘4C]DNA on alkaline sucrose gradients. ‘DNA-membrane complexes’ from E. coli B/r were prepared from bacteria labelled with [l4C]thyrnidine (for genome DNA). Aliquots were pulse-labelled for (A) 1 rnin and (B) 10 min and layered onto linear 5 - 20 % (w/w) alkaline sucrose gradients. After 5 h at 1OOOOOxg and 10 “C, 15-drop fractions were collected by upward displacement and precipitated with ice- cold trichloroacetic acid (5 ”/,). Acid-precipitable radioactivity was assayed. The recoveries of radioactive material from the gradients were between 85 and 100%. Sedimentation in the figure is from left to right. Nascent [’HIDNA (+-+) genome [14C]DNA (&---O)

1-min pulse). The peak at fraction 7 was still present and about 70 % of the new DNA appeared in the first 10 fractions. Thus there was no shift of DNA into larger molecular weights. On the contrary, there had been a shift of the larger material to smaller molecular weights. The effectiveness of the chase was shown by incubating the ‘complexes’ for 21 min with the nucleotide mixture which had been previously supplemented with the 1000-fold unlabelled dCTP. Under these circumstances less than 60 counts/min of the radioactive precursor was incorporated. This showed that the dilution of the isotope with the unlabelled precursor had adequately stopped the incorporation of the radioactive nucleotide.

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Fig.2. The fate of new DNA synthesized hy ‘DNA-memhrane complexes’. ‘Complexes’ were prepared as described in Materials and Methods. Aliquots were pulse-labelled (A) for 1 min or (B) for 10min and incubated for a further 10min in the presence of a 1 000-fold concentration of unlabelled dCTP. Centrifugation conditions and treatment of fractions were as for Fig. 1

To clarify the observation of the concomitant release of genome DNA (and its DNA-synthesizing activity) from its membrane association immediately on the onset of synthesis, neutral sucrose gradients were used. Under these conditions, ‘DNA-membrane complexes’ remained intact at least while they were not synthesizing new DNA.

Two suspensions of ‘complexes’ were prepared. They were labelled with [14C]thymidine (for DNA) and [3H]glycerol (for membrane) respectively. [‘4C]- DNA-labelled ‘complexes’ were incubated with the 3H-labelled nucleotide mixture for 0.25,1,2 and 5 min. A parallel experiment was performed using ‘complexes’ where the membrane was labelled with [3H]glycerol and the nucleotide mixture contained no [3H]dCTP but was otherwise identical to that used earlier. For the control in both experiments, the nucleotide mixture was replaced with 0.02 M Tris/5 mM Mg2’/ 1 mM ATP for 5 min, the same as the longest pulsing time used. The samples were then analysed on neutral sucrose gradients.

In the control experiments (Fig. 3A) the genome DNA and the membrane remained associated and sedimented at fraction 22, on the 60% sucrose shelf. DNA would not sediment to this fraction in 2 h


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Fig. 3. The concomitant release of genome DNA from 'DNA-membrane complexes' during the synthesis of DNA; analysis on 5-20% ( w / w ) neutral sucrose gradients. E. coli BJr were grown in the presence of ['4C]thymidine or [3H]glycerol. 'DNA-membrane complexes' were isolated and were either (A) unpulsed or (B- E) pulsed for (B) 15 s, (C) 1 min, (D) 2 min or (E) 5 min. In 'complexes' where the membrane was labelled with [3H]glycerol, pulse-labelling was performed in the absence of [3H]dCTP. Pulsed samples were then layered on neutral sucrose gradients and centrifuged at 100000 x g for 2 h at 4°C. Other experimental details are as for Fig.l. (+ __ +) Nascent [3H]DNA; (&---O) genome [14C]DNA; ( G W ) 3H-labelled membrane

unless it was attached to a large rapidly sedimenting structure of at least 130 S. In all the incubations where the full nucleotide mixture was used (Fig. 3 B - E), at least 70 % of .the genome DNA was separated from the membrane, and only sedimented to fraction 8, which was consistent with a molecular weight of about

10'. The membrane label still sedimented as for the control, with the peak at fraction 22. Of the genome DNA, 25 % was also found at fraction 22, coincident with the membrane structure.

At least 90% of the newly synthesized DNA was free from the rapidly sedimenting structure, even after a 15-s reaction (Fig. 3 B). This material was found at fractions 5-6 and was consistent with a molecular weight of between (3.6-6.1) x lo7. With increasing incubation times (Fig. 3 C - E), the main peak of nascent DNA remained at fractions 4-5, but a tail developed of material of increasing molecular weight. By 2 min (Fig. 3 D) a small peak of new DNA appeared at the bottom of the tube at a position coincident with the membrane structure. This peak was enlarged at 5 rnin (Fig. 3 E) although it contributed less than 10 % of the total nascent DNA synthesized.

The reduction in the molecular weight of the heavier new DNA at the extended time in the pulse- chase experiment was strongly suggestive of nuclease activity. This evidence, plus that presented in the experiment described above, led us to investigate this possibility further. 'Complexes', pre-labelled with [14C]thymidine, were prepared and pulse-labelled for 1 min, 20 min and 3 h with the normal nucleotide mixture. A control in which the nucleotide mixture was replaced by 0.02 M Tris/5 mM Mg2+/1 mM ATP was also incubated for 3 h, equivalent to the longest pulse-labelling time. The samples were analysed on alkaline sucrose gradients.

At 1 rnin the genome DNA sedimented to the 60 % sucrose shelf (fraction 21), indicating that most of the material had a molecular weight in excess of lo7 (32 S, Fig.4A). After a 20-min reaction the profile of genome-DNA distribution was much broader than that for 1 min with the peak at fraction 12, consistent with a molecular weight of 3 x lo6 (20 S, see Fig.4B). The genome of the DNA-membrane complexes' pulse-labelled for 3 h had a peak at fraction 10 (17.5 S) which indicated a molecular weight of about 2 x lo6 (Fig.4C). The control gradient, where the 'DNA-membrane complexes' were incubated with buffer and ATP for 3 h, did not show any comparable degradation, although there was a very small shift of the DNA to an average M , of 9 x lo6, sedimenting at fraction 19 (Fig.4D). No loss in total DNA radioactivity was found in the incubated controls in any of the experiments during this study. The profiles of the newly synthesized DNA made after 20-min and 3-h incubations were rather similar, although there was evidence of an increased movement to lower molecular weights at the longer incubation time. This may be seen by the almost complete disappear- ance of the higher-molecular-weight peak after 3 h. This high-molecular-weight material was composed of genome DNA and about 30% of the nascent material. Both the parental DNA and this nascent

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Fig. 4. The concomitant degradation of genome Li4C]DNA during the synthesis of nascent DNA. ‘Complexes’ were isolated from bacteria labelled with [‘4C]thymidine (for genome DNA). Aliquots were pulse-labelled for (A) 1 min, (B) 20 min or (C) 3 h. The profile for unpulsed material incubated for 3 h in 20 mM Trisl5 mM Mg2+/ 1 mM ATP is shown in (D). Other experimental details are as for Fig. 1. (O---O) Genome [14C]DNA; (+-+) nascent [3H]DNA

DNA appeared to be degraded in a similar manner during synthesis, a fact strongly suggestive that the two types of DNA were covalently attached to each other. There was no apparent degradation of the new DNA which originally sedimented at fraction 7 (in-

The ‘DNA-Membrane Complex’ of E. co/i B/r

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Fig. 5. Distribution of nascent and genome DNA on neutral and af- kaline cuesium chloride density gradients. ‘Complexes’ from [14C]- thymidine-labelled bacteria were pulsed with [3H]dCTP for 2 min, and stopped with 0.04 M EDTA. After digestion of RNA (by RNase) and protein (by pronase), samples were centrifuged in (A) neutral and (B) alkaline caesium chloride solutions for 48 h at 1 O O O O O x g and 25 “C. (+-+) Nascent [3H]DNA; (G---o) genome [14C]DNA

dicating a molecular weight of 800000). Even after the 3-h pulse-label, when no new DNA had been synthesized for at least 2.5 h, there was no change in the size of the small-molecular-weight nascent DNA.

In parallel experiments where the nucleotide mixture lacked ATP, the molecular weight distribution of the DNA (both nascent and genome) was virtually identical to that in Fig. 4, although the rate of synthesis was reduced by about 30 %.

Analysis of Nascent and Genome DNA on CsCl Gradients

Analysis of pulsed ‘complexes’, containing 14C- labelled genome DNA, on caesium chloride density gradients showed that nascent DNA was double- stranded (Fig. 5A) with a density of 1.715 g/cm3 and contained material of lower density (1.700 - 1.705 g/ cm’). After denaturation a single peak (e 1.765 g/cm3)

W. Robertson and D. Watkins

was obtained for both parental and nascent DNA (Fig. 5 B) ; the smaller size of the latter was emphasized by the broader profile of the tritiated DNA. To differentiate between repair (extension of existing parental strands) and replicative synthesis, E. coli B/r were grown in the presence of br~mo[’~C]uracil (see Materials and Methods), then lysed and pulsed in the normal way. Incorporation of bromouracil into genome DNA was shown on alkaline caesium chloride gradients by a shift in DNA density from 1.765 g/cm3 (Fig.6A) to 1.775 g/cm3 (Fig.6B). The appearance of nascent DNA at the higher density would indicate that it was covalently bound to existing bromouracil-containing material, i.e. a product of repair-type synthesis. When the nascent DNA profile (Fig. 6 B) was extrapolated to coincide with the I4C- labelled genome-DNA peaks, approximately 27 of new DNA was found to be associated with parental material. This figure, allowing for the fact that heavy strands account for only 80 o/, of the total 14C-labelled genome DNA, is very close to that found for heavier DNA in the alkaline sucrose gradients.

DISCUSSION

DNA synthesis by the ‘complexes’ was of limited duration and was about 15 - 20 % of the rate in vivo. Cessation of synthesis (complete at 20 min) was not due to depletion of substrates or to the production or release of inhibitors and has also been observed by other workers using ‘complexes’ [l, 21. At present the reason for it is not known.

The ‘complexes’ isolated from E. coli B/r had a major difference from those isolated from K12 strains of E. coli. Other workers [1,2] found that the DNA synthesis insensitive to p-chloromercuribenzoate and N-ethylmaleimide (presumed to be due to polymerase I) of ‘complexes’ derived from polA+ K12 bacteria was lost during isolation. In the present work the insensitivity of DNA synthesis to p-chloromercuribenzoate, coupled with only 14% inhibition by N- ethylmaleimide, indicated that most of the synthesis by ‘complexes’ of E. coli B/r was mediated through polymerase I which does not require a functional - SH group for its activity. In the presence of dithiothreitol, an - SH-group-protecting agent, there was a 20 % increase in activity, suggesting that a functional -SH group was necessary for the full expression of DNA synthesis. This may indicate that a polymerase (or polymerases) other than polymerase I was active to some extent. However, it has been shown that isolated polymerase I11 was totally inhibited by KCl at a concentration of 0.2 M [15], a concentration which has no effect upon the DNA synthetic activity of E. coli B/r ‘complexes’. Moreover, polymerase I11 had only 10 % activity in 0.08 M KC1, the salt concen-

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Fig. 6. Distribution of nuscent and 5-brornouracil-labelled genome D N A on alkaline caesium chloride densizy gradients. Complexes from bacteria labelled with (A) [‘4C]thymidine or (B) 5-broiiiouracil/ [14C]thym~dine were pulsed with [3H]dCTP for 2 min, stopped and digested as in Fig.5. Samples were then centrifuged in alkaline caesinm chloride gradients. In (A) thymine was substituted for 5-bromouracil. (0- ---O) Genome [14C]DNA; (O----O) genome br5U-containing [I4C]DNA; (+ ~ +) nascent [3H]DNA

tration in the nucleotide mixture used in this study and in the work of Knippers and Stratling [l] and Okazaki et al. [2]. These observations indicate that polymerase 111 was unlikely to be active in E. coli B/r ‘complexes’.

The partial dependence of DNA synthesis on ATP (Table 1) was similar to that obtained with an isolated polymerase I complex [19] and reinforces our conclusion that a major part of DNA synthesis by ‘complexes’ of E. coli B/r is mediated by DNA polymerase I. An essential role for this enzyme in the replication of DNA has now been proved [20,21].

The response of the ‘complexes’ to either mild or severe shearing forces was of interest; in the former, DNA synthesis was unaffected by the treatment (Table 3) although it has been shown by Robertson et al. [22] that up to 20% of genome DNA could be removed from the membrane by such treatment. In their experiments [22] polymerase activity was found to be primarily associated with the DNA rather than the membrane, a result in agreement with the findings of Stratling and Knippers [18]. After sonication, the breakdown of membrane and DNA such that it was no longer acid-precipitable was inversely related to stimulation of DNA synthesis. This enhanced ‘repair’

14 The ‘DNA-Membrane Complex’ of E . coli Bjr

synthesis was not evident after mild shear sufficient to detach up to 20% of the genome DNA [22] from the membrane (Table 3). Moreover, the isolation of the ‘complexes’ by gentle lysis involved less detach- ment of DNA bound to membrane and fewer strand breaks than that found after mild shear. This suggests that a substantial part of the DNA synthesis mediated by the ‘complex’ is unrelated to repair.

Alkaline sucrose gradient analysis showed that nascent DNA synthesized by the E. coZiB/r ‘complexes’ was of two distinct sizes, one of molecular weight 600 000 - 800 000 and a much heavier component prob- ably covalently attached to parental DNA. The low- molecular-weight DNA was of similar size to Okazaki pieces [23] but was not a precursor of the high-molecular-weight material, as shown by pulse-chase experiments (Fig. 2) in which polynucleotide ligase activity was not detected.

It is possible that the ligase enzyme was present but inactive towards the nascent DNA synthesized under our experimental conditions. It is unlikely that post-synthesis nuclease activity was masking a ligase function, as such activity would have to be similar in rate to the ligase and specific for the newly joined Okazaki pieces. In all experiments the lower- molecular-weight nascent DNA was present as a con- stant discrete peak. However, even though nuclease activity was demonstrably active on the heavier nascent material (e.g. Fig.2B), the DNA of this size was only slightly degraded during the first 10 min of DNA synthesis (Fig. 1).

It was clearly demonstrated (Table 4 and Fig. 3) that the onset of DNA synthesis coincided with the dissociation of most of the genome DNA from the ‘complexes’. Moreover, the observation that newly synthesized DNA was separated from the membrane at the initiation of DNA synthesis is in contrast to previous findings. Stratling and Knippers [l, 171 found that most of the DNA synthesized in vitro sedimented with the ‘complexes’ and Okazaki and his co-workers [2] observed that all the nascent DNA was rapidly sedimentable. It is possible that this separation of both new and old DNA upon the initiation of synthesis was linked with the nucleolytic attack on the genome DNA which occurred concomi- tantly with DNA synthesis (Fig. 4). Degradation was not ATP-dependent, and was unlikely to be due to endonuclease I which is competitively inhibited by tRNA [24], present in the nucleotide mixture used in this study. Furthermore, endonuclease I is not dependent on DNA synthesis and in the present work nuclease activity was only observed when DNA synthesis was apparent (Fig. 4D). Inherent nuclease activity was shown to be at a very low level by the profile of the genome DNA obtained from incubated (3 h) ‘complexes’ (Fig. 4D). This profile was similar to that (not shown) of genome DNA in ‘complexes’

prepared and held at 0 - 2 “C until denaturation and centrifugation. It has recently been demonstrated [25] that 90% of genome DNA could be released from the membrane of gently prepared E. coli lysates using a single-strand-specific endonuclease. The released DNA had an average molecular weight of about 1.2 x lo8, very similar to that observed in the present study. The specific nature of the synthesis-induced DNA release from the membrane can be seen from Fig. 3B; after only 15 s all detectable nascent DNA was detached from the complex, whilst breakdown of prelabelled genome DNA was negligible up to 10 min (Fig. 1 B). During the next 10 min, when synthesis had virtually ceased, substantial breakdown of genome DNA was observed (e.g. Fig.4B, 20-min pulse). It should be emphasized that in the present work the release of genome DNA (and the nascent DNA attached to it) during synthesis occurred without fail over many experiments.

Analysis of 5-bromouracil-substituted DNA on caesium chloride gradients indicated that a major part of the DNA synthesis mediated by the ‘complex’ was replicative in nature. Lengthening of genome DNA in which replication had been completed (5-bromouracil was ‘grown-out’ for 1 h prior to lysis) accounted for less than 30% of synthesis in the first 2 min. In these studies a substantial proportion of the nascent DNA was found at anomalously low densities (Fig. 6B). This phenomenon was even more apparent in experiments (not shown) in which bromodeoxy- urjdine triphosphate replaced dTTP in the pulsing medium. The spread of tritiated nascent DNA in these gradients made interpretation impossible. This spread of DNA to lighter densities is typical of that found for sonicated DNA [26] and may be due to 5-bromouracil distortion causing breaks or more initiation points in the replicating DNA.

In conclusion, the synthesis of new DNA by ‘DNA-membrane complexes’ of E. coli Bjr has been shown to be mediated primarily by an enzyme with the properties of polymerase I. Most of the new DNA (70 %) was synthesized in short strands (approximately 2000 nucleotides) and was not covalently attached to genome DNA. Of prime importance was the observation that, concomitant with synthesis, the new DNA and its template were specifically detached from the membrane. The presence of sites at the membrane susceptible to an endonuclease has been reported [25] but has not previously been linked to DNA synthesis itself. Such a mechanism is of obvious interest if it is accepted that replication can only occur at or near DNA-membrane-associated sites.

REFERENCES

1. Knippers, R. & Stratling, W. (1970) Nature (Lond.) 226, 713- 717.


2. Okazaki, R., Sugimoto, K., Okazaki, T., Imae, Y. & Sugino,

3. Cramp, W. A,, Watkins, D. K. & Collins, J. (1972) Nut. New

4. Cramp, W. A,, Watkins, D. K. & Collins, J. (1972) Int. J .

5. Robertson, W. R. (1976) Ph.D. Thesis, University of London. 6. Godson, G. N. QL Sinsheimer, R. L. (1967) Biochim. Biophys.

7. Smith, D. W., Schaller, H. & Bonhoeffer, F. (1970) Nature

8. Daniels, M. J. (1969) Biochem. J . 115, 697-701. 9. Baxter-Gabbard, K. L. (1972) FEES Left. 20, 117-119.

A. (1970) Nature (Lond.) 228, 223 - 226.

Bid. 235, 76 - 77.

Radiat. Bid. 22, 379- 387.

Acta, 149, 476-488.

(Lond.) 226,711-713.

10. Barber, E. T. (1966) Nut1 Cancer Inst. Monogr. 21, 219-239. 11. McEwen, C. R. (1967) Anal. Biochern. 20, 114-149. 12. Studier, F. W. (1 965) J . Mol. Bid. 1 I , 373 - 390. 13. Gillies, N. E. (1966) J. Gen. Microbiol. 45, 97-111. 14. Fry, M. QL Artman, J. (1969) Biochem. J . 115, 287-294. IS. Kornberg, R. QL Gefter, M. L. (1972) J . B i d . Chern. 247,5369-

16. Laipis, P. J., Olivera, B. M. & Ganesan, A. T. (1969) Proc. 5315.

Natl Acad. Sci. U.S.A. 62, 289-296.

17. Stratling, W. & Knippers, R. (1971) J . Mol. Bid. 61,471 -487. 18. Stratling, W. & Knippers, R. (1971) Eur. J . Biochem. 20, 330-

19. Hendler, R. W., Pereira, M. & Scharff, R. (1975) Proc. Natl

20. Konrad, G. B. & Lehman, I. R. (1974) Proc. Natl Acad. Sci.

21. Olivera, B. N. s( Lehman, I. R. (1974) Nature (Lond.) 250,

22. Robertson, W. R., Cramp, W. A. & Watkins, D. K. (1978) Int. J . Radiat. Biol. 34, 101 -118.

23. Okazaki, R., Okazaki, T., Sakabe, K., Sugimoto, K., Kainuma, R., Sugino, R. QL Iwatsuki, N. (1968) Cold Spring Harbour Symp. Quant. Bid. 33, 129- 143.

24. Lehman, I. R., Roussos, G. G. & Pratt, E. A. (1962) J . Bid. Chern. 237,819-828.

25. Abe, M., Brown, C., Hendrickson, W. G., Boyd, D. H., Clif- ford, P., Cote, R. H. & Schaechter, M. (1977) Proc. Nut/ Acud. Sci. U.S .A . 74,2756-2760.

26. Billen, D. & Hellermann, G. R. (1974) Biochim. Biophys. Acta,

339.

Acad. Sci. U.S.A. 72, 2099-2103.

U.S.A. 71,2048-2051.

513-514.

361, 166-175.

W. Robertson, Division of Cellular Biology, Kennedy Institute of Rheumatology, Bute Gardens, London, Great Britain, W6 7DW

D. Watkins*, Division of Biochemistry, Faculty of Science, North East London Polytechnic, Romford Road, London, Great Britain, E l5 4LZ

* To whom correspondence should be addressed.

The ‘DNA-Membrane Complex’ of Escherichia coli B/r : Its Composition and Properties and the Fate...

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