Adenosine Triphosphate-Dependent Synthesis of Biologically Active DNA by Azide-Poisoned Bacteria

Adenosine Triphosphate-Dependent Synthesis of Biologically Active DNA by Azide-PoisonedBacteriaAuthor(s): A. T. GanesanSource: Proceedings of the National Academy of Sciences of the United States of America,Vol. 68, No. 6 (Jun., 1971), pp. 1296-1300Published by: National Academy of SciencesStable URL: http://www.jstor.org/stable/60329 .

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Proc. Nat. Acad. Sci. USA Vol. 68, No. 6, pp. 1296-1300, June 1971

Adenosine Triphosphate-Dependent Synti Azide-Poisoned Bacteria

(Bacillus subtilis/gene markers/detergent Brij-58)

A. T. GANESAN

Lt. J. P. Kennedy, Jr., Laboratories for Molecular Medicine, Departmer School, Stanford, California 94305

Communicated by Joshua Lederberg, April 5, 1971

ABSTRACT Nonviable cells of Bacillus subtilis, when made permeable by treatment under controlled conditions with nonionic detergent, can be shown to replicate DNA normally and to repair certain regions of the chromosome. The former process is stimulated by ATP in the presence of dATP, dCTP, dGTP, and dTTP. The product, a result of semiconservative replication, is biologically active; synthesis of the newly-formed regions of the chromosome appears to be sequential.

Extensive biochemical and morphological evidence suggests that the bacterial chromosome is replicated in an orderly sequential mode, possibly by an enzyme complex bound to cell wall membrane (1-3). Attempts to isolate a structurally intact, active complex that would promote such a process have not been very successful. Here we report a method of making nonviable cells permeable to externally added substrates. These cells have lost most of their DNA polymerase activity and also significant amounts of nucleases and cellular proteins, but are capable of continued DNA synthesis in the presence of four deoxyribonucleoside triphosphates. This synthesis is stimulated by the addition of ATP. A similar response has been reported recently in toluene-treated cells of Escherichia coli (4, 5). The Bacillus subtilis system has the advantage that we can characterize the biological activity of the product and the direction of replication.

MATERIALS AND METHODS

Actively growing cultures of B. subtilis, SB 168 (try-2) and SB 19 (wild type), were frozen in liquid nitrogen. These stocks were thawed, washed, and resuspended at 109 cells/ml of 5% sucrose solution containing 0.01 M Tris HCI buffer, pH 7.5; 0.001 M MgCl2; 0.001 M l-mercaptoethanol; and sodium

azide, 0.05 M. These cells were then made permeable by the addition of Brij-58 (6), a nonionic detergent. The time required to reach this permeable stage was monitored by observing the cells under a phase-contrast microscope or by staining with Giemsa (7). During a period of 1-3 hr, 55% of the cellular proteins, containing more than 95% of the DNA polymerase activity [assayed with poly(dA) .poly(dT) as substrate (3) ], and a large portion of the nucleases were released into the medium and were discarded after centrifugation. The permeable cells were resuspended in the azide buffer.

Abbreviations: HH, HL, LL, and DH DNA: respectively heavy (d = 1.756 g/ml), hybrid (1.729), light (1.703), and denatured heavy (1.810) DNA.

1,:

tesis of Biologically Active DNA by

t of Genetics, Stanford Medical

For characterization of the products of synthesis, cells were

grown in a medium containing 15NH4+, D20, and [i4C]thymine (3) to give the DNA a buoyant density of 1.756 instead of 1.703 g/ml in CsCl. The three types of assay conditions for DNA polymerase (3) were (a) equimolar mixtures of the deoxyribonucleosides of adenine, guanine, cytosine, and thymine (referred to as dN) plus ATP; (b) an equimolar mixture of the four deoxyribonucleoside triphosphates (referred to as dNTP) of the above bases; and (c) the same as (b), plus ATP. These and radioactive substrates were obtained from Schwarz BioResearch. DNA was purified carefully to minimize shear effects that might confuse repaired regions with semiconservatively replicated sequences in density gradients. This was accomplished by lysing the Brij-treated cells with sodium dodecyl sulfate (4 mg/ml) and digesting the lysate with Pronase (Calbiochem) (1 mg/ml) overnight at room temperature (3). Cesium chloride density gradients were centrifuged in a Spinco No. 50 angle-head rotor at 34,000 rpm for 66 hr at 4?C. Density calculations were made with un- labeled B. subtilis DNA of normal density as a. referenice; this DNA carries mutations that do not interfere with the assay of the DNA species under study. Alkaline CsCl gradients were used at pH 12.5 (8). In all the figures, the density positions of heavy, hybrid, light, and denatured heavy DNA molecules, corresponding to densities of 1.756, 1.729, 1.703, and 1.810 g/ml in CsCl are designated as HH, HL, LL, and DH.

RESULTS

Live cells, when incubated with ['H]thymidine and the other three deoxyribonucleosides, exhibit continued linear incorporation into the DNA. However, when inhibitors such as sodium azide are added, the incorporation stops, as is implicit in the earlier kinetic experiments (1). During the next few minutes the cells gradually lose their viability. These cells are not permeable to triphosphates. When such cells are treated with Brij-58 and assayed for DNA synthesis in the presence of dN and ATP, incorporation is resumed after 2 hr (Fig. la). The rate of tracer incorporation in different experiments is similar to that observed in live cells. These Brij- treated cells are now permeable to externally added Pronase, DNase-1, deoxy- and ribonucleoside phosphates. The extent of ATP stimulation in different experiments is 8- to 25-fold, compared to the control. Similar stimulation could be observed with other ribonucleoside triphosphates, but not by monophosphates and only partially by diphosphates. The proportion of viable cells in these experiments was 0.008% or

m96



Proc. Nat. Acad. Sci. USA 68 (1971)

less, and too low to interfere with the assay systems, as verified in reconstruction experiments.

Optimal synthesis depends on the presence of cellular DNA, all four deoxyribonucleosides, and ATP; it is sensitive to Proniase and DNase (Table 1). When the time of incubation is increased beyond the 3 hr, the ability of the treated cells to incorporate dN in the presence of ATP gradually ceases, possibly because of loss of kinases, but the cells can imncorporate dNTP, to an extent that can be increased by mild treatment with DNase-1. This mode of synthesis is not inhibited by N-ethylmaleimide (Table 2) and has been interpreted as a repair process (4). The rate of incorporation is rapid initially, but decreases on continued incubation at 37?C because of nuclease degradation (Fig. lb). With ATP this initial rapid increase is inhibited somewhat, but the extent of DNA synthesis on prolonged incubation is stimulated anywhere from 3.5- to 15-fold (Fig. lb). This process is inhibited by N-ethylmaleimide and has been interpreted as normal DNA synthesis (4). Since the amount of repair depends on the extent of DNA breakage caused in the preparation of permeable cells, one has to take this incorporation into account in evaluating the extent of stimulation observed with ATP. Details concerning the process of this repair synthesis form the subject of a subsequent communication.

In an analysis of the nature of the observed incorporation, permeable cells were made from cells grown in heavy medium. In certaini experiments, these cells were washed and allowed to grow in a light medium for 15 min, so that 10% of the DNA molecules were in hybrid form (Fig. 2a). Under these conditions, extensive replication is marked by the production of hybrid DNA molecules, as judged by a density shift in a CsCl gradient as well as by the amount of substrate used in synthesis. On the other hand, repair synthesis should be reflected by poor incorporation, with no significant change in density profiles.

Permeable cells, when incubated with dN, and without ATP, did not show any incorporation. In the presence of ATP, a significant proportion of heavy DNA approaches the

TABLE 1. DNA synthesis (incorporation of deoxynucleosides) in permeable cells of B. subtilis

System Activity (%)

Complete 100 - ATP 4.8 + Pronase, 100 jg/ml 4.9 + Bacterial alkaline phosphatase (2 U/

ml) + ATP, 2mM 4.6 -any one deoxynucleoside 27.0 +AMP, 2mM 4.8 + A)P, 2 mM 28.0 + A1)P, 1 mM + ATP, 1 mM 64.0 - AI)P, I mM, + AMP, I mM 34.0 + DNase, 10 mg/ml 4.4 + heated cells 3.6

Background 2. 2

Details of the complete system are given in Fig. 2 and ref. 3. O.l-ml aliquots were precipitated with cold trichloroacetic acid onto a Millipore filter soaked in 0.2 M sodium pyrophosphate. The precipitate was dried and counted after extensive washing. 100% represents 8200 cpm of 3H. Cellular DNA was not labeled. Azide was present in all the reactions at 0.1 M.

DNA Synthesis by Detergent-Treated Bacteria 1297

7 / /

I) S c dNTP+ ATP 0 /

dNAdN ...

I, * i

- - ~ Vt dNTP

' IAI"N I I

0 1 2 35 3 10 20 50 Hours /Minutcs

FIG. 1. Effect of ATP on incorporation of four deoxynucleosides (dN) and deoxynucleoside triphosphates (dNTP). 3 X 109 of azide-poisoned cells per ml were incubated at 0?C with Brij-58, and aliquots of 0.1 ml were taken at the indicated time and centrifuged. The sedimented cells were assayed at 37?C for 30 min with dN, dN + ATP, and dNTP. Substrates for DNA synthesis and cell concentrations were the same as in the legend of Figs. 2 and 3 except that cellular DNA was not labeled. After the reaction, the mixture was acid-precipitated and counted for radio- activity (3). a, Permeable cells obtained as above after 3 hr of incubation at 0?C were used in this assay. b, 0.1 ml of cells were incubated at 37?C for different lengths of time with dNTP or dNTP + ATP and assays were performed as above, with substrates as indicated in Fig. 3.

hybrid level (Fig. 2b). Under these conditions the template DNA was found to undergo degradation. In these experiments it is difficult to distinguish repair from normal synthesis. In the presence of dNTP, and the absence of ATP, there was poor synthesis as reflected by the DNA density and amount of substrate incorporation, and very few light molecules were synthesized (Fig. 3a). With ATP, the resulting profile (Fig. 3b) showed the generation of hybrid and some completely light molecules. In addition there were molecules of inter- mediate density present between hybrid and heavy DNA. The overall distribution of [14C]DNA has moved towards lighter strata. The incubation with dNTP alone, there was a

TABLE 2. DNA synthesis (incorporation of deoxynucleotides) in permeable cells of B. subtilis

System Activity (%)

Complete 100 - ATP 31 - any one deoxytriphosphate 11 - ATP + N-ethylmaleimide, 2 mM (ref. 4) 32

- Pronase 1.9 + DNase 1 . 6

-Mg++ 3.8 + Pyrophosphate, 25 mM 2.2

Complete system as in Table 1 and Fig. 2. 100% corresponds to 13,000 cpm of 'H. Cellular DNA was not labeled. Substituting NAD or NADP for ATP at similar concentrations did not stimulate synthesis.



1298 Biochemistry: A. T. Ganesan

HH HL LL

- \ Control !l

2- I

o0 1 '? ???

C) ^ dN+ATP

ft - b'

I k,

Ha

15 25 35 45 55 Fractions

FIG. 2. DNA synthesis with four deoxynucleosides (dN) and ATP. The reaction mixture, I ml, contained 3 X 109 permeable heavy cells, conitaining 10% of hybrid (HL) DNA molecules having 45 nmol of [l4C]thymidine-labeled DNA (930 cpm/nmol) and 45 nmol of each of four deoxynucleosides, of which thymidine was labeled with 3H at 30,000 cpm/nmol. ATP, when used, was at 2 mM. Other reagents were at concentrations described before (3). After 60 min of reaction at 37?C, the isolated DNA contained 80,000 and 150,000 cpm of 14C and 'H in acid-precipitable form. Recovery from the gradient was always more than 80% of the input. 70 fractions were collected from a gradient volume of 8 ml and analyzed as described before (3) for acid-precipitable counts and transforming activity. 1 jg of standard light B. subtilis DNA was added to each gradient. a, Profile of a control sample having 10% hybrid material. b, Profile of synthesis obtained with dN + ATP.

TABLE 3. Density distribution of origin, middle, and terminus gene activities in DNA after synthesis with dNTP and t TP

Time of synthesis (minm)

0 20 40

Density positionl

HH HL LL HH HL LL HH HL LL

ade6 + 0.94 0.06 0.0 0.84 0.12 0.04 0.44 0.39 0.17 leu+ 0.97 0.03 0.0 0.80 0.19 0.01 0.39 0.55 0.07 met'+ 0.96 0.04 0.0 0.75 0.24 0.01 0.37 0.66 0.03

HH, HL, and LL refer to heavy, hybrid, and light DNA density strata in CsCl gradients. Transformation assays were performed at limiting DNA concentrations, with a 'multiple auxotroph (adem6 leu met') as recipient bacterium. The values represent the proportion of colonies observed at different density positions.

Proc. Nat. Acad. Sci. USA 68 (197.1)

4 dNPA " 6

4-dNTP HH HL LL

X \ 1n Ox 04- a . 1

loss of 3 of the Oqp l DNA. The amon 0 X

1t inve tigat th - 1 '

20 0 0 50

Fractions FIG. 3. DNA synthesis with deoxynucleoside triphosphates

and with ATP. Assays as for Fig. 2. Of the four, dATP contained 3H at 30,000 cpm/nmol. Without ATP (a), the total amount of incorporation was equivalent to 10 nmol of DNA; with the cofactor (b), it was 35 nmol.

loss of 33% of the template DNA. The amount of imicorpora- tion reflected an amount of syinthesis equivalent to 20% of the template DNA. With ATP and dNTP, there was 7% loss of template DNA and the extent of synthesis here was 3.5 times greater than without ATP. Without ATP, 3.2 ag amid with ATP, 11.5 gol of DNA was synthesized.

In order to investigatemplat ure n of the products formioed in the presence and absence of ATP, I made commpletely heavy, permeable cells and performed above syn the sis. The isolated DNA was denatured at alkaline pH anid sub- jected to centrifugation in alkaline CsCl. In the presence of ATP a significant proportion of light DNA strands, relatively free of template atoms, was produced (Fig. 4a). There also appear to be heavy chains covalently linked to long stretches of light atoms and heavy chains linked to short stretches of light atoms. The former may be the product of semiconservative replication and the latter the products of repair replication. In the control without ATP, a 5-fold lower synthesis was observed as well as loss of template (Fig. 4b).

If the synthesis in the presence of ATP represents normial replication, at least in part, the lighter species of molecules should be biologically active. Completely heavy SB 168 cells were made permeable and allowed to synthesize D)NA for 40 min in the presence of either dN + ATP or of dNTP + ATP. The DNA was fractionated in a CsC1 gradiemnt and assayed by transformation for genes that are located at the origin, middle, and terminus of the chromosome (adce6, leu, and mets) (9). Profiles for adeus+ and metm+ are presented in Fig. 5; all three marker activities and their distributions in




5 -I , - - 10

a^ A^ldNTP+ATP 43'

D2 H

0 2 d I' ; 6

C_)v I t I I t.

Fractions FIG. 4. Pyenography of the denatured DNA after synthesis

with dNTP in the presence (a) and the absence (b) of ATP. Permeable cells made from completely heavy cells were used. The DNA was labeled with [14C] thymine and had a specific activity of 4000 cpm//Ag. Synthesis was performed as for Fig. 3. The product was denatured at pH 12.5, centrifuged in alkaline CsCl at the same pTI, and analyzed for 3H and 14C after acid precipitation.

the gradient are given in Table 3. In Fig. 5a, the control (incubation. without substrates) shows the expected unimodal distribution for two of the genetic activities. With dNTP alone there was no change in the profile of genetic activity. With dN plus ATP (Fig. 5b) more met5+ activity is f'ound near the hybrid position compared to adel6+. The ratio of bio-

logical activity of molecules with fully heavy density, which was originally 3.6 for ade/met, had decreased to 0.9, which

suggests that under these conditions previously initiated chromosomes complete the replication cycle (as 'judged by tlhe shift of met5+ activity to a hybrid level), but that new iinitiations are not favored. The amount of synthesis observed here was always 2 to 8 times less than the one with dNTP

plus ATP. The behavior of other markers close to the terminus was similar.

The pattern of activity with dNTP + ATP was different. In gene4. Pyral, both the adenatur and metDNA aft gensynthes have moved to

hybrid densities, which suggests not only that certain chromosomes have finished replication but also that others have initiated ew rounds of synthesis. There is also adeific activity near the light DNA, which doed as not have a significa. 3. Thet amount

wasThe position occupiured byat pH 12.5, centrifuged in alkaline CsCid at the exactly with the position of light stand analyzed for H andard DNA. af inter acid precipitation.

this as the result of a second initiation in a, t contromosome that

had the growing point aheadtwo of the ade genetic activities. Withe cellsdNTP were madlone pthermee was no change inetic analysise of gesynthesis at 0, 20,ivity.

athe hybrid pos40 mition comis presented in Table 3. The distributo of bion pattern

of thromosomee genes wicomplete the rent chromosomal location cycle (as judgedis con-by

of three genes with different chromosomal locations is con-

DNA Synthesis by Detergent-Treated Bacteria 1299

HH LL

16'_ Control

12 -

p) -1f < o?-o met +

D I I

4 I 4

Q160 C o dN, ATP-

4-

dN + ATP and dN TP, ATP

4b, with completely heavy permeable cells, but for 40 min. DNA

from this reaction mixture was centrifuged in CsCl, and 0.01 ml of each fraction was assayed for biological activity (3) by means of competent cells containing mutations for adel6 and met5. Radio- activity profiles (not given) were similar to 4b. 100% of adeG+ and met5+ represents 2000 and 700 colonies in a, 3500 and 1800 for b, and 15,000 and 6300 in c.

sistent with the interpretation that one is observing both initiation and termination of chromosomal replication at least in a large proportion of permeable cells.

DISCUSSION

DNA synthesis, by a mechanism not yet clarified, in this azide-poisoned, pyrophosphate-permeable system is pro- moted several-fold by ATP. Pyridine nucleotides, tested because NAD is a cofactor for polynucleotide ligase in B. subtilis (10), do not replace ATP in this reaction. It is possible that yet another enzyme activity that is coupled to ATP is involved in DNA synthesis. If replication normally depends on ATP generation, this would explain the observation that



1300 Biochemistry: A. T. Ganesan

in E. coli a cyanide- and CO-sensitive step is required at all stages of replication (11). In the presence of these inhibitors DNA polymerase and ligases function normally (12), but extensive synthesis at the growing point is blocked. It was shown earlier (1) that the addition of azide prevents DNA synthesis in B. subtilis. In E. coli, similar inhibitors not only block DNA synthesis, but also prevent nucleases from degrading the DNA; this suggests that in vitro these systems are energy- dependent (13, 14). The ATP-stimulated synthesis of DNA is predominantly dissociated from soluble DNA polymerase activity, as 95% of the latter is leached out during the process of making cells permeable.

Synthesis in the absence of ATP results in unimodal distribution of several gene activities, consistent with repair replication of template DNA. In contrast, as shown here, synthesis with ATP results in an asymmetric distribution of gene activities that depends on their respective location on the genetic map. Extensive synthesis in the presence of dNTP and ATP indicate both initiation and termination of chromosome replication. We do not know, however, whether this synthesis reflects the normal in vivo type of chromosome replication.

The author acknowledges the critical comments anid advice of Profs. Joshua Lederberg, Charles Yanofsky, and R. L. Baldwin and the expert assistance of Mrs. Rae Ellen Syverson. This work


was aided by grants from the National Institute of General Medical Sciences, GM-14108, 2 T01- GM-295 and GB- 8739 from National Science Foundation. The author was a recipient of a U.S. Public Health Service Research Career Program Award, GM-50199.

1. Ganesan, A. T., and J. Lederberg, Biochem. Biophys. Res. Commun., 18, 824 (1965).

2. Ryter, A., Current Topics in Microbiology and Immunology (Springer-Verlag, New York, 1969), Vol. 49, p. 151.

3. Ganesan, A. T., Proc. Nat. Acad. Sci. USA, 61, 1058 (1968). 4. Moses, R. E., and C. C. Richardson, Proc. Nat. Acad. Sci.

USA, 67, 674 (1970). 5. Mordoh, J., Y. Hirota, and F. Jacob, Proc. Nat. Acad. Sci.

USA, 67, 773 (1970). 6. De Lucia, P., and J. Cairns, Nature, 224, 1164 (:1969). 7. Ganesan, A. T., Compt. Rend. Lab. Carlsberg, 31, 149 (1959). 8. Vinograd, J., J. Morris, N. Davidsoni, and W. F. D)ove, Jr.,

Proc. Nat. Acad. Sci. USA, 49, 12 (1963). 9. Yoshikawa, H., and N. Sueoka, Proc. Nat. Acad. Sci. USA,

49, 559 (1963). 10. Laipis, P. J., B. M. Olivera and A. T. Ganesaun, Proc. Nat.

Acad. Sci. USA, 62, 289 (1969). 11. Denhardt, D. T., and A. B. Burgess, Cold Spring Harbor

Symp. Quant. Biol., 33, 449 (1968). 12. Cairns, J., and D. T. Denhardt, J. Mol. Biol., 36, 335 (1968). 13. Davern, C., J. Cairns, P. De Lucia, and A. Gunmsalus, Sym-

posium on Organizational Biosynthesis (Academic Press, New York, 1967), Vol. 49.

14. Cairns, J., and C. Davern, J. Mol. Biol., 17, 418 (1966).



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Adenosine Triphosphate-Dependent Synthesis of Biologically Active DNA by Azide-Poisoned Bacteria